Investigation of stress biomarkers in human peripheral blood ...

Investigation of stress biomarkers in human peripheral

blood mononuclear cells in response to chronic

isoproterenol treatment

Dissertation zur Erlangung des

akademischen Grades eines Doktors der Naturwissenschaften

(Dr. rer. nat.)

vorgelegt von

Palombo, Philipp

an der

Mathematisch-Naturwissenschaftliche Sektion

Fachbereich Biologie

Konstanz, 2018

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-1l2hvr1cqeoof1

Tag der mündlichen Prüfung: 14.12.2018

1. Referent/Referentin: Alexander Bürkle

2. Referent/Referentin: Markus Christmann

“It is not stress that kills us, it is our reaction to it„

-Hans Selye-

“Houston, We`ve Had a Problem”

-James A. Lovell-

Table of content

I

Table of content

CHAPTER I ......................................................................................... 1

1 Introduction ...................................................................................... 1

1.1 Biological background of stress .............................................................................................. 1

Medical significance of stress ......................................................................................... 3 1.1.1

1.2 Adrenergic receptor family...................................................................................................... 6

Signaling of the β2-AR .................................................................................................... 7 1.2.1

1.3 Catecholamines ..................................................................................................................... 13

Effects of catecholamines on immune cells .................................................................. 14 1.3.1

Isoproterenol .................................................................................................................. 16 1.3.2

Poly(ADP-ribose) polymerases and poly(ADP-ribosyl)ation ....................................... 18 1.3.3

1.4 Cellular senescence ............................................................................................................... 21

Senescence marker and characteristics of senescent cells ............................................. 23 1.4.1

β-adrenergic signaling and genomic stability ................................................................ 26 1.4.2

2 Objective ........................................................................................ 28

3 Material and Methods .................................................................... 30

3.1 Material ................................................................................................................................. 30

Chemicals ...................................................................................................................... 30 3.1.1

Laboratory equipment ................................................................................................... 32 3.1.2

Consumables ................................................................................................................. 34 3.1.3

Buffers and solutions ..................................................................................................... 35 3.1.4

Cell lines and cell culture reagents ................................................................................ 39 3.1.5

Antibodies and dyes ...................................................................................................... 40 3.1.6

Kits ................................................................................................................................ 40 3.1.7

Software ......................................................................................................................... 41 3.1.8

3.2 Methods ................................................................................................................................. 41

Table of content

II

General aspects of cell culture ....................................................................................... 41 3.2.1

PBMC isolation ............................................................................................................. 41 3.2.2

Isoproterenol treatment of PBMCs ................................................................................ 42 3.2.3

Analysis of cellular cAMP levels .................................................................................. 43 3.2.4

Analysis of cellular ROS ............................................................................................... 45 3.2.5

NAD+ Cycling assay ...................................................................................................... 46 3.2.6

Analysis of gene transcription by real-time PCR .......................................................... 47 3.2.7

PARP1 activity under NAD+ saturated conditions ........................................................ 53 3.2.8

Sample preparation for Western blotting ....................................................................... 54 3.2.9

4 Results ............................................................................................ 60

4.1 Isoproterenol mediated DNA damage ................................................................................... 61

cAMP-signaling of the β-AR after repeated isoproterenol stimulation ......................... 61 4.1.1

Quantification of the intracellular NAD+

content in PBMCs during and after the 4.1.2

repeated isoproterenol treatment ................................................................................... 63

PAR formation after isoproterenol treatment under NAD+ saturated conditions .......... 64 4.1.3

Formation of intracellular ROS in PBMCs during the repeated isoproterenol treatment . 4.1.4

....................................................................................................................................... 65

4.2 Repeated isoproterenol treatment induced senescence like phenotype ................................. 69

Gene expression in PBMCs after the repeated isoproterenol treatment ........................ 69 4.2.1

p16 protein expression after repeated isoproterenol treatment ...................................... 72 4.2.2

4.3 Degradation of isoproterenol under cell culture conditions .................................................. 73

Detection of isoproterenol by absorbance detector ....................................................... 73 4.3.1

Detection of isoproterenol by the fluorescence detector ............................................... 77 4.3.2

Detection of isoprenochrome by the absorbance detector ............................................. 78 4.3.3

Isoproterenol stability in cell culture media at 4 °C ...................................................... 80 4.3.4

Isoproterenol stability in cell culture media at 37 °C .................................................... 82 4.3.5

Isoproterenol concentration after the single dose treatment of PBMCs ........................ 84 4.3.6

Isoproterenol concentration during and after the four-fold treatment of PBMCs ......... 85 4.3.7

Isoproterenol concentration during and after the eight-fold treatment of PBMCs ........ 87 4.3.8

Isoproterenol concentration in cell culture media after a single administration ............ 90 4.3.9

Table of content

III

Isoproterenol concentration in cell culture media during and after the four-fold 4.3.10

administration ................................................................................................................ 91

Isoproterenol concentration in cell culture media during and after the eight-fold 4.3.11

administration ................................................................................................................ 93

5 Discussion ...................................................................................... 96

5.1 Isoproterenol mediated DNA damage ................................................................................... 98

Formation of intracellular ROS ................................................................................... 100 5.1.1

Intracellular cAMP and NAD+ content after repeated isoproterenol treatment of PBMCs5.1.2

..................................................................................................................................... 103

5.2 Repeated isoproterenol treatment induced senescence like phenotype ............................... 105

5.3 Degradation of isoproterenol under cell culture conditions ................................................ 109

6 Conclusions and outlook .............................................................. 116

CHAPTER II .................................................................................... 118

7 Introduction .................................................................................. 118

7.1 DNA damage and DNA damage repair ............................................................................... 118

7.2 DNA strand break detection ................................................................................................ 120

Molecular methods ...................................................................................................... 121 7.2.1

Fluorescence methods ................................................................................................. 121 7.2.2

7.3 Automated fluorometric detection of alkaline DNA unwinding (FADU) assay ................. 123

8 Material and Methods .................................................................. 125

8.1 Material ............................................................................................................................... 125

Chemicals .................................................................................................................... 125 8.1.1

Laboratory equipment ................................................................................................. 126 8.1.2

Consumables ............................................................................................................... 127 8.1.3

Buffers and solutions ................................................................................................... 128 8.1.4

Cell lines and cell culture reagents .............................................................................. 129 8.1.5

8.2 Methods ............................................................................................................................... 129

Table of content

IV

Freezing of cells .......................................................................................................... 129 8.2.1

Thawing of cells .......................................................................................................... 129 8.2.2

Sub-culturing of suspension cells ................................................................................ 130 8.2.3

Sub-culturing of adherent cells .................................................................................... 130 8.2.4

MTT assay ................................................................................................................... 130 8.2.5

Pre-validation of the TOXXs Analyzer ....................................................................... 135 8.2.6

9 Results .......................................................................................... 141

9.1 Pre-validation of the TOXXs Analyzer ............................................................................... 141

Determination of the cytotoxicity of the chemical test compounds ............................ 147 9.1.1

Modification of the neutralization buffer of the automated FADU assay ................... 150 9.1.2

Genotoxicity of test compounds .................................................................................. 151 9.1.3

10 Discussion .................................................................................... 153

Pre-validation of the TOXXs Analyzer ....................................................................... 153 10.1.1

11 Conclusions and outlook .............................................................. 156

12 Appendix ...................................................................................... 157

12.1 Supplementary figures ......................................................................................................... 157

12.2 Genes analyzed by qPCR .................................................................................................... 165

12.3 Contribution......................................................................................................................... 167

12.4 Publications ......................................................................................................................... 168

12.5 Oral presentations ................................................................................................................ 168

12.6 Participation in courses within the teaching program of the Konstant Research School

Chemical Biology ................................................................................................................ 169

12.7 List of abbreviations ............................................................................................................ 170

13 References .................................................................................... 175

Danksagung ....................................................................................... 201

Abstract

V

Abstract

Chronic stress is associated with a higher risk for carcinogenesis as well as age-related diseases and

immune dysfunction. There are several indications that repeated or elevated release of stress

hormones, such as catecholamines, can affect the genomic stability of cells. Therefore, catecholamines

appear to be involved in the occurrence of some diseases. Studies with posttraumatic stress disorder

(PTSD) patients have shown an accelerated aging of these patients. Moreover, accumulated DNA

strand breaks and a shortening of telomeres could be observed in peripheral blood mononuclear cells

(PBMCs) of these patients. On a molecular and cellular level it was demonstrated that catecholamines

can induce the formation of reactive oxidative species (ROS) and the formation of DNA strand breaks

via two pathways. On the one hand, catecholamines stimulate the β2-adrenergic receptor and activate

the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling cascade. On the other

hand, catecholamines undergo oxidative degradation processes that involve the formation of free

radicals and ROS. Moreover, it was shown that the repeated treatment of myocardial cells with

catecholamines induce a senescence like phenotype. In a pilot study, our laboratory established an

ex vivo model to simulate the effect of the elevated and repeated release of catecholamine caused by

chronic stress. Therefore, PBMCs of healthy donors were repeatedly treated 1-fold, 4-fold or 8-fold

with isoproterenol, an epinephrine analog. Our results showed that the repeated administration of

isoproterenol induced the formation of DNA strand breaks in PBMCs, 6 hours after the beginning of

the treatment. These DNA strand breaks could be partially inhibited by the β-blocker propranolol.

24 hours after the first isoproterenol administration, a part of these DNA strand breaks remained

unrepaired. The protein level of poly(ADP-ribose) polymerase-1 (PARP1), an important DNA repair

enzyme which is activated by DNA strand breaks, was reduced by the repeated isoproterenol

treatment. Moreover, the formation of poly(ADP-ribose) (PAR) was reduced in some cells. Also the

intracellular content of the PARP1 substrate nicotinamide adenine dinucleotide (NAD+) decreased in

response to the repeated isoproterenol treatment. However, it was not possible to detect the formation

of intracellular ROS. Additional, the cAMP dependent receptor signalling was also reduced by the

repeated isoproterenol treatment. Therefore, an iterated activation of the β2-adrenergic receptor could

be excluded, at least for the cAMP dependent signalling pathway. In a second parallel performed study

the expression of senescence markers in PBMCs after the repeated isoproterenol treatment was

investigated. The induction of senescence markers in response to a repeated isoproterenol treatment

was observed previously in myocardial cells. The following effects of the repeated isoproterenol

treatment were induced in PBMCs: expression of senescence-associated β-galactosidase, an

enlargement and flattened cell morphology, decreased expression of CCND1, up-regulation of the

expression of VCAN and an inhibition of phytohaemagglutinin (PHA) induced cell proliferation.

Abstract

VI

However, no expression of the senescence markers p16 and p21 could be observed. Taken together,

the results suggested an isoproterenol induce senescence like phenotype in human PBMCs. To our

knowledge, there was no previous investigation of the isoproterenol fate under cell culture conditions.

However, it is known that the degradation of isoproterenol induces the formation of ROS and free

radicals. Therefore, the stability of isoproterenol under cell culture conditions was investigated in third

study. It is known that isoproterenol can be oxidized to isoprenochrome, a cytotoxic aminochrome.

The concentration of isoproterenol and isoprenochrome were measured in three different cell culture

media over a period of 8 h. The determined half-life of isoproterenol ranged between 30 min and 6 h

which were at least about 6-times longer than reported in human studies. The results also showed that

the stability of isoproterenol and the formation of isoprenochrome were influenced by the cell culture

medium. In a second project an inter-laboratory-validation study of a new developed FADU system

was carried out in cooperation with the Swiss Federal Laboratories for Materials Science and

Technology (EMPA) in St.Gallen and the Cetcis GmbH, Esslingen. In a first phase the technical test

of the FADU system was performed. In a second phase the ability of the FADU assay to identify

genotoxic chemicals that induce DNA strand breaks should be proofed. The new FADU platform

should also be used to investigate the influence of isoproterenol on the genomic stability. This task

could never be finished, because of technical difficulties of the new FADU platform.

Zusammenfassung

VII

Zusammenfassung

Chronischer Stress führt zu einem erhöhten Risiko von Karzinogenese, altersbedingten Krankheiten

und Immunschwäche. Es gibt einige Befunde, dass die wiederholte oder erhöhte Freisetzung von

Stresshormonen, wie Katecholaminen, die genomische Stabilität von Zellen beeinträchtigt.

Katecholamine scheinen an der Entstehung von Krankheiten beteiligt zu sein. Studien mit Beteiligung

von Patienten mit posttraumatischer Belastungsstörung (PTSD) zeigten das diese Patienten schneller

altern. Zudem scheinen sich in mononukleäre Zellen des peripheren Blutes (PBMCs) dieser Patienten

DNS Strangbrüche zu akkumulieren, sowie kann eine Verkürzung der Telomere in diesen Zellen

nachgewiesen werden. Auf molekularer und zellulärer Ebene konnte gezeigt werden das

Katecholamine an der Entstehung von reaktiven Sauerstoffverbindungen (ROS) und der Entstehung

von DNS Strangbrüchen beteiligt sind. Dabei spielen zwei Mechanismen eine zentrale Rolle.

Einerseits können Katecholamine den β2-Adrenergen Rezeptor stimulieren und damit die cyclisches

Adenosinmonophosphat/ Proteinkinase A (cAMP/PKA) abhängige Signalkaskade aktivieren. Auf der

anderen Seite können Katecholamine oxidative Abbaumechanismen durchlaufen, bei denen freie

Radikale und ROS gebildet werden. Außerdem wurde gezeigt, dass die wiederholte Behandlung von

Herzmuskelzellen mit Katecholaminen einen Seneszenz-ähnlichen Phänotypen induzieren kann. In

einer Pilotstudie in unserem Labor wurde ein ex vivo Modell zur Simulierung der wiederholten und

erhöhten Freisetzung von Katecholaminen durch chronischen Stress entwickelt. Dafür wurden PBMCs

von gesunden Spendern wiederholt mit einer, vier oder acht Dosen Isoproterenol, einem Adrenalin

Analog, behandelt. Die Ergebnisse zeigten, dass die wiederholte Gabe von Isoproterenol die Bildung

von DNS Strangbrüchen, 6 Stunden nach dem Beginn der Behandlung in PBMCs induzierte. Die

Bildung der DNS Strangbrüche konnte teilweise durch die Gabe des β-Blocker Propranolol inhibiert

werden. 24 Stunden nach der ersten Isoproterenol Gabe war ein Teil der DNS Strangbrüche weiterhin

vorhanden. Die Proteinmenge des DNS Reparaturproteins Poly(ADP-ribose) polymerase-1 (PARP1),

das durch DNS Strangbrüche aktiviert wird, wurde durch die Isoproterenol Behandlung in dem

PBMCs reduziert. Außerdem war die Bildung von poly(ADP-ribose) (PAR) in einigen Zellen und der

intrazelluläre Gehalt des PARP1 Substrates Nicotinamidadenindinukleotid (NAD+) durch die

wiederholte Gabe von Isoproterenol reduziert. Es war aber nicht möglich die Bildung von

intrazellulärem ROS zu messen. Des Weiteren nahm die cAMP vermittelte Signalweiterleitung ab.

Dies deutet darauf hin, dass keine wiederholte Stimulierung des β2-Adrenergen Rezeptors stattfand,

zumindest nicht für den cAMP/PKA Signalweg. In einer zweiten parallel durchgeführten Studie wurde

untersucht ob die wiederholte Isoproterenol Behandlung zur Induktion von Seneszenzmarkern in

PBMCs führt. Da dies bereits in Mäusen beobachtet wurde und persistierende DNS Strangbrüche

Seneszenz auslösen können. In PBMCs konnten folgende Beobachtungen gemacht werden, die

Zusammenfassung

VIII

wiederholte Behandlung mit Isoproterenol führt zu einer Expression von Seneszenz assoziierter β-

Galaktosidase, zu einer abgeflachten und vergrößerten Zellmorphologie, zu einer verminderten

Expression von CCND1, zu einer Erhöhung der Expression von VCAN und zu einer Inhibierung der

durch Phytohämagglutinin (PHA) stimulierten Proliferation. Jedoch konnte keine Expression der

Seneszenzmarkern p16 und p21 beobachtet werde. Dies deutet darauf hin, dass Isoproterenol

möglicherweise einen Seneszenz-ähnlichen Phänotypen in PBMCs induziert. Nach unserer Kenntnis

gibt es keine Daten zur Stabilität von Isoproterenol in der Zellkultur. Es ist aber bekannt, dass sowohl

der enzymatische Abbau als auch die chemische Oxidation von Isoproterenol zu Bildung von freien

Radikalen und ROS führt. Daher wurde in einer dritten Studie die Stabilität von Isoproterenol unter

Zellkulturbedingungen untersucht. Es ist bekannt, dass Isoproterenol zu Isoprenochrome, einem

zytotoxischen Aminochrome, oxidiert werden kann. Daher wurden die Konzentrationen von

Isoproterenol und Isoprenochrome während einer Zeitspanne von 8 Stunden in drei unterschiedlichen

Zellkultur Medien gemessen. Die Halbwertszeit von Isoproterenol lag circa zwischen 30 Minuten und

6 Stunden. Dies entspricht einer mindestens 6-fach längeren Halbwertszeit als die in humanen Studien

gemessene Halbwertszeit. Dabei zeigte sich auch, dass die Zusammensetzung des Zellkulturmediums

einen Einfluss auf den Abbau von Isoproterenol und die Bildung von Isoprenochrome hatte. Im

Rahmen dieser Dissertation wurde noch ein zweites Projekt bearbeitet. Dabei handelte es sich um eine

Inter-Labor-Validierungsstudie eines neu entwickelten FADU-Systems. Diese Studie wurde in

Kooperation mit dem Swiss Federal Laboratories for Materials Science und Technology (EMPA) in

St.Gallen und der Cetcis GmbH in Esslingen durchgeführt. In einer ersten Phase ist eine technische

Testung des FADU-Systems erfolgt. In einer zweiten Phase sollte gezeigt werden das, dass neue

FADU-System DNS Strangbrüche zuverlässig detektieren kann. Das neue FADU-System sollte auch

dazu verwendet werden, um Einflüsse von Isoproterenol auf die genomische Stabilität zu untersuchen.

Dies konnte allerdings nie durchgeführt werden, da die neue FADU-Plattform technische Mängel

aufwies.

Introduction

1

CHAPTER I

1 Introduction

1.1 Biological background of stress

In a biological view, stress is a process that influences the physiology and induces the adaption of

many body functions such as heart rate, blood pressure, blood sugar, respiratory rate, muscle tone,

digestion and immune system. However, the origins of these global adaptions take place on a

molecular and cellular level. Stress can be defined as the response of an organism to stimuli that

represent a threat of the homeostasis [1]. Such stimuli are called stressors. All stressors represent a

challenge for the complex and dynamic equilibrium of the organism, also called homeostasis [2]. Their

origin can be external or internal. A stressor can be physical, like a chemical or biological agent, a

physical force or environmental conditions. But it can also be imaginary, like an idea or emotion [3,

4]. The body needs to respond and induce internal adjustments to the stress signal which induce

adaptions of the body functions to the changed conditions to restore homeostasis. The central nervous

system (CNS) controls and regulates this response. Two pathways, the sympathetic-adrenal-medullary

axis (SAM) and the hypothalamic-pituitary-adrenal axis (HPA), are the most important ones, see

Figure 1-1 [5-7]. There is also a crosstalk between both axes, which is important for a correct stress

reaction of the body [5, 8, 9]. The SAM triggers the release of the catecholamines such as epinephrine

and norepinephrine by the adrenal medulla into the bloodstream [10]. Epinephrine and norepinephrine

are two important stress hormones that are indispensable for the “fight-or-flight” response of the body

[11, 12]. The HPA induces the release of glucocorticoid hormones from the adrenal cortex [13]. The

most important glucocorticoid hormone in humans is cortisol. Cortisol and other glucocorticoids

regulate many body functions [14, 15]. Stress hormones also regulate many functions of the immune

system. For example, glucocorticoids have an anti-inflammatory and immunosuppressive function. In

general, the CNS, the endocrine system and the immune system form together a complex network with

reciprocal interactions [16]. All three systems are highly adaptive and can be adjusted as it is required.

The immune system and the CNS use a wide range of chemical messengers for communication.

Therefore, cells and tissues of both systems share the identical receptors for those messengers. This

allows a crosstalk between these two systems. For instance, during infections the immune system uses

cytokines to influence the CNS which leads to a sickness behavior [17]. Moreover, lymphocytes can

produce a variety of hormones and neurotransmitters [18]. On the other way round the CNS can

Introduction

2

regulate the immune system [19]. Almost all immune cells express receptors for at least one of the

stress hormones. Glucocorticoid receptors can be found in T- and B-cells, neutrophils, monocytes and

macrophages. Receptors for epinephrine and norepinephrine can also be found in T- and B-cells,

monocytes, macrophages and neutral killer cells. Additional, nerve fibers of the sympathetic nerve

system innervates primary and secondary immune tissues [20, 21].

Figure 1-1: Stress induced modulation of immune cells by the release of stress hormones. A stressor is perceived by the

brain and induces a response of the body to counteract the stressor and restore homeostasis. This results in the stimulation of

the HPA (red) and the SAM (blue). The hypothalamus produces corticotrophin releasing factor (CRF). CRF stimulates the

pituitary gland and induces secretion of the adrenocorticotropic hormone (ACTH). ACTH stimulates the adrenal cortex and

induces the release of corticosteroids. The brainstem innervates sympathetic nervous fibers which stimulates the adrenal

medulla to release epinephrine and norepinephrine. Norepinephrine is also released from synapses of the sympathetic nervous

system (SNS). The SAM also innervates lymphoid tissues which influence the amount and the types of circulating

leukocytes. Leukocytes express receptors for different stress hormones. The receptors for epinephrine and norepinephrine are

adrenergic receptors which are localized at the cell surface. The most important adrenergic receptor on leukocytes is the β2-

adrenergic receptor. Binding of stress hormones to their respective receptors modulates the function of the leukocytes.

Introduction

3

Medical significance of stress 1.1.1

It is well known that stress impairs the function of the immune system [16]. In addition, stress-induced

processes are also important in the development of cancer [22, 23]. Cancer is the genus for a big

heterogeneous group of diseases. There are many different causes that induce development of cancer

such as chemical and physical agents, diet, physical inactivity, infections, radiations, heredity and

hormones. Some types of cancer can be affected by stress, others not or only slightly. The research

that deals with stress and its molecular and cellular impact on the genomic stability, cancer

development, DNA damage and DNA damage repair is at the beginning. Results of studies that

investigated the influence of stress on the incidence and progression of cancer are inconsistent [24,

25]. The current data suggest that carcinogenesis is less influenced by stressors than the progression of

cancer [26-28].

1.1.1.1 The influence of stress on tumor growth and metastasis

Tumor growth and metastasis are complex processes of high medical relevance. Particularly metastasis

is important, because it is the most common cause of death in cancer patients [29]. Metastasis is a

process of a serial and contiguous, complex steps which include: formation of a primary tumor,

proliferation and angiogenesis, invasion into host tissue, detachment and circulation, followed by

embolization of tumor cells, attachment of circulating tumor cells at new sides of blood vessels,

extravasation into host tissue, proliferation and thereby formation of metastases [29]. Already in the

year 1979 Sklar and Anisman showed in a xenograft mouse model that stress has an influence on the

growth of P815 mastocytoma cells which were transplanted into mice [30]. Mice stressed by

inescapable electroshocks have a faster tumor growth, bigger tumors and a reduced survival time [30].

Rats stressed by electroshocks after a tumor implantation have a 50% lower rejection rate of the tumor

compared to control animals that were not shocked [31]. Social isolation stress could enhance the

tumor metastasis in mice and suppresses the immune response [32]. Furthermore, social isolation

stress increases metastases formation and stressed mice respond weaker to a chemotherapy than

unstressed mice [33]. Human studies showed a correlation between stress and the development of

cancer. A large study in Israel with a cohort of 6284 participants showed that stress has an influence

of the cancer incidence [34]. Stress caused by the dead of an adult son as a result of war or an accident

increases the risk for the development of lymphatic- and hematopoietic malignancies, melanomas or

respiratory cancer in the following life time. These findings are supported by cellular and molecular

biology findings which link the impact of stress to important steps of tumor progression [35].

Angiogenesis is an essential process for the growth of tumors and metastasis. Growth factors such as

vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), transforming growth factor alpha

(TGF-α), transforming growth factor beta (TGF-β) and tumor necrosis factor alpha (TNF-α) are

Introduction

4

important in this process. Expression of VEGF in tumors and in serum of ovarian carcinoma patients

correlates with social stress [36, 37]. Epinephrine, norepinephrine and isoproterenol, a synthetic

sympathomimetic, could induce the expression of VEGF in cell lines. The higher expression of VEGF

could be inhibited by the β-blocker propranolol [38]. The blood concentration of IL-6 positively

correlates with social stress in patients with ovarian cancer [39]. Moreover, stress has also an influence

on tumor and metastasis invasion into the host tissue. For instance, matrix metalloproteinase are

important in the turnover of the extracellular matrix [40]. Norepinephrine could increase the

expression of matrix metalloproteinases in nasopharyngeal carcinoma tumor cells and propranolol

could inhibit the expression of these matrix metalloproteinases [41].

1.1.1.2 Stress mediated DNA damage

On a cellular and molecular level it was demonstrated that stress could induce DNA damage and also

impair the DNA damage repair. Rats which suffer from behavioral stress 24 h prior their scarification

have significantly more sister chromatid exchanges in their bone marrow cells compared to unstressed

animals [42]. A follow-up study demonstrated that rats which are stressed by different stressors have

a significant increase in sister chromatid exchange and also in chromosomal aberrations in their bone

marrow cells [43]. It was also shown that different behavioral stressors can induce chromosomal

alterations. However, the degree of chromosomal alternations depends on the type of the behavioral

stressor. Stress is also linked to oxidative DNA damages. In psychological stressed rats, the amount of

8-hydroxydeoxyguanosine (8-OH-dG) in the DNA isolated from the liver cells is increased compared

to unstressed animals [44]. Rats of a conditioned taste aversion study showed an increase of 8-OH-dG,

a marker for oxidative DNA damage, after receiving the conditioned stimulus compared to rats that

received the unconditioned stimulus [45]. Different types of psychological stressors could induce

oxidative DNA damage in human PBMCs. Although there is no general correlation between

psychological stress and oxidative DNA damage [46]. Some psychological stress parameters

positively correlate with the amount of 8-OH-dG in human PBMCs, for example: the depression-

rejection score, the profile of mood states (POMS) and the center for epidemiological studies

depression scale (CES-D). Moreover, stress can influence the expression of DNA repair enzymes. For

example, the expression of O6-methylguanine DNA methyltransferase is reduced in spleens of stressed

animal after induction of carcinogenic damage [47]. The DNA repair in lymphocytes of stressed non-

psychotic psychiatric inpatients is lower after X-ray irradiation compared to the DNA repair of

lymphocytes of unstressed non-psychotic psychiatric inpatients. Moreover, the depressed inpatients

have a poorer DNA damage repair compared with less depressed inpatients [48]. In contrast, some

studies showed a positive effect of stress on DNA repair. Two studies measured a higher DNA repair

capacity of the nucleotide excision repair pathway in blood cells of students during stress phases

(exam period) compared to unstressed phases (holidays) [49, 50]. Posttraumatic stress disorder

(PTSD) is an example for the link between psychological stress, stress hormones and genomic

Introduction

5

instability. PTSD is a mental disorder that can develop in the aftermath of severe traumatic events

[51]. Today, PTSD is defined as a trauma- and stressor-related disorder according to the “Diagnostic

and Statistical Manual of the Mental Disorder” (fifth edition) (DSM)-5 [51]. The hallmark of a PTSD

diagnosis is the experience of an extraordinarily threatening and distressing event of the person’s own

life or the life of a closely related person [52]. PTSD can be caused by a single traumatic event or by a

prolonged trauma exposure [53]. The prevalence for the PTSD during the lifetime is around 1.9% to

8.8% [51, 54, 55]. In contrast, to normal trauma, PTSD is characterized by a cluster of three long-term

persistent types of symptoms: reminder or re-experience symptoms of the trauma (flashbacks,

nightmares, intrusive images), activation (hyperarousal, insomnia) and deactivation (avoidance of

reminders and withdrawal) [51, 52]. Additionally, to distinguish PTSD from other mental diseases,

these symptoms must not be present prior to the trauma exposure and must persist longer than 1 month

after the traumatic event. Some of the pathophysiology features of PTSD are associated with changes

in the neurobiology including anatomical and endocrinal changes [56]. The endocrinal changes have

an impact on the stress response of the body. For instance, the HPA axis is dysregulated which

elevates the catecholamine and CRF levels in the brain. Furthermore, a sustained hyperactivation of

the sympathetic branch of the autonomic nervous system (ANS) is a cardinal marker for PTSD.

Studies have observed that the catecholamine concentrations as well as the concentrations of their

metabolites are elevated in the blood plasma and in the urine of PTSD patients [57-61]. In contrast, the

cortisol concentrations are lowered in the blood plasma and the urine [58, 62-64]. Studies have also

shown that at the time of exposure to the trauma, the peripheral epinephrine excretion can be used to

predict the possibility of the development for PTSD [65]. Also the administration of propranolol, a β-

adrenergic receptor antagonist, shortly after the exposure to psychological trauma can reduce PTSD

symptoms [66]. PTSD patients have a higher prevalence for somatic comorbidities like: type-2

diabetes, cardiovascular- , respiratory- , gastrointestinal- , inflammatory- and autoimmune diseases

[67-72]. Furthermore, PTSD is also associated with a higher risk of cancer [67, 73-75]. Many of these

comorbidities can be associated with inflammatory processes, genomic instability, increased aging and

senescence of the immune system. The increased stimulation of the sympathetic nervous system (SNS)

together with the dysregulation of the HPA increases the cytokine production, forcing a low-grade

chronic inflammatory character in PTSD patients [76, 77]. On a cellular level a change in the T cell

subset could be observed in PTSD patients [78-80]. PTSD is associated with an aged immune

phenotype of T cells [81]. The N-glycosylation profile of plasma of PTSD patients showed an

accelerated aging process [82]. In addition, it has been shown that PBMCs of PTSD patients have

accumulated DNA strand breaks that can be reversed by narrative therapy [83].

Introduction

6

1.2 Adrenergic receptor family

Adrenergic receptors belong to the large family of G protein couple receptors (GPCRs) [84, 85]. The

adrenergic receptor family consists of two alpha receptors (α1 and α2) and three beta receptors (β1, β2

and β3). The natural agonists of the adrenergic receptors are the catecholamines, epinephrine and

norepinephrine. All adrenergic receptor display similar structural features: a single polypeptide chain

with three extracellular and three intracellular loops and seven highly conserved hydrophobic

transmembrane domains, see Figure 1-2 [86]. The α-adrenergic receptors (α-ARs) are important signal

mediator of the CNS and peripheral nervous systems. The β-adrenergic receptor (β-AR) subtypes have

a 65-70% sequence homology [87]. The β1-adrenergic (β1-AR) is predominant and the most important

adrenergic receptor in the heart [88]. The β3-adrenergic receptor (β3-AR) is mainly located in adipose

tissue and involved in the controlling of lipolysis [89]. In contrast, the β2-adrenergic receptor (β2-AR)

is expressed ubiquitous in the most human tissues. The β2-AR induces relaxation of the smooth

muscles. Therefore, β2-sympathomimeticas are used for the treatment of asthma and chronic

obstructive pulmonary disease (COPD).

Figure 1-2: Structure of the β2-adrenergic receptor. A) Schematic representation of the β2-AR, green: consensus positions

for N-glycosylation, blue: homologues residues with rhodopsin, orang: consensus sequence for PKA-phosphorylation, red:

residues for G protein coupled receptor kinase-phosphorylation, adapted from [90].

Introduction

7

Signaling of the β2-AR 1.2.1

As a prototypical GPCR the β2-AR signals via a guanine nucleotide-binding protein (G protein). The

β2-AR is coupled to a heterotrimeric Gs protein build up by three subunits αs, β and γ. The Gαs subunit

is involved in hydrolysis of guanosine triphosphate (GTP), binding to Gβγ subunit and downstream

effectors [91, 92]. The β- and γ-subunits are tightly associated to each other and build a dimer.

Activation of the β2-AR induces conformational changes of the receptor and involves several

intermediates of the receptor structure [93, 94]. The type of ligand is essential as full-, partial- and

inverse-agonist stabilize different conformations of the receptor [95, 96]. The stabilization of the

active state of the β2-AR requires the binding of an agonist as well as the coupling to a Gs protein to

form a ternary complex [97-99]. The agonist binding pocket is formed by residues of the three

transmembrane domains 3, 5 and 6 which bind catecholamines [93]. The binding of the agonist

induces small changes of the receptor structure at the ligand binding pocket. As a consequence the

guanosine diphosphate (GDP) which is bound to α subunit is exchanged to GTP. This triggers the

dissociation of the G protein into its α subunit and a complex of the βγ subunits. Gs proteins induce

downstream signaling via the second messenger cyclic adenosine monophosphate (cAMP). The

signaling pathway via cAMP and PKA is the classical, canonical signal transduction pathway of the

β2-AR, see Figure 1-3. It is known that the β2-AR also engage additional signaling pathways which

can be seen as non-classical, non-canonical pathways [100-102]. The Gs protein binds to adenylate

cyclase (AC) which is present in the cytoplasm or bound at the lipid rafts. Binding of the Gs protein

activates the catalytic activity of AC and leads to the formation of cAMP from adenosine triphosphate

(ATP). Nine different membrane bound and one soluble isoform of AC are expressed in mammals.

Immune cells express high levels of the isoform 7 and low amounts of the isoforms 3, 6 and 9. Each

AC isoform can influence cell functions in a specific manner [103]. cAMP binds to the regulatory

subunits of PKA. The PKA holoenzyme is a tetramer build up by two regulatory- and two catalytic

subunits. Two types of the regulatory subunits, each with two isoforms, have been identified so far.

Lymphocytes express all four isoforms. The regulatory subunits are important for the localization of

PKA to specific cellular compartments, by binding to A-kinase anchor proteins (AKAPs). AKAPs also

guide the localization of PKA to ACs [104, 105]. Most important, each regulatory subunit binds two

cAMP molecules which induce conformational changes in the regulatory subunits. Subsequently, the

tetramer dissociates and the catalytic subunits are activated. The catalytic subunits phosphorylate

target proteins at serine and threonine residues, using ATP as substrate. A variety of PKA target

proteins exist which are involved in many different signaling pathways. Therefore, PKA mediates

further downstream signaling and regulates the expression of several thousands of genes [106]. cAMP

also activates gated ion channels and exchange proteins activated by cAMP (EPAC).

Introduction

8

Figure 1-3: The “classical” cAMP/PKA signaling pathway of the β2-AR. Binding of catecholamines to the β2-AR

induces conformational changes of the receptor. Active receptor conformation is stabilized by the bound G protein. The

coupled Gs protein gets also activated and an exchange of GDP to GTP is initiated. This induces the dissociation of the Gs

protein in its α- and βγ-subunits. The Gαs subunit binds to the AC and stimulates the formation of cAMP from ATP. cAMP

binds to the PKA holoenzyme, which consists out of two catalytic subunits (C) and two regulatory subunits (R) and induces

the dissociation and activation. Each regulatory subunit binds two cAMP molecules. The activated catalytic subunits

phosphorylate target proteins. On the one hand, this leads to the activation of transcription factors and modifications of the

gene expression. On the other hand, the β2-AR gets also phosphorylated. Phosphorylation by PKA induces further

phosphorylation of the receptor by G protein coupled receptor kinases (GRKs). These phosphorylations are important for the

recruitment of β-arrestins. Besides the activation of PKA, cAMP also activates EPACs which activates downstream signaling

via the mitogen-activated protein kinase (MAPK) pathway. cAMP gets rapidly degraded within minutes by

phosphodiesterases (PDEs) and leads to termination of the cAMP dependent signaling.

Introduction

9

EPACs are guanine nucleotide exchange factors which induce signaling via the mitogen-activated

protein kinase (MAPK) pathway [100, 107-109]. However, the main signaling pathway is mediated by

PKA. The signaling via cAMP is only transient, because cAMP is degraded by phosphodiesterases

(PDEs). Real time cAMP dynamics measurements showed that the maximum of cAMP formation is

reached after about 0.5-1 min and return back to basal level within a few minutes [110]. PDEs

hydrolyze cAMP to adenosine monophosphate (AMP) by cleaving the phosphodiester bond. The PDE

superfamily contains 11 PDE families, the PDE families 4, 7 and 8 hydrolyze specifically cAMP.

PDE4 is the major expressed PDE family in leukocytes [111, 112]. PDEs are recruited by β-arrestins

to the activated β2-AR [113, 114]. By hydrolyzing the cAMP, PDEs reduce the local cAMP levels and

terminate the second messenger signaling [104]. Additional, the Gα subunit hydrolyses GTP to GDP,

inducing reassociation of the heterotrimeric G protein and termination of the signal transduction. After

activation of the receptor and signal transduction, the signaling process must be terminated to allow

the resensitization of the cell. Different processes are involved in the termination of the β2-AR

signaling, see Figure 1-4. Moreover, at nearly every stage of the signaling pathway the signal can be

down-regulated or terminated. One of these processes is the receptor desensitization. This is a rapid

process which reduces the signaling, although the receptor is occupied by a ligand. Desensitization of

the β2-AR is a multistep process, involving phosphorylation of the receptor by PKA and GRKs. These

phosphorylations induce the recruitment of β-arrestin to the β2-AR and its internalization [114-116].

Phosphorylation of the receptor induces uncoupling from the Gs protein, forming a negative-feedback

loop. Moreover, phosphorylation of β2-AR serves as a “switch”, because phosphorylated β2-AR

couples predominantly to Gi proteins [117-119]. In contrast to Gs proteins, Gi proteins inhibit the AC.

In addition, a “second signaling wave” can be induced by the Gβγ dimer of the Gi protein, see Figure

1-5. Activating a MAPK signaling pathway, that involves the proto-oncogene tyrosine-protein kinase

Src (Src) and the G protein rat sarcoma (Ras) and results finally in the activation of the extracellular

signal-regulated kinases (ERKs) [117, 119]. PKA mediated phosphorylation of the receptor also

induces further phosphorylation of the β2-AR by GRKs [120, 121]. These phosphorylations then

promote the binding of β-arrestin to the carboxy-terminal tail of the receptor [122, 123]. Mammals

express four arrestin subtypes: arrestin 1, arrestin 2, arrestin 3 and arrestin 4. The expression of

arrestin 1 and arrestin 4 is limited to the retinal rods and cones. In contrast, arrestin 2 and 3 are

ubiquitously expressed. They are also called β-arrestin 1 and 2 [124-126]. The binding of β-arrestins to

the receptor sterically inhibits the coupling to a G protein [127, 128]. Binding of β-arrestins to the

receptor initiates also the internalization of the β2-AR via clathrin-coated vesicles [129]. However, the

binding of β-arrestin to the receptor is only transient, β-arrestin dissociates from the β2-AR. It is

excluded from the endocytic vesicles that sequestered the receptor from the cell membrane [130-132].

After the internalization, the β2-AR can either be recycled or degraded, see Figure 1-4. The duration of

the agonist treatment determines the fate of the receptor. A short-term stimulation, up to 1 h, leads to

sequestering of the receptor from the plasma membrane and its internalization into endocytic

Introduction

10

compartments [133]. There, the receptor can be dephosphorylated which allows its resensitization and

recycling to the cell membrane [134]. Long-term treatment with an agonist for several hours or days,

induces receptor breakdown and down-regulation [133]. Agonist concentrations in µM ranges can also

reduce sensitivity to the agonist and reduce the maximum response to the agonist stimulus [135]. The

β2-AR has a recycling half-life of about 7.5 min [87]. Additional, β-arrestin acts as scaffold protein

and recruits several further proteins. Β-arrestin directly interacts with mouse double minute 2 homolog

(MDM2) which is an E3 ubiquitin ligase. MDM2 ubiquitylates β-arrestin as well as the β2-AR which

is a further mechanism for termination of the signaling [133].

Figure 1-4: Termination of the β2-AR signaling, receptor internalization and recycling of the receptor. β-arrestin binds

to the phosphorylated receptor and inhibits the coupling to G proteins and the subsequent signaling. In addition, β-arrestin

acts as scaffold protein for the binding of the adaptor related protein complex 2 (AP2) and clathrin. Both are needed for the

internalization of the receptor into clathrin coated-pits. After internalization the β2-adrenergic receptor can be either recycled

or be degraded. During recycling, the receptor gets dephosphorylated and recycled back to the cell surface. Degradation of

the receptor and β-arrestin is induced by MDM2 by polyubiquitination.

Besides the role in receptor desensitization, β-arrestin is also involved in non-classical, non-canonical

signal transduction pathways of the β2-AR, see Figure 1-5. β-arrestin recruits and activates Src which

triggers the activation of ERK mitogen-activated protein kinase pathway [136]. The kinase activity of

Src seems to be important for the receptor internalization. Since it is involved in the phosphorylation

of proteins which are involved in the internalization process [137]. Hence, the β2-AR can induce ERK

Introduction

11

signaling via two pathways. One pathway involves PKA. Phosphorylation of the β2-AR mediated by

PKA induces a switch from Gs proteins to Gi proteins which results in the activation of the ERK

signaling cascade. The other pathway is β-arrestin dependent. Both pathways can be distinguished

from each other. The PKA/Gi protein mediated pathway induces a rapid, 2-5 min, but transient

activation of ERK. Moreover, this activation is sensitive to the PKA inhibitor H-89. The β-arrestin

dependent activation of ERK is slower, 5-10 min, less robust, prolonged and insensitive to H-89 [138].

Besides the activation of ERK also p38 another mitogen-activated protein kinase can be activated by

the β2-AR. p38 signaling is important during oxidative stress [109, 139]. The activation is biphasic, the

early phase is mediated by β-arrestin 1 and the late phase is mediated by PKA [140]. The β2-AR can

also induce signaling via the Gi/PI3K/Akt pathway; thereby the signal is transmitted by the Gβγ-

subunit [141-144]. The non-classical signaling pathways are cell-type dependent and have so far been

mainly studied in cell lines and non-immune cells.

Introduction

12

Figure 1-5: “Second wave signaling” of the β2-AR. In addition to the cAMP mediated receptor signaling pathway, the β2-

AR is coupled to several other signaling pathways. β-arrestin also binds the protein kinases Src and p38 which induce various

transcription factors via the ERK pathway of the MAPK. The phosphorylated receptor also couples to Gi proteins instead of

Gs proteins. The βγ heterodimer of the Gi protein activates the PI3K/AKT signaling pathway and also the MAPK pathway

via the Src protein kinase. At the same time, the αi subunit of the G protein inhibits the AC and the cAMP mediated

signaling.

There is one more fact that further increases the complexity of the β2-AR signaling, a phenomenon

known as “biased agonism”. This describes the fact that ligands that bind to receptors which are

Introduction

13

coupled to several signaling pathways can activate only distinct pathways and not all pathways. On the

one hand, ligands can be inverse agonist for one signaling pathway and on the other hand, the same

ligands can activate another signaling pathway. This phenomenon can be observed for the β2-AR [145-

148]. For instance, the β-blockers propranolol and ICI118551 are inverse agonists for the Gs protein

pathway but both are agonists for the β-arrestin dependent activation of ERK [147]. In contrast, the

agonist isoproterenol activates both pathways and no biased agonism is known [148, 149].

1.3 Catecholamines

The catecholamines epinephrine and norepinephrine are stress hormones and mediate the stress

response through the whole body. Both are required for the induction of the “fight-or-flight” stress

response. Together with dopamine, epinephrine and norepinephrine they are the most important

naturally occurring catecholamines in the human body. All three catecholamines are synthesized from

the amino acid tyrosine in a serial synthesis pathway of 4 steps [150]. The human plasma contains,

under resting conditions, mainly the following catechols: dopamine, epinephrine, norepinephrine, 3,4-

dihydroxyphenylalanin (DOPA) their precursor and the two metabolites dihydroxyphenylacetic acid

(DOPAC) and dihydoxyphenylgycol (DHPG) [151]. Catecholamines have only a short half-life of a

few minutes after the secretion into the bloodstream. Several enzymes are involved in the degradation

of catecholamines. The expression of these enzymes varies between different tissues and cells. Hence,

the degradation products are different, depending on the cell type and tissue type that metabolizes the

catecholamines [151]. In the following, the focus will be on the degradation of catecholamines that

are secreted into the bloodstream during stress. These are the two catecholamines epinephrine and

norepinephrine. Under resting conditions, the main sources of noradrenalin secretion are the

sympathetic nerves. Under stress conditions, the secretion of norepinephrine is increased by an

additional release of norepinephrine by the adrenal medulla. The plasma concentration of

norepinephrine depends on different factors, such as the rate of release, the body site of sampling, the

reuptake of norepinephrine and the modulation of α2-adrenoreceptors [151]. The human resting plasma

concentration of norepinephrine ranges between 1-1.5 nmol/l [152-154]. In contrast, epinephrine is

released mainly by the adrenal gland. The plasma concentration of epinephrine under resting

conditions is low; it ranges from 0.2 nmol/l to 0.5 nmol/l [152-154]. During stress about 80% of the

chromaffin cells of the adrenal medulla release and synthesize epinephrine. The remaining 20% of the

chromaffin cells release and synthesize norepinephrine [155, 156]. The increase of the epinephrine and

norepinephrine plasma concentrations is influenced by the stress intensity and type of the stressor

[157-159]. After the release catecholamines must be metabolized. This is essential for the organism to

return back to resting conditions. Two enzymes are the key player in the metabolism of

catecholamines, catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) [160].

Introduction

14

COMT transfers a methyl group of S-adenosylmethionine to one of the hydroxyl groups of the

catechol ring [161]. COMT is expressed in most human tissues but is mostly expressed in the liver,

kidney and gastrointestinal tract [162]. The second key enzyme of the catecholamine metabolism is

MAO [160]. MAO is a mitochondrial enzyme and located at the outer mitochondrial membrane [163,

164]. It is expressed in most cell types but the highest expression, outside of the brain, can be found in

the liver and the kidney [165]. MAOs catalyze the oxidative deamination of primary, secondary and

some tertiary amines [166]. During the oxidative deamination of amines, MAO produces reactive

oxygen species (ROS) in the form of hydrogen peroxide [167, 168]. COMT as well as MAO can

catalyze the initial step of the catecholamine metabolism. But also other enzymes are involved in the

degradation. Besides the breakdown, catecholamines can also be conjugated with sulfate and

glucuronic acid. Both conjugates and metabolites are inactive and cannot further activate adrenergic

receptors (AR).

Effects of catecholamines on immune cells 1.3.1

Catecholamines are important messengers between the immune system and the CNS. Therefore, both

systems need receptors for catecholamines. Indeed receptors for catecholamines can be found in

different types of leukocytes. Moreover, enzymes for the synthesis and degradation of catecholamines

can be found in leukocytes [169]. Sympathetic nerve fibers directly innervate lymphoid organs [170,

171]. Catecholamines can influence immune cell proliferation, differentiation and cytokine production

[150, 172, 173]. The receptor expression on immune cells is dynamic and the expression pattern can

vary. It is known that immune cells such as T cells, B cells, neutral killer cells, monocytes and

macrophages express AR. B cells express approximately a 2.5- to 4-fold bigger amount of β-ARs than

T cells [174, 175]. The different T cell subpopulations show different densities of β-ARs on their cell

surfaces. The most β-ARs are present on T-suppressor cells with about 2900 receptors/cell, followed

by cytotoxic T cells with about 1800 receptors/cell and T-helper cells with about 750 receptors/cell

[176]. Monocytes show a β-AR density of about 2400 receptors/cell [177, 178]. Natural killer cells

express about 1900 β-ARs/cell. During the differentiation of monocytes into macrophages the cells

lose their β-ARs. This is associated with insensitivity to catecholamines [179, 180]. The receptor

densities which are listed above are only approximations. The used methodologies can influence the

determination of receptor density. Also biological process can influence the measured densities. For

example, the density of β2-ARs on the T cells surface can be influenced by IL-2 and

phytohaemagglutinin (PHA) [181, 182]. Agonists of the β2-AR such as epinephrine and

norepinephrine lead to a decrease of the β2-AR density on T cell surface [183]. A reduced receptor

expression can also be observed by culturing the cells without stimulation of the β2-AR [183]. IL-2

prevents this loss of β2-ARs on the cell surface of T-helper cells. Moreover, IL-2 increases the density

of β2-ARs on the plasma membrane of cytotoxic T cells [183]. Treatment of human PBMCs with

Introduction

15

IL1-β induces a transient increase of β2-ARs density within the first 6 hours compared to untreated

cells [184]. The dynamic of β2-AR density can also be seen during physical stress which induces a

significant increase of β2-ARs in T cells, B cells and monocytes. The amount of β2-AR at the cell

surface return back to basal levels after 30 min of the rest [177]. Although, lymphocytes express AR

on their cells surface, there are even more indications for the importance of catecholamines, see Figure

1-6. Lymphocytes can uptake, store, release and synthesis catecholamines [185]. Human PBMCs

contain dopamine, epinephrine and norepinephrine and several of their metabolites [185-189].

Different human hematopoietic cell lines like NALM-6, Jurkat and U937 also contain endogenous

catecholamines [188]. Pharmacological manipulation of the tyrosine hydroxylase and dopamine-β-

hydroxylase can influence the intracellular catecholamine levels of PBMCs. Incubation with a tyrosine

hydroxylase inhibitor, tyrosine hydroxylase is the rate limiting enzyme in the catecholamine synthesis

pathway, lead to a significant decline of intracellular norepinephrine and dopamine levels and their

metabolites [186]. Moreover, expression of tyrosine hydroxylase mRNA, in human PBMCs can be

stimulated with PHA [190, 191]. PBMCs also express the enzymes for the degradation of

catecholamines. For this purpose, cells need a reuptake-system for catecholamines and enzymes like

COMT and MAO for the breakdown. Indeed, human PBMCs have a monoamine uptake mechanism

which shows similarity to the monoamine transporter that could be found in neuronal tissues [192,

193]. PBMCs express also a vesicular monoamine transporter of the type-1 and -2 in their plasma

membrane and cytoplasm [194]. Lymphocytes also express COMT and MAO [175, 195-197].

Inhibition of MAO increases the intracellular concentrations of dopamine, epinephrine and

norepinephrine [186]. Additionally, PBMCs seem to have storage vesicles for catecholamines.

Treatment of PBMCs with reserpine reduces the intracellular dopamine and norepinephrine

concentrations and their metabolites [186, 188]. Incubation with a monoamine uptake blocker

increases the level of norepinephrine and dopamine in culture medium [186]. Immune cells can also be

stimulated by catecholamines in an autocrine or paracrine manner [198, 199]. All these findings

demonstrate that catecholamines can influence immune cells.

Introduction

16

Figure 1-6: Summary of catecholamine receptors, uptake mechanisms, synthesis processes and degradation

mechanisms in lymphocytes, adapted from [169]. Lymphocytes contain all important factors that are needed for the

synthesis and metabolism of catecholamines, including MAO and COMT.

Isoproterenol 1.3.2

Isoproterenol is a synthetic catecholamine and a sympathomimetic drug [200]. It is a nonselective β-

AR agonist and an analog of epinephrine. The chemical structure differs from epinephrine by the

substitution of the methyl group at the nitrogen atom by an isopropyl group. This substitution makes it

selective for β-ARs. Furthermore, the isopropyl group inhibits the degradation of isoproterenol by

MAOs [201]. Isoproterenol was used in the medicine for the treatment of bradycardia and heart block

[202]. As aerosol it can be used for the treatment of asthma by relaxing the smooth muscles leading to

bronchodilation via activation of the β2-AR [203, 204]. The route of administration determines the

dose and influences the metabolism and the plasma half-life of isoproterenol. Different routes of

administration in dogs show significant differences in the onset of the heart rate increase, time to

maximal heart rate and duration of the effects. The fastest increase of the heart rate and the highest

response is induced by intravenous injection [205]. Isoproterenol is bound by plasma proteins. The

plasma protein bound fraction of the administered dose ranges from about 40% to 70% [206, 207].

The measured plasma half-lives vary between a few minutes until 7 h [200]. The elimination half-life

of isoproterenol is 2-10 min in rats, measured by micro dialysis sampling [206]. In children a plasma

half-life of about 4.2 min can be detected after intravenous infusion [208]. Studies in adults and

children using tritiated isoproterenol as radioactive tracer have shown that intravenous injected

Introduction

17

isoproterenol exhibits a biphasic elimination profile in the plasma [201, 209]. The first, rapid phase

shows a plasma half-life of about 2.5-5 min. The second, slower phase shows a plasma half-life of

about 2.5-7 h [201, 209, 210]. Follow-up analysis of the plasma radioactivity in the next 6.5 h have

shown the largest amount of the remaining radioactivity is caused by unchanged isoproterenol [201].

The smaller part is caused by 3-O-methyl isoproterenol. No further compounds like conjugated

isoproterenol could be detected [201]. In contrast, after oral dosage only a small amount of unchanged

isoproterenol or 3-O-methyl isoproterenol could be detected [201]. Further analyses of blood plasma

or urine samples have shown that isoproterenol is conjugated with sulfate and this conjugated

isoproterenol is the major metabolite [201, 211]. Inhalation of isoproterenol results in a quite similar

metabolite pattern with sulfated isoproterenol as major compound [211-213]. Isoproterenol is

metabolized primarily in the liver, lung and intestines by COMT and excreted in the urine and bile,

either in conjugated form or free form [205, 208, 214]. The main part, about 50-60%, of the

intravenous applied isoproterenol dose is excreted unchanged [201]. The remaining part, 40-50%, of

the dose is free isoproterenol or conjugated 3-O-methyl isoproterenol. Catecholamines including

isoproterenol undergo also chemical degradation processes [215]. These oxidation processes seem to

be involved in neuro- and cardiotoxicity of catecholamines [216-220]. Oxidized isoproterenol can

induce myocardial necrosis [221, 222]. The toxicity is mainly caused by oxidations of the

catecholamines to aminochromes which can occur in vivo and in vitro [223-229]. Catecholamines are

first oxidized to ortho-semiquinones which than undergo further oxidation to ortho-quinones, see

Figure 1-7 [229-231]. Ortho-quinones can undergo a 1,4-intramolecular, irreversible cyclisation

reaction. Induced by deprotonation of the amine nitrogen atom and a nucleophilic attack to the 6-

position of the quinone ring. The result is an unstable leukoaminochrome which is again oxidized to a

leukoaminochrome-ortho–semiquinone. Finally, the leukoaminochrome-ortho–semiquinone is

transformed into an aminochrome of the corresponding catecholamine. Aminochromes can undergo

further oxidation processes which result in polymeric pigments [228]. The oxidation of

catecholamines can be caused by autoxidation or by enzymes like xanthine oxidase, peroxidase,

tyrosinase, lipoxygenase, catechol oxidase, cytochrome c oxidase [232-235]. But also several metal

cations such as Cu2+

, Mn2+

, Co2+

and Ni2+

can induce the oxidation of catecholamines [233].

Polymorph nuclear leukocytes can oxidize adrenaline to adrenochrome [236, 237]. Isoproterenol is

stable in water and normal saline solution in an pH-range of 1.9 to 7.4 at 22 °C for 24 h [200]. An

increase of the pH to 8 or 9 at 22 °C in aqueous solution leads to a decrease of the initial isoproterenol

content to 94% or 50% after 24 h. Also an increase of the temperature to 37 °C leads to a reduction of

the initial isoproterenol concentration to 50% after 24 h. These data suggest that isoproterenol also

degrades under cell culture conditions. Moreover, it might be possible that isoproterenol is oxidized to

isoprenochrome under these conditions.

Introduction

18

OH

HO

HO

HN

OH

O

HO

HN-1e-,1H+

-1e-,1H+

OH

NH

O

O

OH

HO

HO N

-1e-,1H+

OH

O

HO N

-1e-,1H+

OH

O

O N

OH

O

O N

isoproterenol isoproterenol-O-semiquinone

isoproterenol-O-quinoneleukoisoprenochrome

leukoisoprenochrome-O-semiquinone

isoprenochrome

Figure 1-7: Isoproterenol oxidation to isoprenochrome, adapted from [228].

Poly(ADP-ribose) polymerases and poly(ADP-ribosyl)ation 1.3.3

The human Poly(ADP-ribose) polymerases (PARPs) are important DNA repair enzymes. PARPs are

involved in the repair of various types of DNA lesions, such as oxidative damage and DNA strand

breaks [238]. As described above, these types of DNA lesions can be caused by several types of

stressors. The PARP enzyme family consists of 17 members which share a conserved catalytic domain

[239]. PARPs use NAD+ as a substrate to catalyze the formation of poly(ADP-ribose) (PAR). PAR is

an important posttranslational modification that is especially important under genotoxic stress. The

best known member of this enzyme family is PARP1, which is responsible for about 90% of the PAR

formation in cells under genotoxic conditions. PARP1 is highly conserved and constitutively

expressed. It has a molecular size of about 113 kDa and consists of six major domains, see Figure 1-8

[240, 241]. The activity of PARP1 is regulated by various mechanisms. However, unusual DNA

structures like single-strand breaks, double-strand breaks, three- and four-way junctions and hairpins

are the most important PARP1 activators [242-244]. After the formation of DNA single- or double-

strand breaks, the PAR content in cells increases dramatically to about 100-fold [245, 246]. PARP1 is

Introduction

19

mainly responsible for that increase, but also PARP2 and PARP3 can be activated by DNA damages

[247]. Besides DNA strand breaks also posttranslational modifications modulate the activity of

PARP1 [241]. The most important posttranslational modification of PARP1 is the covalent

PARylation, the result of auto-PARylation [248]. Auto-PARylation inhibits the DNA binding and

catalytic activity of PARP1 [249, 250]. PARP1 is also a substrate for SUMOylation and acetylation

[251-253]. Another important posttranslational modification of PARPs is phosphorylation. PARP1

interacts with several cell signaling kinases, including protein kinases which are involved in beta-

adrenergic signaling. For instance, phosphorylated ERK2 can directly activate PARP1 via protein-

protein interactions [254]. Phosphorylation of PARP1 at serine 372 and threonine 373 by ERK1/2 is

required for maximal PARP1 activation after DNA damage formation [255]. PKA can directly

phosphorylate PARP1 in vitro at serines 465, 782 and 785 [256]. During H2O2 induced cell death c-Jun

N-terminal kinase1 (JNK1) interacts directly via protein-protein interactions and phosphorylates

PARP1 [257]. Phosphorylation of PARP1 by protein kinase C (PKC) has an inhibitory effect on

PARP1 [258]. After activation, PARP1 covalently attaches ADP-ribose moieties mainly on glutamate,

aspartate, lysine and arginine residues on target proteins [248, 259, 260]. PARP1 is the main acceptor

of PAR but also several hundreds of other proteins are targets for PARylation [261, 262]. The

formation and degradation of PAR is a highly dynamic process, characterized by an immediate but

transient PARylation of proteins. After genotoxic stress the polymer has a half-life between 1-6 min

[263, 264]. Poly(ADP-ribose) glycohydrolase (PARG) is the counter player of PARPs and degrades

PAR with its exo- and endoglycosidase activity [265-268]. Besides the covalent attachment of PAR to

proteins, proteins can also interact with PAR in a non-covalently manner [269-275]. PARP1 can

influence protein function either by direct protein-protein interactions or covalent PARylation of

proteins or by non-covalent PAR binding. In this way various cellular functions can be influenced by

PAR [238]. PARP1 is an important factor for the maintenance of genomic stability including DNA

damage response and DNA damage repair [276, 277]. With exception of the direct removal of DNA

damage by the O-6-methyguanine-DNA methyltransferase (MGMT) and the DNA mismatch repair

(MMR) pathway it is involved in all DNA repair pathways [238]. PAR plays an important role in the

modeling of the chromatin structure [278, 279]. PARP1 is also important for the maintenance of the

telomeres. PARP1-/-

mice have shorter telomeres, already in the first generation [280]. Restoration of

PARP1 in telomerase positive cells leads to a recovery of telomere length [281]. Moreover, PARP1

interacts and modifies telomeric repeat-binding factor 2 (TRF2) a core component of the shelterin

complex which protects the telomere ends from unwanted DNA repair [282]. PARP1 is also involved

in the regulation of the cell cycle [238]. One of the most important interaction partners with regards to

the cell cycle control is the “guardian of the genome” p53. PARP1 and p53 interact directly via

protein-protein interactions but also by covalent PARylation of p53 and non-covalent PAR binding of

p53 which modulates the function of p53 [283-288]. Chronic stress is associated with an increased

level of inflammation and oxidative stress, which in turn are associated with an increased risk for type-

Introduction

20

2 diabetes, cardiovascular- and inflammatory diseases [69, 70, 289]. These findings provide a link

between the interactions of chronic stress and PARP1. Since PARP1 contributes to inflammation and

the development of related pathologies by interaction with nuclear factor kappa-light-chain-enhancer

of activated B cells (NF-κB) [238]. NF-κB is an important transcription factor for the regulation of the

gene expression after proinflammatory stimuli [290]. PARP1 interacts with both major NF-κB

subunits, p65 and p55, and is required for the NF-κB induced gene transcription [291]. Acetylation of

PARP1 by the histone acetylase p300/CBP upon inflammatory stimuli, leads to a stronger binding to

NF-κB [253]. PARP1 -/-

mice have an impaired expression of the NF-κB controlled proinflammatory

mediators, such as TNF-α, IL-6 and iNOS [292, 293]. NF-κB signaling is also important for the

promotion of senescence [294]. Several studies have demonstrated that a PARP1 knockout or PARP

inhibitors can be protective against inflammatory conditions and oxidative stress [269, 295, 296]. The

association between PARP1 and chronic stress is further supported by the finding that PARP

inhibition might be a new therapeutic instrument for the treatment of stress related diseases. A mice

study has shown that PARP1-/-

mice are protected against stress induced immune-compromisation

[297]. Mice treated with the PARP inhibitor 3-aminobenzamide are also protected against stress

induced reduction of antibody production in response to a novel antigen [298]. A recent study showed

the potential use of PARP inhibitors as a new class of antidepressants. 3-aminobenzamide and 5-

aminoisoquinolinone were used to treat the effects of repeated physiological (swim test) and

psychological (social defeat stress and chronic unpredictable stress) stress. The results showed an

antidepressant activity and mitigation of the stress symptoms by both PARP inhibitors. Moreover, the

effects were comparable with a fluoxetine treatment. Fluoxetine is a commonly used antidepressant for

the treatment of major depressive disorder [299]. Finally, PARP1 is an important switch between cell

survival and cell death [241]. NAD+ is an essential cofactor of the cellular metabolism and needed for

the maintenance of the redox state of a cell [249]. It is also the substrate of PARPs. PARylation seem

to be the master regulator of the NAD+ catabolism in mammalian cells [300, 301]. Under genotoxic

stress and hyperactivation of PARP1, the half-life of NAD+ decreases from 1 h to 5-15 min. Moreover,

the NAD+ level decrease to about 20% of the basal level [302-304]. Treatment of cells with various

DNA damaging agents have shown that low or moderate doses induce a reduction of the cellular

NAD+ level to 65-75% of the basal level. In contrast, high doses can induce nearly a complete

depletion of the cellular NAD+ pools [303-308]. The NAD

+ depletion results also in a depletion of

ATP pools. The result is an energy crisis which results in necrosis [309].

Introduction

21

Figure 1-8: Schematic representation of PARP1 protein structure. PARP1 is build up by 3 major domains. The DNA

binding domain contains the two homologous zinc fingers (ZnF1 and ZnF2) and the WGR (tryptophan-glycine-arginine)

domain. The nuclear localization sequence (NLS) is localized between ZnF2 and ZnF3. ZnF3 has a different structure

compared to the two other zinc fingers. It is involved in interdomain contacts. The BRCA1 C terminus (BRCT) domain is

important for protein-protein interactions and contains several residues for the automodification. The catalytic domain built

up the helical domain (HD) which has regulatory functions and the ADP-ribosyltransferase (ART) domain. The ART domain

is the catalytic center of the enzyme.

In addition, PAR can induce also cell death. After cleavage, PAR molecules leave the nucleus and

induce the release of apoptosis inducing factor (AIF) from mitochondria. AIF induces a caspase

independent chromatin condensation and large scale DNA fragmentation, this kind of cell death is

called parthanatos [310].

1.4 Cellular senescence

Chronic stress can be linked with an increased risk of several somatic diseases. The same changes can

be observed during aging. Aging can be defined as a process of progressive deterioration of

physiological function at a cellular, tissue and body level [311]. Several hints suggest that stress may

promote earlier onset of age-related diseases that might also be associated with cellular senescence

[312]. Persons with elevated levels of stress hormones have shorter telomeres [313, 314]. PTSD

caused by childhood trauma or rape is associated with an increased telomere shortening in leukocytes

[315, 316]. A study with 650 veterans of US army special operation units deployed during Iraq or

Afghanistan wars showed that participants with PTSD had shorter telomeres than participants without

PTSD [317]. A further study with veterans of the Croatian war could confirm these findings [318].

Additionally, it was shown that the percentage of proliferating cytotoxic T cells and T-helper cells is

lower in an elderly control group. In PTSD patients only the percentage of proliferating cytotoxic T

cells is lowered [318]. A recent study with 3000 participants in south Germany showed shortening of

the telomeres has a dose-dependency. Since the telomeres of subjects with partial PTSD are longer

than telomeres of subjects with full PTSD [319]. One possible explanation for telomere shortening in

PTSD patients is the increased inflammatory activity accompanied with increased oxidative stress.

That is caused by the dysregulation of the HPA and the SNS. Cellular senescence is characterized by

an irreversible arrest of the cell proliferation. It can explain, at least partly, the age-related phenotypes.

Senescent cells may contribute to the imbalance between the rate of cell loss and the rate of cell

Introduction

22

renewal. Insufficient cell renewal leads to deterioration of tissue and organ function and finally results

in their failure [320]. This growth arrest was first described by Hayflick in 1961 as “replicative

senescence” [321, 322]. Replicative senescence is age-related and induced by the shortening of

telomeres. At a critical telomere length, the telomeres become dysfunctional and a persistent DNA

damage response is activated, which results in an inhibition of the cell cycle [323-325]. Human

senescent fibroblasts show a colocalization of phosphorylated γ-H2AX and p53-binding protein 1

(53PB1) in distinct foci, a maker for DNA double-strand breaks, at telomeres [323]. Also

phosphorylated ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related

protein (ATR) kinases as well as their phosphorylated downstream effector kinases could be detected

in senescent cells [326, 327]. For example, the telomeres of human leukocytes show an age related

shortening [328]. In addition to replicative senescence caused by telomere shortening during aging,

premature senescence is known which is independent from telomere shortening. Premature senescence

can be caused by DNA damage, genotoxic stress, mitogen signalling and activation of tumor

suppressor genes [329-333]. The mitogen signalling and tumor suppressor gene induced senescence is

also called “oncogene-induced senescence” (OIS). The pathways of OIS are complex and not fully

understood. But it is known that the p53 and retinoblastoma protein (RB) pathways are essential [334].

Senescence can also be induced by a persistent DNA damage response (DDR) induced by many

different sub lethal stresses. In this case the senescence is called “stress-induced premature

senescence” (SIPS) [335]. SIPS and replicative senescence share some of the biological features [320].

Both engage the p53/p21 pathway. The DDR that causes SIPS is induced by DNA damage primarily

induced in telomeric DNA and is independent from telomere shortening. It is induced by telomeric

foci containing multiple DNA damage response factors [323, 325, 336]. Cellular senescence is

controlled mainly by two pathways which cross talk to each other, see Figure 1-9 [337-339]. The key

players in these pathways are p53 and p21 on the one hand and p16 and RB on the other hand [340,

341]. p14, also known as alternate reading frame (ARF), is an important linker between both pathways

[342]. p53 and RB are important tumor suppressors, while p16 and p21 are cyclin-dependent kinase

(CDK) inhibitors [340, 343-346]. However, cellular senescence can be induced by one of these two

pathways, which pathway is induced depends also on the cell type and the senescence inducing stimuli

[347]. An age related increase of p53 can be explained by telomere shortening which induces a

persistent DDR [325, 348]. OIS also activates the p53/p21 pathway [332, 349, 350]. The main

regulator of the p53 protein level in cells is MDM2 [351-353]. Breakdown of p53, mediated by

MDM2, suppresses its function as transcription factor. This subsequently inhibits the cell cycle arrest

because p53 is an important regulator for the expression of the cell cycle inhibitor p21 [325, 354]. p21

inhibits all CDK-cyclin complexes and induces a cell cycle arrest [355, 356]. The p16/RB pathway is

the second main pathway that controls cellular senescence [357]. During senescence the RB is

hypophosphorylated. This is the active form of RB and triggers cell cycle arrest at G1 phase. RB

controls DNA replication by binding to transcription factors of the E2F-family [358]. The E2F

Introduction

23

transcription factors control the expression of essential cell cycle regulators, like cyclin E and B [359].

The binding of hypophosphorylated RB to E2F suppresses the activity of E2F. Primarily by the

recruitment of additional co-repressors like histone deacetylases (HDACs) [360]. Phosphorylation of

RB by the CDK4/6/cyclin D complex induces the dissociation of the E2F transcription factors from

the RB protein. This subsequently induces the transcription of genes that drive the cell cycle form G1

phase to S phase [361]. Senescent stimuli like DNA damage response engage also the RB/p16

pathway but only secondarily [355, 362, 363]. However, p21 and p16 are not equivalent [345]. p16

binds only to CDK4 and CDK6 and inhibits the interaction with cyclin D by inducing conformational

changes [364]. Thereby, RB is kept in its active state because its phosphorylation by CDK4/6 is

inhibited [365].

Senescence marker and characteristics of senescent cells 1.4.1

Cellular senescence is not characterized by a single hallmark. Although, cell cycle arrest is evident for

senescence it is not exclusively found in senescent cells. Therefore, senescent cells need to be

identified not only by a single marker, but rather by a combination of markers at the same time. The

most important senescence markers are listed below:

Morphological changes

Cellular senescence is often accompanied by morphological changes of the cell. Most common

changes are enlargement, flattening and multi-nucleation of the cell. The senescence trigger often

determines the morphological changes of the cell. Senescence induced by genotoxic stress or DNA

damage is normally accompanied by a flattened and enlarged phenotype [333, 366].

Senescence-associated heterochromatin foci

Senescence-associated heterochromatin foci (SAHFs) are altered chromatin structures that are

associated with cellular senescence [347]. Normal proliferating cells show a homogenous staining of

the nuclear DNA with DNA-binding dyes. Whereas, senescent cells show an inhomogeneous and

punctual staining of heterochromatin by DNA-binding dyes. SAHFs are found at E2F target genes

[367]. The RB/p16 pathway is essential for the formation of SAHFs and the formation requires several

days.

Senescence-associated β-galactosidase

Senescence-associated β-galactosidase (SA-β-GAL) is a common and often used biomarker to detect

senescent cells [368, 369]. SA-β-GAL is active at a pH of 6.0. This allows the discrimination between

SA-β-GAL and normal β-galactosidase, because normal β-galactosidase activity can be detected at a

pH of 4.0.

Introduction

24

Activation of tumor suppressors

As described above, p21 and p16 are important cell cycle inhibitors and involved in senescence related

cell cycle arrest. Therefore, the expression of them can be used as a marker for senescence.

Senescence-associated secretory phenotype

The senescence-associated secretory phenotype (SASP) is an important and interesting feature of

many but not all senescent cells. The reason for that is, SASP could explain how senescence could be

on the one hand tumor suppressing and on the other hand tumor growth promoting [370]. Several

studies showed that senescent fibroblast could promote the growth of tumors derived from different

tissues [371-373]. SASP is characterized by the secretion of a variety of soluble and insoluble factors

[370, 374]. These factors can influence the local microenvironment of the senescent cell, or even act

on a systemic scale. Components of the SASP are interleukins, inflammatory cytokines, growth

factors, matrix metalloproteinases, serine proteases and their inhibitors [374-378]. The specific

composition of SASP-factors can vary and is dynamic, depending on the cell type and the origin of the

senescence.

Introduction

25

Figure 1-9: Overview of senescence inducer, senescence controlling pathways and senescence markers. Cellular

senescence is caused by various stimuli. These stimuli include dysfunctional telomeres, DNA damage, chromatin

perturbations, active oncogenes and stress. The consequences of senescence are diverse and can be beneficial or detrimental.

Senescence causes a cell cycle arrest. This could have protective effects for a multicellular organism because it suppresses the

growth of degenerated cells. Thus, senescence has a tumor suppressing function. However, senescence can also induce tumor

progression because some senescent cells express a SASP and release growth factors. The senescence inducing signals

engage either the p53/p21 pathway or the p16/RB pathway. Some stimuli also engage both pathways. Senescence stimuli that

activate p53 induce the expression of p21. p21 is a CDK inhibitor, its expression inhibits all CDK/cyclin complexes and

induces a cell cycle arrest. Senescence stimuli can also induce the expression of p14 which is a negative regulator of MDM2.

Under normal conditions p53 is ubiquitylated by MDM2 and degraded. Hence, the p21 expression is suppressed and the cell

cycle is not inhibited. Senescence stimuli can also induce the expression of p16, a further CDK inhibitor. p16 only inhibits

CDK4/6/cyclin D complexes. This inhibits the phosphorylation of RB. The hypophosphorylated RB inhibits the release of the

transcription factor E2F which induces a G1 cell cycle arrest. Senescent cells can be identified by a combination of various

types of senescent markers.

Introduction

26

β-adrenergic signaling and genomic stability 1.4.2

The above described interactions between stress, stress hormones, DNA damage, DNA damage repair,

senescence and AR signaling suggest that β-adrenergic signaling can influence the genomic stability

of cells including leukocytes. β2-ARs can be found in various tissues and their signaling is important

in the regulation of several cellular processes that are essential in carcinogenesis, such as

inflammation, angiogenesis, apoptosis, cell motility and DNA damage repair. Various cancer models,

cellular studies and epidemiologic studies demonstrated these circumstances [379]. However, β-

adrenergic signaling seems to affect mostly the progression of cancer and less the carcinogenesis

[380]. β-adrenergic signaling plays an important role in tumor progression of human prostate cancer

cells in a xenograft mouse model [381]. Moreover, mouse models of breast cancer demonstrated an

increased metastasis induced by β-adrenergic signaling after the exposure to a stressor [382]. Also

behavioral stress increases the growth of malignant melanoma in a dose-dependent manner [383].

Environmental stress or administration of epinephrine increases the mortality rate of rats that were

transplanted with CRNK-16 leukemia cells [384]. Furthermore, all four studies demonstrated that

beta-adrenergic antagonists could be used to diminish or inhibit the stress-related tumor growth or

metastasis. These findings correspond with human studies that showed that a treatment with β-blocker

reduce metastasis, cancer recurrence and mortality in breast cancer patients [385, 386]. A reduced

tumor growth and an increased revival rate of patients with malignant melanoma could be observed

after treatment with β-blocker [387, 388]. Interestingly, it was shown that in ovarian tumor tissue the

norepinephrine concentrations are higher than the norepinephrine concentrations in the blood plasma

of the same patients. Moreover, the catecholamine concentrations in the tumor tissue correlate

positively with psychosocial risk factors, but the catecholamine concentrations in the plasma do not

show this correlation [389, 390]. Besides the xenograft animal models and the epidemiological studies

also molecular biological studies demonstrated that β-adrenergic signaling is involved in the tumor

growth. For instance, invasion of macrophages into the tumor [382], elevated expression of pro

inflammatory cytokines, IL-6 and IL-8, in tumor cells [391-393], vascular endothelial growth factor

(VEGF) mediated increase of angiogenesis of the tumor [41], increased expression of

metalloproteinases accompanied by increased tumor invasion in surrounding tissue [41, 394, 395],

resistance against apoptosis induce by chemotherapeutics [396] are stimulated by β-adrenergic

signaling. The important transcription factor cAMP response element-binding protein (CREB) is

activated by stress hormones and influences the proliferation, migration and angiogenesis of tumor

cells [397, 398]. Epinephrine can induce DNA damage in human lymphocytes [399]. Hara et al.

proposed the following model for the induction of DNA damage in human cancer cell lines and mice

by chronic stress, see Figure 1-10 [400, 401]. Stress hormones, like the catecholamines epinephrine

and norepinephrine but also the synthetic homolog isoproterenol activate the β2-AR and downstream

signaling. Two different signaling pathways get activated. On the one hand, the Gs protein/PKA

Introduction

27

dependent pathway and on the other hand signaling via β-arrestin 1 is induced. Activation of the β2-

AR induces the formation of ROS by NAD(P)H oxidases and signaling via the G protein/PKA

pathway suppresses anti-oxidative mechanisms. At the same time, β-arrestin 1 mediates the activation

of MDM2 by the protein kinase B (AKT). Moreover, β-arrestin 1 facilitates the interaction of MDM2

and p53 which induces the degradation of p53. The degradation of p53 compromises the DNA damage

repair. The interplay of both signaling pathways results in the accumulation of DNA damage [400,

401].

Figure 1-10: Schematic representation of the catecholamine induced, β2-adrenergic receptor signaling that mediates

the stress response and leads to the accumulation of DNA damage. According to Hara et al. adapted from [401].

Objective

28

2 Objective

Clinical and animal studies have shown that chronic psychological stress impairs the immune system

[16, 77, 79, 402]. For instance, chronically stressed humans have an increased risk to suffer from an

infectious disease. Also the recovery time after an infection is prolonged and the recurrence rate of

latent virus infection is higher [403, 404]. On the other side, chronic stress also induces DNA damage

and affects the DNA damage repair [43, 47, 48, 400, 401]. Previous studies have shown that immune

cells of PTSD patients have an accumulation of DNA strand breaks [83]. Psychotherapy reversed the

PTSD symptoms as well as the accumulation of this DNA strand breaks. Further studies have shown

that PTSD patients have an increased rate of telomere shorting, an accelerated aging rate and an

increased risk for cancer [28, 73, 82, 316, 319]. Moreover, there are some indications that PTSD may

be associated with a phenotype of accelerated senescence [312]. At the molecular level, stress

hormones like catecholamines can induce DNA damage in human cell lines and in mice via the

activation of the β2-AR [400, 401]. On the other hand, the degradation of catecholamines induces the

formation of ROS and free radicals [167, 168, 233]. All results together give an indication that chronic

stress may impair the genomic stability. Therefore, the following hypothesis was raised:

“Catecholamines can induce biomarkers of stress and may impair the genomic stability of human

PBMCs via the β2-AR”. An ex vivo model was established to mimic the repeated release and action of

catecholamines during chronic stress [405]. Pilot experiments have shown that the repeated treatment

of PBMCs with isoproterenol induces DNA strand breaks which were in parts unrepaired after 24 h.

These DNA strand breaks could be partial inhibited by the β-blocker propranolol. Additional, the

protein level of PARP1 and the formation of PAR were reduced by the repeated isoproterenol

treatment [405]. PBMCs incubated over a longer time period (48 h and 72 h) after the isoproterenol

treatment showed the expression of SA-β-GAL. The presented thesis had four main objectives.

The first objective was to complement the pre-existing results. Since cAMP and the downstream

signaling cascade are involved in generation of ROS, the formation of cAMP in response to the

repeated isoproterenol treatment was analyzed. DNA strand breaks induce the activation of PARP1

which depletes cellular NAD+ pools by the formation of PAR. Hence, the cellular NAD

+ and ATP

content were measured. Since the formation of ROS appeared to be the most probable cause for the

induction of DNA strand breaks, the formation of intracellular ROS during the repeated isoproterenol

treatment was analyzed.

The second objective was to complement the pre-existing results with regards to the observed

induction of senescence markers in PBMCS after the repeated isoproterenol treatment. Therefore, the

mRNA expression of several genes which were involved in the cell cycle regulation, DNA damage

response, oxidative stress response, β-adrenergic signaling and in the controlling of senescence was

Objective

29

measured by real-time PCR. The protein level of p16, an important biomarker of aging and inducer of

cellular senescence, was measured.

The third objective was to investigate the stability and degradation of isoproterenol in the used cell

culture media. As the ligand binding pocket of the β2-AR is located extracellular, the concentration of

the ligand in the surround medium is crucial for its stimulation. Therefore, the time course of the

isoproterenol concentration during the treatment and the degradation of the isoproterenol after the

treatment were measured by HPLC. The isoproterenol concentration was measured by its specific

absorbance at 280 nm and at its fluorescence signal at an excitation wavelength of 280 nm and an

emission wavelength of 310 nm. Additional, the formation of isoprenochrome, an oxidation product of

isoproterenol, was measured at its specific absorbance at 490 nm.

The fourth objective was the pre-validation of a new developed FADU system, called TOXXs

Analyzer. The technical pre-validation should be performed in cooperation with two partners, the

EMPA in St. Gallen and the Cetcis GmbH in Esslingen, in the course of an inter-laboratory study. The

aims of this pre-validation were to test and to improve the reproducibility, sensitivity and intra-

laboratory variability of the TOXXs Analyzer.

Material and Methods

30

3 Material and Methods

3.1 Material

Chemicals 3.1.1

Substance Supplier

acetonitrile Carl Roth, Karlsruhe, Germany

acryl-/bisacrylamid Carl Roth, Karlsruhe, Germany

alcoholdehydrogenase Sigma-Aldrich, Steinheim, Germany

ammonium acetate Sigma-Aldrich, Steinheim, Germany

APS Serva, Heidelberg, Germany

bicine Sigma-Aldrich, Steinheim, Germany

Bicoll Biochrome, Berlin, Germany

bromphenol blue Sigma-Aldrich, Steinheim, Germany

BSA Serva, Heidelberg, Germany

caffeine Sigma-Aldrich, Steinheim, Germany

calcium chloride Fluka, Buchs, Switzerland

calf thymus DNA Sigma-Aldrich, Steinheim, Germany

CasyClean OMNI Life Science GmbH, Bremen, Germany

CasyTon OMNI Life Science GmbH, Bremen, Germany

citric acid Sigma-Aldrich, Steinheim, Germany

complete protease inhibitor cocktail Roche, Mannheim, Germany

DCFDA Sigma-Aldrich, Steinheim, Germany

DHE Sigma-Aldrich, Steinheim, Germany

DMSO Merck, Darmstadt, Germany

DNaseI Roche, Mannheim, Germany

ECL solution Lumigen Inc., Michigan, USA

EDTA Sigma-Aldrich, Steinheim, Germany

ethanol pa VWR, Darmstadt, Germany

ethanol tech. Riedel-de Haen, Seelze, Germany

FCS Biochrome, Berlin, Germany

formic acid Merck, Darmstadt, Germany

forskolin Sigma-Aldrich, Steinheim, Germany

glycerol Acros, Geel, Belgium

glycine Carl Roth, Karlsruhe, Germany

Hoechst 33342 Invitrogen, Karlsruhe, Germany

hydrogen chloride Riedel-de Haen, Seelze, Germany

hydrogen peroxide Merck, Darmstadt, Germany

IBMX Sigma-Aldrich, Steinheim, Germany

IMI Sigma-Aldrich, Steinheim, Germany

isopropanol VWR, Darmstadt, Germany


31

isoproterenol Sigma-Aldrich, Steinheim, Germany

Jurkat cell lysate Cell Signaling Technology

luminol Fluka, Buchs, Switzerland

magnesium acetate Merck, Darmstadt, Germany

magnesium chloride Acros, Geel, Belgium

magnesium sulfate Riedel-de Haen, Seelze, Germany

magnesiumchloride-6-hydrate Merck, Darmstadt, Germany

MEN Sigma-Aldrich, Steinheim, Germany

methanol (p.a.) VWR, Darmstadt, Germany

milk powder Rapilait, Migros, Switzerland

MOPS Sigma-Aldrich, Steinheim, Germany

MTT Sigma-Aldrich, Steinheim, Germany

NAD+ Sigma-Aldrich, Steinheim, Germany

octansolfonic acid Sigma-Aldrich, Steinheim, Germany

PageRuler Thermo Scientific, Schwerte, Germany

paraformaldehyde Merck, Darmstadt, Germany

PBS Biochrome, Berlin, Germany

p-coumaric acid Fluka, Buchs, Switzerland

penicillin/ streptomycin (5000 units/ml) Gibco Life Technologies, Karlsruhe, Germany

perchloric acid Riedel-de Haen, Seelze, Germany

phenazine ethosulfate Sigma-Aldrich, Steinheim, Germany

phosphodiesterase Affymertrix

phosphoric acid Riedel-de Haen, Seelze, Germany

potassium dihydrogen phosphate Riedel-de Haen, Seelze, Germany

potassium hydroxide Merck, Darmstadt, Germany

potassium hydroxide Riedel-de Haen, Seelze, Germany

propranolol Sigma-Aldrich, Steinheim, Germany

proteinase K Roche, Mannheim, Germany

RNase A Sigma-Aldrich, Steinheim, Germany

RPMI-1640 Gibco Life Technologies, Karlsruhe, Germany

RPMI-1640 without phenol red Gibco Life Technologies, Karlsruhe, Germany

sodium azide Merck, Darmstadt, Germany

sodium chloride Carl Roth, Karlsruhe, Germany

sodium dodecyl sulfate (SDS) Sigma-Aldrich, Steinheim, Germany

sodium hydroxide Merck, Darmstadt, Germany

sodium periodate Merck, Darmstadt, Germany

sodium-deoxycholat Merck, Darmstadt, Germany

SYBR Green I Invitrogen, Karlsruhe, Germany

TBHP Fluka, Buchs, Switzerland

TEMED Carl Roth, Karlsruhe, Germany

TexMACS Miltenyi Biotec, Bergisch Gladbach, Germany

trichloracetic acid Carl Roth, Karlsruhe, Germany

Tris-HCl Sigma-Aldrich, Steinheim, Germany

Trisma Base Sigma-Aldrich, Steinheim, Germany

trypan blue Sigma-Aldrich, Steinheim, Germany

trypsin-EDTA Gibco Life Technologies, Karlsruhe, Germany

Tween 20 Sigma-Aldrich, Steinheim, Germany


32

urea Carl Roth, Karlsruhe, Germany

β-mercaptoethanol Merck, Darmstadt, Germany

Laboratory equipment 3.1.2

Object Type Supplier

-80°C Fridge Hera freeze Heraeus Instruments

-80°C Fridge -86Frezzer Thermo Scientific

1100 Well-plate Autosampler G1357A Agilent

1100/ 1200 Column Thermostat G1316A Agilent

1100/ 1200 Fluorescence Detector G1321A Agilent

1100/1200 Binary Pump G1312A Agilent

1100/1200 Diode Array Detector G1315A Agilent

1200 Sample Thermostat G1330B Agilent

suction system cell culture Vacusafe IBS Integra Biosiences

Alliance (HPLC) Waters 2695 Waters

bacterial incubator Minitron INFORS AG

benchtop Centrifuge Biofuge pico Heraeus Instruments

benchtop Centrifuge Heraeus Fresco 17 Thermo Scientific, Schwerte,

Germany

benchtop Centrifuge 5810 R Eppendorf

benchtop Centrifuge Pico17 Hereaus

biological safety cabinet S1 Nanc

biological safety cabinet S2 HeraSafe Heraeus Instruments

biological safety cabinet S2 Lamin Air HB 2448 Heraeus

C18-column length 15 cm,

inner diameter 4.6 mm,

particle size 5 µm,

column volume 2.5 ml

Macherey-Nagel

C18-column UPLC

cell counter Casy CellCounter TT Innovatis

cell culture microscope Axiovert40C Zeiss

centrifuge 5415R Eppendorf

chemiluminescence detector Image Quant LAS 4000 mini GE Healthcare

condensation trap of

Vacuum concentrator

RVT5105 Thermofisher scientific

ELISA Reader SLT Spectra Tecan

FACS BD LSRII BD Biosciences

fluorescence microscope Axiovert 200M

Plan-APOCHROM

63X/1.4 oil

Zeiss


33

fridge

Premium

Liebherr

glassware Schott

guard column Security Guard Phenomenex

halogenlamp ebq 100 isolated

hemocytometer Casy Innovartis

ice maker AF206 Scotsman

incubator Hera Cell 240 Heraeus Instruments

incubator Hera Cell Heraeus Instruments

magnetic Stirrers IKAM Häberle Labortechnik

magnetic Stirrers MR3001K Heidolph

mass spectrometer Quattro micro Waters

mass spectrometer Xevo TQ-S Waters

micro clear plate 96-well Cellstar ®,96well-Platten,

clear, flat bottom

Greiner Bio-One

micro plate black bottom FluotracTM 200, 96W-

Microplate, medium

binding, black, flat bottom

Greiner Bio-One

micro scales CP2202S Sartorius

micro scales CP225D Sartorius

micropalte reader SLT Tecan

micropalte reader Infinite F200 PRO Tecan

micropalte reader Varioscan flash Thermofisher scientific

microscope Leitz DK IL Leica

MilliQ Reference A+ Millipore

minisaker Duomax 1030 Heidolph

minisaker MTS4 IKA

orbital shaker ROTAMAX 120 Heidolph

PCR thermal cycler Flex Cycler Analytik Jena

pH meter knick

pH meter 605pH Meter Metrohm

pipetboy Pipetboy Comfort IBS Integra Biosiences

pipettes 0,1-2 µl Eppendorf


pipettes 2-20 µl Eppendorf




power supplies Power Pac 200 Bio-Rad, München, Germany

power supplies Electrophoresis power

supply EPS301

Amersham Biosciences

printer C3760dn Dell

pump MS-Reglo Ismatec

Real-time PCR system CFX96 Bio-Rad, München, Germany

roller mixer RS-TR05 Phoenix Instrument

scale AG 204 Delta Range Mettler

scale PM2000 Mettler

semi dry blotting system Bio-Rad, München, Germany


34

sonicater Sonorex Super RK102H Bandelin

sonicater TK52 Bandelin

spectrometer Ultraspec 2100pro Amersham

spectrometer NanoDrop Thermofisher scientific

thermomixer Thermomicer Comfort Eppendorf

UPLC Acquity UPLC CLASS H Waters

vacuum concentrator UNIVAPO100ECH UNIVAPO

vacuum pump PC3004 Vario Vacuubrand

vortexer Vortex-Genie 2 Bender & Hobein AG

water bath 1083 GFL

water bath 1002 GFL

wet blotting Hoefer miniVE Vertical

Electrophoresis

System

Amersham Biosciences

X-ray system XRAD 225IX PXI PRECISION X-RAY

Consumables 3.1.3

Product Supplier

384-well microplate for cAMP assay PerkinElmer, Hamburg, Germany

bottle-top vacuum filters, pore size 0.22 µm Corning, Schiphol-Rijk, Netherlands

Casy cup

OMNI Life Science GmbH, Bremen,

Germany

cell culture 96-well microplate Corning, Schiphol-Rijk, Netherlands

cell culture dish Corning, Schiphol-Rijk, Netherlands

cell culture flask T 175 Corning, Schiphol-Rijk, Netherlands



cell scraper Corning, Schiphol-Rijk, Netherlands

cryovials Corning, Schiphol-Rijk, Netherlands

FACS vials Starlab, Hamburg, Germany

conical tube (15 ml) Corning, Schiphol-Rijk, Netherlands


glassware Schott, Mainz, Germany

gloves (Latex) MaiMed, Neuenkirchen, Germany

gloves (Nitril) VWR, Darmstadt, Germany

HPLC sample vials Phenomenex, Aschaffenburg, Germany

HPLC sample vials inserts Phenomenex, Aschaffenburg, Germany

parafilm Pechiney Plastic Packing

serological pipette (10 ml stripette) Corning, Schiphol-Rijk, Netherlands



reaction vessel (SafeSeal 0.5 ml) Sarstedt, Nürnbrecht, Germany


reaction vessel (SafeSeal 2 ml) Sarstedt, Nürnbrecht, Germany


35

Safety-Multifly-canula G21/0.8 mm Sarstedt, Nürnbrecht, Germany

S-Monovettes, EU color code: green

citrate 3.2% (1:10)

Sarstedt, Nürnbrecht, Germany

tips (1000 µl) Sarstedt, Nürnbrecht, Germany



tips long (200 µl) VWR, Darmstadt, Germany

Buffers and solutions 3.1.4

1.5X SDS-Page high-urea sample buffer

93.75 mM Tris-HCl (pH 6.8)

9 M urea

7.5% (v/v) β-mercaptoethanol

15% (v/v) glycerol

3% (w/v) SDS

0.01% (w/v) bromphenol blue

add MilliQ water

10X Laemmli buffer

250 mM Tris-HCl (pH 7.4)

1.92 M glycine

1% (v/v) SDS

add MilliQ water

10X SDS-Page sample buffer

583 mM Tris HCl pH (8.0)

8.5% (w/v) SDS

60% (v/v) glycerol

10% (v/v) 2-mercaptoethanol

0.01% (w/v) bromphenol blue

Alcohol dehydrogenase (NAD

+ cycling assay)

10 mg/ml alcohol dehydrogenase

add 0.1 M bicine in NaOH (pH 8.0)

Buffer A

10 mM Tris HCl pH 7.8

1 mM EDTA

4 mM magnesium chloride

14.3 mM β-mercaptoethanol

add MilliQ water


36

cAMP-Glo detection solution

0.35% PKA

add cAMP-Glo reaction buffer

Complete protease inhibitor cocktail

1 pill

add MilliQ water

DCFDA stock solution

20 mM 2′.7′-dichlorofluorescin diacetate (DCFDA)

add DMSO

DHE stock solution

40 mM dihydroethidium (DHE)

add DMSO

Diluent (NAD

+ cycling assay)

0.25 M phosphoric acid

0.5 M sodium hydroxide

add MilliQ

DNase I on column-degradation solution

10 µl DNase I stock solution

70 µl RDD buffer

DNase I stock solution

1500 Kunits DNase I

550 µl RNase-free water

ECL solution A

4.4 ml MilliQ water

500 µl Tris-HCl pH (8.5)

50µl luminol

22µl coumaric acid

ECL solution B

4.5 ml MilliQ water

500 µl Tris-HCl pH (8.5)

3 µl hydrogen peroxide

FACS blocking buffer

0.3 M glycine

10% fetal calf serum

add PBS


37

FACS buffer

2 mM NaN3

2 mM EDTA

3% fetal calf serum

add PBS

FACS fixation solution

4% paraformaldehyde

add PBS

FACS permeabilization/washing buffer

2 mM sodium azide

2 mM EDTA

3% fetal calf serum

0.1% Tween 20

add PBS

HPLC eluent

95 mM citric acid

0.35 mM EDTA

0.46 mM octane sulfonic acid

5.7% (v/v) acetonitrile

94.3 (v/v) MilliQ water

HPLC oxidation buffer

10 mM ammonium acetate

2 mM sodium periodate

add MilliQ water

Induction buffer

0.1 mM imidazolidin

0.5 mM 3-isobutyl-1-methylxanthin

add PBS

Modified high-salt-radio-immunoprecipitation assay (RIPA) buffer

500 mM sodium chloride

1% (v/v) Triton-X-100


0.1% (w/v) SDS

1% Na-deoxycholat

1 X complete protease inhibitor cocktail

add MilliQ water

MTT solution

5 mg/ml MTT

add PBS


38

NAD

+ (NAD

+ cycling assay)

10 mM NAD+

add diluent

PARP1 reaction buffer

50 mM Tris HCl pH 7.8



13.5 ng/µl GGAATTCC

0.3 mM NAD+

PBS (pH 7.4)


2.7 mM potassium hydrogen phosphate

8.1 mM disodium hydrogen phosphate

1.8 mM potassium dihydrogen phosphate

add MilliQ water

Perchloric acid (NAD

+ cycling assay)

3.5 M perchloric acid

add MilliQ

Premix (NAD+ cycling assay)

0.48 M bicine in NaOH (pH 8.0)

4 mg/ml BSA

20 mM EDTA

2 mM MTT

2.4 M ethanol

Reactionmix (NAD

+ cycling assay)

5 parts premix

1 part phenazine ethosulfate 40 mM

1 part alcohol dehydrogenase 1mg/ml

RLT buffer

1% β-mercaptoethanol

add RLT buffer

SDS-Page resolving gel buffer

1.86 M Tris-HCl (pH 7.4)

7 mM SDS

adjust pH 8.8

add MilliQ water


39

SDS-Page stacking gel buffer


7 mM SDS

adjust pH 6.8

add MilliQ water

TCA solution

20% TCA

add MilliQ water

TNT buffer



0.5% (v/v) Tween20

add MilliQ water

Towbin buffer


192 mM glycine

0.1% (w/v) SDS

20% (v/v) methanol

add MilliQ water

Cell lines and cell culture reagents 3.1.5

Cell line Basal medium Supplements

PBMCs RPMI-1642

100 units/ml penicillin,

100 µg/ml streptomycin

PBMCs RPMI-1643

10% (v/v) FCS,



PBMCs TexMACS




40

Antibodies and dyes 3.1.6

Primary Antibodies

Antibody Origin Purpose Dilution Supplier

anti-PARP1 (CII10) monoclonal mouse WB 1/300 CII10 hybridoma

cells

from G. G. Poirer,

Quebec,

Canada

anti-PARP1 (FI-23) monoclonal mouse FACS/WB 1/300 CII10 hybridoma

cells from

G. G. Poirer,

Quebec,Canada

anti-p16 monoclonal rabbit FACS 1/320 Abcam

anti-PAR (10H) monoclonal mouse FACS 1/300 10H hybridoma

cells from M. Miwa

and T. Sugimura,

Tokyo Japan

anti-H1 monoclonal mouse WB 1/200 Santa Cruz

Biotechnology

anti-p21 monoclonal rabbit FACS 1/100 Abcam

anti-Actin monoclonal mouse WB 1/50000 Millipore

Secondary Antibodies

Antibody Conjugation Purpose Dilution Supplier

goat anti mouse IgG HRP WB 1/2000 Dako

goat anti rabbit IgG Alexa Flour 488 FACS 1/1000

Invitrogen,

Karlsruhe,

Germany

goat anti mouse IgG Alexa Flour 488 FACS 1/1000

Invitrogen,

Karlsruhe,

Germany

Kits 3.1.7

Kit Supplier

cAMP-Glo assay Promega, Madison, USA

Allprep RNA/DNA/Protein Mini Kit Qiagen, Hilden, Germany

RNase-Free DNase Set Qiagen, Hilden, Germany

iScript Advanced cDNA Synthesis kit Bio-Rad, München, Germany

iScript Advanced Universal Sybr Green Supermix Bio-Rad, München, Germany

PrimePCR assay Bio-Rad, München, Germany

High pure miRNA isolation kit Roche, Mannheim, Germany


41

Software 3.1.8

Software Source

Agilent ChemStation Rev. B.02.01 Agilent

Bio-Rad CFX Manager 3.1 Bio-Rad

ChemOffice2004 CambridgeSoft Corporation

EndNote X7.7.1 Clarivate Analytics

GraphPad Prism 6 GraphPad Software

ImageJ 1.6.0_20 Wayne Rasband

MassLynx MS-Software Waters

Microsoft Office 2010 Microsoft

3.2 Methods

General aspects of cell culture 3.2.1

Cell lines were cultured according to the “American Type Culture Collection” (ATCC) guidelines. All

cell culture working steps were performed under a laminar flow cabinet (safety class 2). In the

beginning, the laminar flow cabinet was turned on at least 15 min before the actual cell culture work

was started. Then the surface of the cabinet was cleaned with 70% ethanol (v/v) in the beginning as

well as at the end of the cell culture work. All working steps were performed under aseptic conditions.

A cell culture coat and latex or nitrile gloves were worn the whole time. Cell culture plastics were

bought as ready to use sterile packages. Buffers, media and chemicals which were used for culturing

of the cells were bought sterile with cell culture grade. All items and materials used under the laminar

flow cabinet were pre-cleaned with 70% ethanol (v/v) immediately before they were put in the laminar

flow cabinet. Only individual cell lines were processed under the laminar flow cabinet, to avoid cross

contaminations. Between the processing of two or more cell lines the laminar flow cabinet was

cleaned after each processing of a cell line. Cell lines were cultured at 37 °C with 5% CO2 .

PBMC isolation 3.2.2

Venous blood samples were taken from the forearm of healthy volunteers with a 21 G cannula and

collected in S-Monovettes (filled with trisodium citrate-solution 1 ml per tube (0.106 mol/l) as

anticoagulant). For each donor a maximum sample size of ten S-Monovettes with blood were taken.

These samples were combined in three 50 ml conical tubes. PBMCs were isolated according the

following protocol, see Figure 3-1. Blood samples were centrifuged at 300 g, at RT for 10 min without

breaks. The upper plasma layer was carefully removed with serological pipettes and pre-warmed PBS


42

(37 °C) was added until a total volume of 50 ml was reached. The blood was mixed with PBS by

inverting the conical tubes a few times. The 50 ml of the resulting sample were divided into two equal

parts. Each part was transferred on the top of a 15 ml Biocoll layer in a 50 ml conical tube.

Afterwards, the blood samples were separated by density centrifugation. For that, the samples were

centrifuged for 15 min at RT and 900 g without breaks. The PBMC layer was carefully removed with

a Pasteur pipette. The PBMC-layers of two samples were pooled in a new 50 ml conical tube and

stored on ice. Then the conical tubes were filled up with ice-cold PBS. The samples were centrifuged

for 10 min at 4 °C and 300 g and the supernatant was removed. Next, PBMCs were resuspended in

50 ml ice-cold PBS and centrifuged again for 10 min at 4 °C and 300 g the supernatant was removed

again. The cell pellets were resuspended in 10 ml cell culture medium and stored on ice until further

processing. The cell number was counted with the Casy cell counter and the cell number was adjusted

to 2*106 cells per ml.

Figure 3-1. Isolation of human PBMCs from whole blood by Biocoll density gradient centrifugation.

Isoproterenol treatment of PBMCs 3.2.3

After the isolation PBMCs were resuspended in one of three different cell culture media, either in

RPMI-1640 without fetal calf serum (FCS) supplementation, RPMI-1640 with FCS supplementation

or TexMACS. The cell concentration was adjusted to 2*106 cells/ml. Then the cells were aliquoted,

1 ml cell suspension per 2 ml reaction vessel. Isoproterenol was dissolved, in the same cell culture

medium which was used to resuspend the PBMCs, to a stock solution with a concentration of 10 mM.

The isoproterenol stock solution was prepared fresh for each experiment and stored on ice in the dark


43

until the treatment was finished. Three different treatment types were administered to the cells, see

Figure 3-2. Cells were either treated with a single dose of 10 µM isoproterenol and then every 30 min

only with the solvent for the next seven treatments (1x iso). Or cells were treated with 10 µM

isoproterenol and then every 30 min alternating with solvent or 10 µM of isoproterenol for the next

seven treatments (4x iso). The last treatment was an eight-time isoproterenol administration, each time

a dose of 10 µM with an interval of 30 min (8x iso) was added. Afterwards, cells were incubated for

the indicated time at 37 °C in a shaking water bath. Control cells received every 30 min the pure cell

culture medium which was used to dissolve the isoproterenol.

Figure 3-2. Timetable of repeated isoproterenol treatment of fresh isolated human PBMCs. Human PBMCs were

treated at intervals, with isoproterenol (red arrow) or with solvent w/o isoproterenol (blue arrow) with a time interval of

30 min between each treatment, for 3.5 h.

Analysis of cellular cAMP levels 3.2.4

cAMP was measured with the cAMP-Glo assay from Promega with some modifications of the

protocol. Assay principal is depicted in Figure 3-3. Briefly, PBMCs were isolated as described above.

For the cAMP assays a smaller sample volume had to be used. Therefore, 40 µl of the cell suspension

(2*106 cells/ml) were aliquoted in 0.5 ml reaction tubes. 10 min before the isoproterenol treatment was

started, control cells were treated with 10 µM propranolol dissolved in cell culture medium. Next, the

isoproterenol treatment was started with the first dose of the 8x isoproterenol treatment, 1 µl of a

400 µM stock solution was added to the cell suspension. The 1x and 4x treated samples received 1 µl

of cell culture medium. The following treatment steps were performed every 30 min, as described

before with the adjusted volumes.


44

Figure 3-3: Flowchart of the cAMP-Glo assay. G protein coupled receptors get activated by binding of an extracellular

ligand that stabilizes the active receptor conformation. This induces the dissociation of the G protein into the α subunit and

the βγ heterodimer. The α subunit of a Gs protein activates the AC which induces the formation of cAMP from ATP. The

principal of the cAMP-Glo assay is depicted in the grey box. cAMP binds to the regulatory subunits of PKA. The PKA

holoenzyme dissociates into its two regulatory subunits and two catalytic subunits. The regulatory subunits use ATP to

phosphorylate a PKA substrate. ATP at the same time is also needed for the luciferase catalyzed luminescence reaction.

Hence, the cAMP concentration is inversely related to the luminescence. The more receptors are activated, the more cAMP is

formed which leads to a lower light output. Figure was copied from the handbook [406].

Directly before the last isoproterenol treatment was performed, cells received 100 µM 3-isobutyk-1-

methylxanthine (IBMX) and 500 µM imidazolidin (IMI) for inhibiting the PDEs. Samples were

centrifuged at RT and 900 g for 10 min. The supernatant was removed and cells were resuspended in

30 µl of induction buffer. Samples were transferred in technical triplicates into a 384-well assay plate

(white) 7.5 µl/well. Cells were lysed with 7.5 µl/well of cAMP-Glo lysis buffer. These lysates were

incubated for 15 min at RT on an orbital shaker. Then 15 µl/well of fresh prepared cAMP-Glo

detection solution were added. The samples were placed on an orbital shaker for 1 min. Afterwards,

the samples were incubated for 20 min at RT. 30 µl/well of the kinase Glo-reagent were added and the

samples were placed for 1 min on an orbital shaker. Samples were incubated for 10 min at RT. Then

the luminescence was measured.


45

Analysis of cellular ROS 3.2.5

The formation of intracellular ROS was analysed with the two fluorescence dyes 2′,7′-

dichlorofluorescin diacetate (DCFDA) and Dihydroethidium (hydroethidine) (DHE). DCFDA is a

cell-permeable dye which can be used to determine the overall intracellular oxidative stress [407, 408].

After diffusion into the cell, the acetate groups are cleaved by intracellular esterases. The resulting

product H2DCF is cell membrane-impermeable and can be oxidized to the highly fluorescent 2´,7´-

dichlorofluorescein (DCF). The reactivity of DCFDA is still not completely clarified, but it seems to

have a good specificity for peroxides [408, 409]. DHE is another fluorogenic probe which reacts

rapidly with superoxide anions (O2-)

[407, 410, 411]. After oxidation it intercalates into the DNA

which induces a shift of the fluorescence spectrum. Stock solution of DHE and DCFDA dyes were

prepared in anhydrous dimethyl sulfoxide (DMSO). DHE was solved to a concentration of 40 mM and

DCFDA was solved to a concentration of 20 mM. The stock solutions were frozen and for each

experiment a new aliquot was unfrozen and used only one time. During the use the solutions were

protected from light. Both dyes were used separately to analyse the redox state of PBMCs during the

isoproterenol treatment. Cells were aliquoted in 2 ml reaction tubes, 2*106

cells per tube. First, the

DCFDA staining was performed. The cells were stained with 20 µM of DCFDA, 1 µl of the DCFDA

stock solution was added per reaction tube. The cells without the DCFDA staining received 1 µl of

DMSO. Cells were incubated for 30 min at 37 °C. During this time the cell samples for the DHE

staining were stored on ice. Afterwards, cells of the DCFDA staining were treated with isoproterenol,

as described in section 3.2.3. As positive control, cells were treated with different doses of tert-

butylhydroperoxide (TBHP): 200 µM, 500 µM or 50 mM TBHP. During the isoproterenol treatment

control samples received 1 µl of cell culture medium instead of isoproterenol solution. For the staining

with DHE, control cells were treated with different doses of menadione (MEN): 10 µM, 200 µM,

50 µM and 500 µM MEN. MEN is a free radical generator which can be used to produce O2- in cells

[407]. Samples were treated with isoproterenol as described above; control samples received 1 µl of

cell culture medium instead of isoproterenol solution. After finishing the last isoproterenol treatment,

cells were incubated for further 30 min, reaching a total incubation time of 4 h. This has two reasons:

the formation of ROS may need some time and 4 h after the first isoproterenol dose the formation of

DNA strand breaks began. Furthermore, some experiments were performed with a total incubation

time of 6 hours (2.5 h after the last isoproterenol administration), because at this point in time the

maximum of DNA strand breaks could be observed. Samples were incubated at 37 °C in a water bath,

removed at indicated points of time and centrifuged for 5 min at 600 g. Supernatant was removed and

cells were washed with 1 ml of pre-warmed (37 °C) PBS. Samples of the DHE staining were

resuspended in 1 ml of cell culture medium containing 10 µM DHE (a 1:10 pre-dilution was

performed immediately before the use). Afterwards, the DHE samples were incubated for 30 min at

37 °C. During this time, the DCFDA samples were resuspended in 200 µl of ice-cold RPMI-1640 cell


46

culture medium without phenolred per sample. For the measurement samples were transferred on 96-

well microplate in triplicates of 50 µl per well. Fluorescence was measured with a plate reader at an

excitation wavelength of 485 nm and an emission wavelength of 535 nm. At the end of the incubation

time of the DHE samples, the cells were centrifuged for 5 min at 600 g and the supernatant was

removed. Then the cells were washed with 1 ml pre-warmed (37 °C) PBS. Next, the cells were also

resuspended in 200 µl of ice-cold RPMI-1640 cell culture medium without phenolred per sample. For

the measurement, samples were transferred on a 96-well microplate in triplicates of 50 µl per well.

Fluorescence was measured in a plate reader at an excitation wavelength of 520 nm and an emission

wavelength of 610 nm.

NAD+ Cycling assay 3.2.6

The influence of the repeated isoproterenol treatment on the cellular NAD+

concentration was analysed

using a modified NAD+ cycling assay protocol according to Jacobson and Jacobson [412], modified by

Weidele et al. [413]. Cells were treated with isoproterenol as descripted in section 3.2.3. Additionally,

cells were treated with 500 µM H2O2 (positive control) for 5 min at 37 °C in PBS to lower intracellular

NAD+ levels (positive control). At each point in time the NAD

+ content of 2*10

6 PBMCs was

extracted and measured. For this purpose, the cell culture medium was removed by centrifugation at

1500 g for 5 min at 4 °C. Then cells were washed with 500 µl of ice-cold PBS. PBMCs were

resuspended in 250 µl of ice-cold PBS. For the lysis of the cells, 12 µl of 0.5 M HClO4 (perchloric

acid) were added and mixed by vortexing. The lysates were placed on ice for 15 min. Afterwards, the

samples were centrifuged at 1500 g for 10 min at 4 °C. The supernatant was transferred into a new

reaction tube and mixed with 175 µl of phosphate buffer and incubated on ice. After 15 min the

precipitated KClO4 (white sediment) was removed by centrifugation at 1500 g for 10 min at 4 °C. The

supernatant was collected in a new reaction tube, snap frozen in liquid nitrogen and stored at -80 °C

for further analysis. For the quantification of the cellular NAD+ content, a fresh prepared NAD

+

standard curve (0 µM, 0.01 µM, 0.02 µM, 0.04 µM, 0.08 µM, 0.12 µM, 0.24 µM NAD+ diluted in

diluent) and samples were transferred into a 96-well microplate. Samples were defrosted on ice and

centrifuged at 1500 g for 10 min at 4 °C to remove insoluble KClO4 carryovers. Then the cellular

samples were transferred in triplicates, 40 µl per well, into a 96-well microplate and were mixed with

160 µl of diluent per well. The standard curve was also measured in triplicates, 200 µl per well (NAD+

stock solution was prepared on ice, by solving NAD+ in ice-cooled diluent with a concentration of

10 mM. Aliquots were snap-frozen and stored at -80 °C. For each experiment a fresh aliquot was used.

The reaction mix for the enzymatic determination of the cellular NAD+

level was prepared fresh.

Therefore, five volumes premix were mixed with one volume phenazine ethosulfate (PES) (40 mM)

and one volume alcoholdehydrogenase (ADH) (1 mg/ml). Then 100 µl of this reaction mix were added

to each well and mixed. The samples were incubated for 30 min at 30 °C. The absorbance was


47

measured at a wavelength of 550 nm with a reference wavelength of 690 nm. The absorbance of

NAD+ standards was used to calculate a standard curve, which allowed the transformation of the

absorbance of the cellular samples into a cellular NAD+ concentration.

Analysis of gene transcription by real-time PCR 3.2.7

3.2.7.1 General procedure

The mRNA isolation was performed under a laminar flow cabinet (safety class 1). In the beginning,

the laminar flow cabinet was turned on at least 15 min before the actual work was started and a UV-

lamp was used to irradiate the surface. Then the working surface of the cabinet was cleaned with 70%

ethanol (v/v) and additionally with RNase away solution. A lab coat and latex or nitrile gloves were

worn at all times. The workflow contains five steps: isolation of the mRNA, synthesis of cDNA,

preparation of the real-time PCR reactions, running the real-time PCR, analysing the gene expression

data.

3.2.7.2 Cell lyses and mRNA isolation of PBMCs

The mRNA of PBMCs was isolated with the use of the Allprep RNA/DNA/Protein Mini Kit from

Qiagen, according to the handbook only the steps for the mRNA isolation were performed. Therefore,

PBMCs incubated in TexMACS cell culture medium were treated as described above. 24 h after the

first isoproterenol treatment cells were lysed. For this purpose, samples were centrifuged for 5 min at

500 g and 4 °C. The cell culture medium was removed. Afterwards, cells were lysed in fresh prepared

RLT buffer (one part β-mercaptoethanol was added to 100 parts RLT buffer). For the lysis of

6*106 cells, 600 µl of RLT buffer were used, cells were lysed by pipetting up and down. These lysates

were shock frozen in liquid nitrogen and stored at -80 °C until the next working steps were performed.

Cell lysates were thawed at RT and homogenized with QIAshredder spin columns to achieve a

maximum yield of mRNA. Samples were directly transferred into a QIAshredder spin column, which

was placed in a 2 ml collection tube and centrifuged for 2 min at full speed. Then 600 µl of lysate were

mixed with 400 µl of ethanol p.a. (99%) by pipetting up and down. Samples were completely

transferred on an RNeasy spin column placed in a 2 ml collection tube and centrifuged for 15 s, at

10000 g (two steps were needed, because the RNeasy spin column had only a capacity of 700 µl). The

flow-through was removed. Next, the on-column DNase digestion was performed using the RNase-

free DNase Set form Qiagen. This was done to remove genomic DNA residues. First, 350 µl of RW1

buffer were added to an RNeasy spin column and centrifuged for 15 s, at 10000 g, to wash the spin

column membrane. The flow-through was removed and 10 µl DNase I stock solution (1500 Kunitz

solved in 550 µl RNase-free water) were diluted in 70 µl RDD buffer. This solution was added


48

directly on the RNeasy spin column membrane and incubated at RT for 15 min. 350 µl of RW1 buffer

were added on the RNeasy spin column membrane and centrifuged for 15 s, at 10000 g. The flow-

through was discarded and the RNeasy spin column membrane was washed with 500 µl of RPE

buffer. Samples were centrifuged for 15 s, at 10000 g. Second times, 500 µl of RPE buffer were added

to the RNeasy spin column. The samples were centrifuged for 2 min, at 10000 g, to dry the membrane.

Then the RNeasy spin column was put into a new 2 ml collection tube and centrifuged at full speed for

1 min, to avoid any carryovers of the RPE buffer. The RNeasy spin column was put into a new 1.5 ml

reaction tube and 30 µl of RNase-free water were put on the RNeasy spin column membrane, to elute

the mRNA. Samples were centrifuged for 1 min, at 10000 g. To increase the yield, the 30 µl of eluent

were put again onto the RNeasy spin column membrane and eluted again by centrifugation at 10000 g

for 1 min. The mRNA samples were snap-frozen and stored at -80 °C. Concentration and purity of the

mRNA samples were determined by absorbance measurement using a NanoDrop (ND2000).

Concentration of mRNA was determined by measuring the absorbance at a wavelength of 260 nm.

Purity of the samples was determined by the ratio of the absorbance at 260 and 280 nm (A260/A280). A

ratio of A260/A280 > 2 indicates a high purity of the RNA without protein contaminations.

3.2.7.3 cDNA synthesis

The reverse transcription of the mRNA to cDNA was carried out with the iScriptTM

Advanced cDNA

Synthesis kit from Bio-Rad according to the manufacture instruction. The reaction mix for 1 sample

had the following composition:

Component: Volume [µl]

5X iScript advanced mix 4

iScript advanced reverse transcriptase 1

RNA variable (668 ng)

reverse transcription control 1

nuclease free water variable

total 20

Table 1: Composition of reverse transcription reaction mix.

The reaction mix was prepared on ice. A master mix for all samples was prepared by mixing the 5X

iScript advanced mix, with iScript advanced reverse transcriptase and the reverse transcription control.

Then 6 µl of this master mix were mixed with the RNA sample (668 ng total RNA per sample) and

water was added to adjust the total volume of each sample to 20 µl. The cDNA synthesis was carried

in a thermal cycler (CFX96 from Bio-Rad) using the following protocol: first, samples were heated for

30 min at 42 °C to allow the reverse transcription. Second, the samples were heated to 85 °C for 5 min

to inactive the reverse transcriptase. The cDNA was then stored at -20 °C until further use.


49

3.2.7.4 Real-time PCR using prime PCR arrays

The used PrimePCR assay was a customized PCR array purchased from Bio-Rad in a 96-well format.

Each well contained a lyophilized validated and specific primer pair for one gene. Each plate was

divided into two equal parts, hence, it was possible to analyse two samples on one plate in parallel. For

each sample 41 genes of interest, two housekeeping genes and five technical controls were analysed.

The arrays were used in combination with the iScript Advanced Universal SYBR Green Supermix

from Bio-Rad. The assay plates were taken out from the fridge and used after they reached RT. The

components of the supermix and the cDNA were thawed on ice. The following master mixes were

prepared.

Master mix (genes of interest)

Component Volume [µl]

2x SsoAdvanced universal

SYBER Green supermix 500

cDNA 18

nucleaee free water 482

Total volume 1000

Reverse transcription control




20x PrimerPCR RT control assay 1

cDNA 0.36

nuclease free water 8.64

total volume 20

gDNA control




20x PrimerPCR gDNA control assay 1

cDNA 0.36


total volume 20


50

RQ1 control




20x PrimerPCR RQ1 assay 1

cDNA 0.36


total volume 20

RQ2 control




20x PrimerPCR RQ2 assay 1

cDNA 0.36


total volume 20

PCR control


master mix 19

20x PrimerPCR positive PCR control

assay 1

cDNA 0.36


total volume 20 Table 3-3: Master mixes for real time quantitative PCR.

20 µl of the master mix were transferred to the wells with the primer pairs for the genes of interest.

And 20 µl of the master mix for each technical control have been transferred to the corresponding

well. The plate was sealed with an optical seal and centrifuged for 30 second at full speed to remove

air bubbles.

Figure 3-4: Layout of the of the assay plates. The assay plates had a 96-well format, each box represents one well of the

PCR plate. Green boxes represent 41 genes of interest (GOI) (Wells C3, F5, C9 and F11 were labelled with two genes

because two different assay plates were used. The difference between the two plate setups was only the position of these

genes). Red boxes represent the two housekeeping genes and white boxes represent the five technical controls.

1 2 3 4 5 6 7 8 9 10 11 12

A ADRB2 BLM WRN BRCA1 CDKN1C XRCC1 ADRB2 BLM WRN BRCA1 CDKN1C XRCC1

B AKT3 CDKN1A PARP2 TP53 GALNT6 GAPDH AKT3 CDKN1A PARP2 TP53 GALNT6 GAPDH

C ARRB1 LIG4 CDKN2A/ TERF2 ERCC5 NOS3 HPRT 1 ARRB1 LIG4 CDKN2A/ TERF2 ERCC5 NOS3 HPRT 1

D GRK5 B3GALTL MRE11A BRIP1 PRKDC gDNA GRK5 B3GALTL MRE11A BRIP1 PRKDC gDNA

E GRK6 POMGNT1 XCL1 CAT GALNT7 PCR GRK6 POMGNT1 XCL1 CAT GALNT7 PCR

F CCND1 B3GNT1 VCAN CYGB FGL2/ TANK RQ1 CCND1 B3GNT1 VCAN CYGB FGL2/ TANK RQ1

G POLβ OGG1 S100A8 DHCR24 SOD2 RQ2 POLβ OGG1 S100A8 DHCR24 SOD2 RQ2

H SRC PARP1 RPA1 BRCA2 XPC RT SRC PARP1 RPA1 BRCA2 XPC RT


51

DNA contamination control (gDNA control)

This control was used to check for a contamination of the cDNA with genomic DNA (gDNA). A

primer pair (wells D6 and D12) targeting a non-transcribed region of the human genome was used. A

quantification value (CT) lower than 35 indicated a contamination with gDNA. A CT value above 35

showed the absence of any gDNA in the sample.

Positive PCR control (PCR control)

The positive PCR control was used to test if samples contained inhibitors or other compounds that had

a negative effect on the PCR reaction. A synthetic DNA template (PrimePCR control assay) was

added to the reaction mix before this reaction mix was added to the assay plate. Primers for this

synthetic DNA were lyophilized in the respective wells (E6 and E12). The sequence of the synthetic

DNA is not present in the human genome.

RNA quality control (RQ1 and RQ2 control)

These two controls were used to test the quality and integrity of the RNA. This control was designed

as two primer pairs targeting the cDNA of the same RNA template but at different locations within the

cDNA sequence. If the quality of the RNA was good, meaning the RNA was not degraded or

fragmented, the transcribed cDNA should be intact. Since the cDNA concentration in each well is

equal, the CT value of RQ1 (wells F6 and F12) and RQ2 (wells G6 and G12) should also be equal.

Reverse transcription control (RT control)

This assay served as a control for the reverse transcription of the mRNA into cDNA. Therefore, a

synthetic RNA template with a sequence which is not present in the human genome was added to the

cDNA synthesis reaction mix. A primer pair for the amplification of the respective cDNA was

lyophilized on the PCR plate (wells H6 and H12). CT values above 30 indicated poor reverse

transcription reaction performance.

3.2.7.5 Real-time PCR using p16 and p21 primer pairs

To analyze the expression of p21 and p16 genes, validated primer pairs for real-time PCR from

Biomol were purchased. The p21 and p16 primer pairs were ordered together with a primer pair set of

ten housekeeping genes. The required mRNA and cDNA was isolated and transcribed in the same way

as in 3.2.7.2 and 3.2.7.3., real-time PCR was performed as described in 3.2.7.4. .


52

3.2.7.6 Data evaluation of real time PCR

The real time PCR data were evaluated with the Bio-Rad CFX Manager, data for a gene of interest

(GOI) were calculated according:

(1) efficiency:

𝐸 = (% 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 ∗ 0,01) + 1

% 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = (𝐸 − 1) ∗ 100

(2) relative expression:

𝑟𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 (𝐺𝑂𝐼) = 𝐸𝐺𝑂𝐼

(𝐶𝑇(𝑀𝐼𝑁)−𝐶𝑇(𝑠𝑎𝑚𝑝𝑙𝑒))

CT (MIN): average CT for sample with the lowest CT for GOI

CT (sample): average CT for sample

(3) relative expression of housekeeping genes (∆𝑪𝑻)

relative quantity sample (GOI)= 𝐸𝐺𝑂𝐼

(𝐶𝑇(𝑐𝑜𝑛𝑡𝑟𝑜𝑙)−𝐶𝑇(𝑠𝑎𝑚𝑝𝑙𝑒))

CT (control): average CT of the control

CT (sample): average CT for any sample with GOI

(4) normalization factor

normalization factor sample (GOI)

𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 (𝐺𝑂𝐼) = (𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓1) ∗ 𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓2) ∗ … . 𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓𝑛))1𝑛

= (𝐸𝐺𝑂𝐼


(𝑅𝑒𝑓1)∗ 𝐸𝐺𝑂𝐼


𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓2)

∗ … . 𝐸𝐺𝑂𝐼


𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓𝑛)) 1/𝑛

RQ: Relative quantity

n: Number of reference targets (housekeeping genes)

(5) normalized expression (∆∆𝑪𝑻)

𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝐸𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 (𝐺𝑂𝐼) =𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝐺𝑂𝐼)

(𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓1) ∗ 𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓2) ∗ … . 𝑅𝑄𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓𝑛))1𝑛

=𝐸𝐺𝑂𝐼


(𝐸𝐺𝑂𝐼


(𝑅𝑒𝑓1)∗ 𝐸𝐺𝑂𝐼


𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓2)∗ … . 𝐸𝐺𝑂𝐼


𝑠𝑎𝑚𝑝𝑙𝑒 (𝑅𝑒𝑓𝑛)) 1/𝑛


53

RQ = Relative quantity of sample

REF = Reference target (housekeeping genes)

(6) relative normalized expression

The relative normalized expression was calculated according to formula (5). Therefore, the ΔΔCT

value of the control was set to 1 and the ΔΔCT value of the sample was expressed relative to that.

PARP1 activity under NAD+ saturated conditions 3.2.8

PAR formation, in intact PBMCs after isoproterenol treatment was analysed by flow cytometry,

according to Kunzmann et al. [414] and Weidele et al. [413]. PBMCs were treated with isoproterenol

as described in section 3.2.3. 24 h after the first isoproterenol treatment, PBMCs were pooled in 15 ml

conical tubes and centrifuge for 10 min at 300 g at 4 °C. Supernatant was removed and cells were

resuspended in 1 ml of ice-cold 100% ethanol, for the first fixation. Under these conditions PARP1

can be still activated. Samples were incubated at -20 °C, for at least 20 min. Then samples were mixed

1:10 (1 ml cell suspension + 9 ml buffer A) with buffer A (14.3 mM β-mercaptoethanol were freshly

added). Afterwards, samples were centrifuged for 10 min at 750 g at 4 °C. Supernatant was removed

and cells were resuspended in 1 ml of buffer A. An aliquot of the cell suspension was taken to

determine the cell count. Cells were transferred into a v-bottom 96-well microplate (minimum 500,000

cells per well). Samples were centrifuge for 10 min at 750 g and 4 °C and the supernatant was

removed. Then the cells were resuspended in 23 µl of buffer A. PARP activity was measured at a basal

level and after activation by an oligonucleotide which mimics DNA strand breaks. Therefore, two

reaction buffers were prepared and 37 µl were added to each well. One reaction buffer contained an

oligonucleotide and NAD+

for the activation of PARP, whereas the other reaction mix did not contain

these (50 mM Tris HCl pH 7.8, 60 mM MgCl2, 53 mM NaCl, with or without 13.5 ng/µl

deoxyoligonucleotide (GGAATTCC), 0.3 mM NAD+). Samples were incubated for 10 min at 37 °C to

allow PARP1 activation and PAR formation. Next, cells were fixed with 60 µl of 4%

paraformaldehyde per well, samples were incubated for 20 min at RT. Fixation was stopped with 60 µl

of 100 mM glycine per well. Then samples were centrifuged at 750 g, at 4 °C for 10 min, supernatant

was decanted and cells were washed with 200 µl FACS buffer per well. The FACS buffer was

removed by centrifuged at 750 g, at 4 °C for 5 min. The cell pellet was resuspended in 100 µl/well

primary antibody solution (monoclonal 10H antibody, 1:300 diluted in FACS buffer). Samples were

incubated overnight at 4 °C. Afterwards, samples were centrifuged at 750 g, at 4 °C for 5 min,

supernatant was removed and cells were washed two times with 200 µl of FACS buffer per well. Next,

samples were incubated with the secondary antibody solution (goat anti mouse Alexa 488, diluted

1:1000 in FACS buffer), 100 µl per well for 30 min at 37 °C. Then samples were centrifuged at 750 g,

at 4 °C for 5 min and antibody solution was removed. Cells were washed two times with 200 µl of


54

FACS buffer per well. Finally, cells were resuspended in 200 µl of FACS buffer and transferred into

FACS vials. Samples were stored on ice in the dark until flow cytometric analysis was performed (BD

LSRII). Per sample 10000 events were measured and analysed.

Sample preparation for Western blotting 3.2.9

Cells were treated as described in section 3.2.3. For the lysis of the cells the cell culture medium was

removed and cells were incubated on ice. Then cells were washed with ice-cold PBS to remove cell

culture medium remains. Afterwards, cells were lysed in ice-cold modified high-salt RIPA buffer

supplemented with complete protease inhibitor cocktail. For the analysis of the PARP1 protein

expression two samples were prepared. One sample was lysed in modified high-salt RIPA buffer on

ice and used for the determination of the protein concentration. The second sample was lysed with

high-urea sample buffer, because to avoid the bind of PARP1 to DNA and to inhibit the degradation of

PARP1 by proteases. For this purpose, the high-urea sample buffer was heated to 95 °C for 5 min

before it was added to the cells. All lysates were sheared by passing them through cannula with

different inner diameter sizes until the lysates were homogenized. Afterwards, the samples were snap

frozen in liquid nitrogen and stored at -20 °C for further processing.

3.2.9.1 Determination of protein concentration

Total protein content of cell lysates was measured by the BCA protein assay kit (Pierce) according to

the manual. A standard curve with bovine serum albumin (BSA) concentrations of 0.0, 0.2, 0.4, 0.6,

0.8, 1.0, 1.2 mg/ml were prepared from a stock solution (2 mg/ml) by dilution with MilliQ. Then 5 µl

of each standard and of each protein sample were transferred into a 96-well microplate as triplicates.

The BCA working reagent was prepared by mixing 1 part of reagent B with 50 parts of reagent A.

Next, 95 µl of the BCA working reagent were added per well. Samples were shaken for 30 sec and

then incubated for 30 min at 37 °C. Absorbance was measured at a wavelength of 550 nm. The total

protein concentration of the samples could be calculated using the BSA standard curve.

3.2.9.2 SDS-polyacrylamide gel electrophoresis

Proteins were separated by their electrophoretic mobility using SDS-polyacrylamide gel

electrophoresis (SDS-PAGE). The Hoefer Mini VE system from Amersham Biociences was used to

perform the SDS-PAGE. The electrophoresis module parts were cleaned with water and detergent to

remove old gel residues. The module parts were also cleaned with ethanol, dried and finally put

together. Tightness of the electrophoresis module was controlled with water. Before the use, the water

was discarded and the electrophoresis module was filled with fresh prepared resolving gel solution.

Immediately after the addition of the resolving gel, 1 ml of isopropanol was added to the top, to avoid


55

air bubbles on the gel surface. The polymerization of the gel was completed after about 20 min.

Isopropanol was removed and gel surface was cleaned with MilliQ to remove the isopropanol

completely. Freshly prepared stacking gel solution was added on the top of the resolving gel and a

comb was inserted. After 20 min, the gel was polymerized. The electrophoresis module was

transferred into a tank, which was filled with 1X Laemmli buffer. The comb was removed and the gel

pockets were washed out with 1X Laemmli buffer to remove gel residues. Protein samples were

loaded into gel pockets (50 µg total protein content per pocket). At least one pocket per gel was loaded

with 10 µl PageRuler pre-stained protein ladder. The electrophoresis was started with a current of

10 mA per gel until the loading dye reached the interface of the stacking gel and the resolving gel.

Then the current was set to 20 mA per gel. The electrophoresis was finished within 3 h.

Component: Separation gel Stacking gel

10% 13%

MilliQ 7.2 ml 5.6 ml 2.2 ml

30% acryl-/bisacrylamid 5.2 ml 6.8 ml 1.1 ml

separating gel buffer (5x) 3.2 ml 3.2 ml -

stacking gel buffer (2x) - - 3.2 ml

10% (w/v) APS 132 µl 132 µl 66 µl

TEMED 32 µl 32 µl 12 ml Table 2: Composition of the separation- and the stacking gel for protein separation by SDS-PAGE.

3.2.9.3 Western blot

Western blot is used for the detection of specific proteins in a complex protein mixture like cell

lysates. This requires a separation of the proteins. Proteins were separated by their size by the use of a

SDS-PAGE as described above. After the separation the proteins were transferred electrophoretically

from the gel onto a Hybond-ECL nitrocellulose membrane. Therefore, a transfer stack was assembled

according to the user manual, using a Hoefer Mini Blot Module wet blotting device (Amersham

Biosciences), see Figure 3-5.


56

Figure 3-5: Preparation of the transfer stack, copy from the manual [415].

A pre wetted packing sponge was placed onto the cathode, followed by one filter paper, also pre

wetted. The gel with the protein samples was equilibrated in Towbin buffer and transferred on the

filter paper. The gel surface was wetted with a few drops of Towbin buffer to remove air bubbles.

Then the gel was covered with a nitrocellulose membrane which was also equilibrated in Towbin

buffer. On top of the membrane a second pre wetted filter paper was added. Finally, packing sponges

were added on the top until the transfer stack was stable enough. The blot module was filled up with

Towbin buffer. Then the blot module was transferred into the tank, which was filled with ice-cold

Laemmli buffer. Proteins were transferred on the membrane at a constant current of 300 mA for 2 h.

Afterwards, the membrane was incubated for 1 h in blocking solution (5% milk powder solved in TNT

buffer) to block unspecific binding sides. Marker lines were cut off and incubated in TNT buffer. The

membrane containing the protein samples was incubated with the primary antibody solution for 1 h, at

RT or at 4 °C overnight under constant shaking. The membrane was washed three times, each time for

5 min with TNT buffer. Then the membrane was incubated with the secondary antibody (diluted in 5%

milk powder solved in TNT buffer) for 1 h, at RT under constant shaking. Finally, the membrane and

the marker line were washed three times, each time for 5 min at RT with TNT buffer. The detection of

the proteins of interest was done by chemiluminescence, catalysed by the horse radish peroxidase that

was tagged to the secondary antibody. Therefore, 1 ml of freshly prepared enhanced

chemiluminescence (ECL) solution (1 part solution A and 1 part solution B) was distributed on the

membrane. The light emission was detected with a luminescent image analyser (LAS400). The

exposure time was adapted for each blot. Afterwards, the membrane was washed thrice with TNT

buffer to remove the ECL solution. Then the membrane was incubated with the anti-α-actin (1:50000)

or anti-histone H1 antibody (1:2000) as loading control, diluted in 5% milk powder solved in TNT

buffer. The membrane was incubated with the antibody solution for 1 h, at RT under constant shaking.

Then the membrane was washed thrice, each time for 5 min with TNT buffer, followed by the


57

incubation of the membrane with the secondary antibody for 1 h, at RT under constant shaking.

Finally, the membrane was washed again thrice, each time for 5 min with TNT buffer. The proteins

were detected as described above.

3.2.9.4 p16 and p21 protein expression

p16 and p21 proteins are important in the regulation of cell proliferation. p16 is an inhibitor of the

CDKs. It binds to CDK4/6, this leads to an inhibition of the progression of the cell cycle from the G1

phase to the S phase. PBMCs were isolated and repeatedly treated with isoproterenol as described in

section 3.2.3. The protein expression was measured at indicated points of time after the first

isoproterenol treatment. Afterwards, cells were incubated for 24 h, 48 h in a shaking water bath at

37 °C. At the indicated points of time cells were removed from the water bath and centrifuged at RT

and 600 g for 5 min. The TexMACS cell culture medium was removed and cells were washed with

PBS. Then cells were resuspended in 100 µl PBS. Next, the cells were fixed by adding 100 µl of 4%

PFA to the cell suspension. Cells were incubated for 10 min at RT. Then cells were permeabilized

with the addition of 200 µl of permeabilization buffer to the cell suspension. Cells were incubated in

the dark for 20 min at RT. Cells were centrifuged for 5 min, at 600 g and RT, the supernatant was

removed. Then cells were resuspended in 200 µl of blocking buffer and incubated for 30 min at RT.

Each PBMCs sample was split into two samples. One sample was used for the p16 staining. Cells

were centrifuged for 5 min, at 600 g and RT to remove the blocking solution and washed two times

with permeabilization buffer. Next, the cells were labelled with the primary antibodies. Therefore, the

anti-p16 antibody was diluted 1:320 also in FACS buffer. Cells were resuspended in 100 µl of the

respective antibody solution and incubated for 1 h, at 37 °C. Afterwards, the cells were washed three

times with permeabilization buffer and centrifuged at 600 g for 5 min at RT. Cells were stained with

the secondary antibody, goat anti-rabbit Alexa Fluor 488 diluted 1:1000 in FACS buffer. Therefore,

cells were resuspended in 100 µl of the secondary antibody solution and incubated at RT for 1 h in the

dark. Finally, cells were centrifuged 5 min, at 600 g and RT. The supernatant was removed and cells

were washed three times and resuspended in 200 µl ice-cold FACS buffer and transferred into FACS

vials. Samples were stored on ice in the dark until flow cytometric analysis was performed. Per sample

10000 events were measured and analysed (BD LSRII).

3.2.9.5 Determination of the isoproterenol concentration in cell culture media

In the beginning the dynamic range of the diode array detector and the fluorescence detector was

tested for their suitability for the detection of isoproterenol in cell culture media. For this purpose, a

dilution series of isoproterenol and caffeine was used. Caffeine was chosen, because it is stable under

the used conditions. Hence, the dilution series should be linear. A nonlinearity of the dilution series

would indicate a problem with the HPLC system. The dilution series was prepared by dissolving


58

caffeine 1 mg/ml (5 mM) in ice-cold RPMI-1640 cell culture medium. Afterwards, isoproterenol was

dissolved in this caffeine solution to a concentration of 10 mM. This stock solution was then further

diluted (1:2, 1:10, 1:20, 1:100 and 1: 200) with ice-cold RPMI-1640 cell culture medium on ice. The

solution was stored on ice in the dark until the HPLC measurement could be performed.

3.2.9.6 Oxidation of isoproterenol

Isoproterenol was chemically oxidized to isoprenochrome. Freshly synthesized isoprenochrome was

used for determination of the UV/VIS spectrum and the retention time of isoprenochrome. The

oxidation of isoproterenol was performed in a 10 mM aqueous ammonium acetate buffer the pH was

adjusted to 5.4 with acetic acid. Potassium periodate solution at a concentration of 2 mM was freshly

dissolved in the oxidation buffer immediately before isoproterenol was added. Isoproterenol (10 mM)

was dissolved in oxidation buffer and mixed for 1 min. The solution recolored immediately into red.

The reaction mix was diluted 1:10 (10 µl reaction mix in 90 µl HPLC eluent).

3.2.9.7 Measurements of isoproterenol and isoprenochrome concentrations

The HPLC system (Agilent) which was used contained the following modules: 1100/1200 binary

pump, 1100 wellplate autosampler, 1100/1200 diode array detector, 1100/ 1200 column thermostat,

1100/ 1200 fluorescence detector, 1200 sample thermostat. Chemical compounds were detected by the

photodiode array detector (absorbance detector). In addition, a more sensitive fluorescence detector

was used as second detector and was direct connected to the photodiode array. As analytical column a

commercial available reverse phase C18-column (length 15 cm, inner diameter 4.6 mm, particle size

5 µm, column volume 2.5 ml) from Macherey-Nagel, was used. The column was equipped with a

guard column (Phenomenex, Aschaffenburg, Germany) containing C18 cartridge (3.0 mm inner

diameter) (Phenomenex, Aschaffenburg, Germany). An isocratic elution was performed at a flow rate

of 2 ml/min; the column temperature was not controlled (RT). The samples were cooled to 4 °C. The

mobile phase was a composition of water/acetonitrile (94.3/5.7 %v/v), 0.095 M citric acid, 350 µM

Na2EDTA and 460 µM octansolfonic acid. The pH was adjusted to 2.3 with ammonium acetate. Prior

to use, the buffer was filtered through a bottle-top vacuum filter with a pore size of 0.22 µm. The run

time was set to 20 min. For the detection of isoproterenol the diode array detector operated with the

following parameters: absorbance wavelength 280 nm with a bandwidth of 30 nm. As reference, a

wavelength of 700 nm with a bandwidth of 100 nm was used. For the detection of isoprenochrome the

diode array detector parameters were set to an absorbance wavelength of 490 nm with a bandwidth of

20 nm and a reference wavelength of 700 nm with a bandwidth of 100 nm. For the identification of the

isoproterenol peak an absorbance spectrum from 190 nm to 900 nm was recorded for each peak of the

chromatogram. The fluorescence detector was calibrated for isoproterenol according the handbook.


59

The parameters were set to an excitation wavelength of 280 nm and an emission wavelength of

310 nm.

Absorbance detector

Signals [nm] Bandwidth [nm] Reference [nm] Bandwidth [nm]

490 20 700 100

280 20 700 100

Spectrum Lamps

Range [nm] Steps [nm] UV Vis

190-900 2

Peakwidth Slit [nm]

> 0.2 min (4 s) 16

Fluorescence detector

Excitation [nm] Emission [nm]

280 316 Table 3: Parameter of the HPLC absorbance and fluorescence detector.

3.2.9.8 Sample preparation for HPLC analysis

PBMCs were treated as described in section 3.2.3. Additional, the same measurements were also done

without cells as control experiment. This was done to compare the isoproterenol stability and

degradation processes in cell culture medium with and without cells. The stability measurements were

performed according the following pattern. For each time series, cell suspension (2*106 cells/ml) or

cell culture medium was treated on ice with the isoproterenol stock solution (10 mM) reaching a

concentration of 10 µM isoproterenol. Afterwards, immediately an aliquot of 100 µl was taken and

transferred into a 1.5 ml reaction tube. The sample was taken and centrifuged at 15000 g and 4 °C for

5 min for sedimentation of cells and cell debris. Then 70 µl of the supernatant were carefully removed

and put into a pre-cooled HPLC vial with an HPLC insert for small volumes. Next, 50 µl of the sample

were immediately injected into the HPLC system separated and analyzed, as described in section

3.2.9.7. The first sample of each time series was defined as point in time zero and represents the first

isoproterenol dose. The rest of the sample mixture was aliquoted on ice into 1 ml aliquots. Then the

samples were transferred into a water bath and repeatedly treated with isoproterenol as described in

section 3.2.3. Direct after each treatment one sample was removed randomly. Then 100 µl of the

sample were transferred into a 1.5 ml reaction tube and centrifuged at 15000 g and 4 °C for 5 min.

Again 70 µl of the supernatant were carefully removed and put into a pre-cooled HPLC vial with an

HPLC insert for small volumes, 50 µl were injected into the HPLC system. Samples were analysed

every 30 min, up to 540 min after the first isoproterenol treatment. Data were evaluated with the

Agilent ChemStation software. Peaks were identified according to their retention time and the

absorbance spectrum. The isoproterenol or isoprenochrome content of a sample was determined by the

integration of the peaks by the software.

Results

60

4 Results

Current research indicates that catecholamines can induce the expression of biomarkers of stress. Such

hints can be found in PTSD patients that showed an accelerated aging and an increased amount of

DNA strand breaks [82, 83]. Also on cellular level, catecholamines can induce after repeated dosage,

the accumulation of DNA strand breaks in mice and in human cell lines [399-401]. High doses of

epinephrine can induce DNA damage in human lymphocytes [399]. Additional, repeated dosage of

catecholamines can induce a senescence like phenotype in the myocardium of mice [416]. PTSD

seems also to be associated with a phenotype of accelerated senescence [312]. A pilot study in our

group showed that repeated stimulation of the β2-AR of human PBMCs by isoproterenol induced the

formation of DNA strand breaks. Moreover, the expression of the DNA repair protein PARP1 and the

formation of PAR were affected by the repeated isoproterenol treatment [405]. Based on these

observations an understanding of the responsible mechanisms is needed. To our knowledge

Isoproterenol can induce DNA damage by two different mechanisms. On the one hand, isoproterenol

can induce DNA strand breaks by signaling processes via the Gs protein/PKA pathway of the β2-AR

[400]. On the other hand, isoproterenol can be oxidized which is associated with the formation of free

radicals and ROS [226, 228]. Both mechanisms can induce DNA strand breaks. Moreover, we found

some indications that isoproterenol may induce a senescence like phenotype in human PBMCs [417].

Therefore, a second study investigated whether isoproterenol could induce the expression of

senescence markers in human PBMCs. These experiments required the culturing of PBMCs for

several days. Thus, the RPMI-1640 basal cell culture medium had to be supplemented with FCS or an

optimized cell culture medium, like TexMACS could be used. TexMACS is a FCS-free cell culture

medium developed for the cultivation of immune cells. The use of FCS was avoided because FCS

contains various hormones and growth factors which may influence the PBMCs [418, 419]. FCS as

well as TexMACS contain serum albumin and other factors which may influence the isoproterenol

degradation. To our knowledge no previous studies were performed to investigate the influence on the

stability and degradation of isoproterenol in cell culture. Therefore, a third study was performed to

investigate the isoproterenol stability and degradation under cell culture conditions. Also the influence

of different cell culture media and supplements like FCS were analyzed by HPLC measurements

(Palombo et al., manuscript in preparation) [420].

Results

61

4.1 Isoproterenol mediated DNA damage

cAMP-signaling of the β-AR after repeated isoproterenol stimulation 4.1.1

The β2-AR is a prototypical G protein coupled receptor which is coupled to a Gs protein. After binding

of a ligand such as isoproterenol, the receptor is stabilized in its active conformation. This leads to

formation of the second messenger cAMP, which activates downstream signaling pathways. Signaling

of the β2-AR can be associated with the formation of intracellular ROS which is PKA dependent. After

activation of the AC the cAMP signaling must be terminated. Therefore, the receptor can be uncoupled

from the G protein and internalized into the cell. In the cell, the receptor can either be recycled or

degraded. Varies factors influence the fate of the receptor such as: type of the ligand, concentration of

the ligand, duration of the activation, and cell type. As the ligand binding site of the β2-AR is located

extracellular, the cAMP-dependent signaling pathway can only be stimulated by receptors on the cell

surface. Previous results showed that repeated isoproterenol treatment induced the formation of DNA

strand breaks. These DNA strand breaks could be partial inhibited by pretreatment with the β-AR

antagonist (β-blocker) propranolol, see appendix Figure 12-2 [421]. Propranolol inhibits cAMP-

depended signaling of the β2-AR. The question arises, if the β2-AR can be stimulated and induces

cAMP-dependent signaling after the repeated isoproterenol administration. Thus, the formation of

cAMP in PBMCs after the repeated isoproterenol treatment and the influence of propranolol (prop)

were investigated, see Figure 4-1. Freshly isolated PBMCs were treated either with one dose, four

doses or eight doses of isoproterenol (each dose 10 µM). The receptor was blocked by a pretreatment

of the cells with propranolol. 10 min before the isoproterenol treatment was started cells were treated

with 10 µM propranolol (iso + prop). Since cAMP is degraded within minutes by phosphodiesterases,

the cAMP concentration was measured directly after the last isoproterenol dose. Immediately before

the last isoproterenol dose was applied, cells were treated with phosphodiesterase inhibitors. As

positive control, cells were treated with forskolin, which induces the formation of cAMP, receptor-

independently, by direct activation of the AC. PBMCs were cultured either in RPMI cell culture

medium without FCS (black) or in TexMACS cell culture medium (green). Forskolin induced a

significant increase of the cAMP content in PBMCs, approximately an 8-fold increase of the cAMP

concentration could be observed, see Figure 4-1 B) and D). A single dose of 10 µM isoproterenol

increased the intracellular cAMP content by a factor of approximately two to three. Pretreatment with

10 µM propranolol inhibited the formation of cAMP, see Figure 4-1 A) and C). After the four-fold and

eight-fold administration of isoproterenol, no significant increase of the intracellular cAMP content

compared to control cells could be observed for cells cultured in RPMI cell culture medium. But there

was an increase of the cAMP levels in comparison to the propranolol pretreated cells. PBMCs cultured

in TexMACS cell culture medium showed a three-fold higher intracellular cAMP content after the

Results

62

four-fold isoproterenol treatment compared with the propranolol pretreated cells. Cells which were

treated eight-fold with isoproterenol had a two-fold higher intracellular cAMP content compared with

the propranolol pretreated cells. PBMCs cultured in RPMI-1640 cell culture medium without FCS

showed a minimal increase in intracellular cAMP content of approximately 1.5-fold compared with

propranolol pretreated cells after the four-fold isoproterenol administration. Hence, cells lose the

sensitivity or responsiveness of the cAMP-dependent signaling pathway in the course of the interval

treatment.

n(c

AM

P)

(fm

ol/

15

00

0 c

ell

s)

co

ntr

ol

pro

p

1x iso

1x iso

+ p

rop

4x iso

4x iso

+ p

rop

8x iso

8x iso

+ p

rop

0

1 0 0

2 0 0

3 0 0

4 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0

** ** *

n(c

AM

P)

(fm

ol/

15

00

0 c

ell

s)

co

ntr

ol

fors

ko

lin

0

1 0 0

2 0 0

3 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0***

A ) B )

n(c

AM

P)

(fm

ol/

15

00

0 c

ell

s)

co

ntr

ol

pro

p

1x iso

1x iso

+ p

rop

4x iso

4x iso

+ p

rop

8x iso

8x iso

+ p

rop

0

1 0 0

2 0 0

3 0 0

4 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0

** **

***

n(c

AM

P)

(fm

ol/

15

00

0 c

ell

s)

co

ntr

ol

fors

ko

lin

0

1 0 0

2 0 0

3 0 0

4 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0 ****

C ) D )

Figure 4-1: Signaling of the β2-AR via cAMP after the repeated isoproterenol treatment. Freshly isolated PBMCs were

cultured either in RPMI-1640 cell culture medium w/o FCS (black) or in TexMACS cell culture medium (green). Cells were

treated either with a single dose (1x iso) of isoproterenol or repeatedly with four (4x iso) or eight (8x iso) doses of

isoproterenol. Each isoproterenol dose had a concentration of 10 µM. As a negative control, cells were pretreated with

10 µM of propranolol (prop), 10 min before the isoproterenol treatment was started. As positive control cells were treated

with 250 µM of forskolin to induce cAMP formation. Before the last administration of isoproterenol, cells were treated with

two phosphodiesterase inhibitors, IBMX (500 µM) and imidazolidin (100 µM), to avoid the degradation of cAMP.

Intracellular cAMP content was quantified with the help of a cAMP standard curve, according to the cAMP Glo handbook.

(Figure C) and D) were performed in cooperation with Anith Grath, master student, Measurement of cAMP concentration in

PBMCs after repeated isoproterenol treatment in TexMACS cell culture medium). Data represent means with SEM of six

experiments for PBMCs cultured in RPMI-1640 cell culture medium w/o FCS (black) and seven experiments (three

experiments for 4x iso and 4x iso + prop) for PBMCs cultured TexMACS cell culture medium. Statistical analysis was

performed using paired t-test (*), **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05.

Results

63

Quantification of the intracellular NAD+

content in PBMCs during 4.1.2

and after the repeated isoproterenol treatment

The repeated isoproterenol treatment not only induced DNA strand breaks it also led to an increase in

the percentage of cells which had a lower PAR formation ability, see appendix Figure 12-4 [421].

DNA strand breaks are known to activate PARP1. Activated PARP1 uses NAD+ as a substrate for the

formation of PAR. Hence, DNA strand breaks induce the depletion of intracellular NAD+ pools to a

significant degree. The decrease of cellular NAD+ content correlates with the amount of DNA strand

breaks and the resulting PAR formation. The formation of intracellular cAMP was reduced after the

repeated isoproterenol treatment. This could be caused by the internalization of the receptor or by the

uncoupling of the receptor from the Gs protein. These processes were involved in the termination of

the receptor signaling. However, reduced cAMP formation could be also caused by a cellular energy

crisis. Since, the formation of cAMP requires ATP. The intracellular ATP content of isoproterenol

treated cells was negatively correlated with the applied amount of isoproterenol doses, see appendix

Figure 12-3 [421]. Thus, the NAD+ content of PBMCs was analyzed during and after the isoproterenol

treatment by the NAD+ cycling assay. The strongest effects of the isoproterenol treatment could be

observed after the 8-fold isoproterenol treatment. Therefore, the NAD+ content of PBMCs was

measured after each dose during the 8-fold isoproterenol treatment. The NAD+ content was normalized

to an untreated control sample at each point in time, see Figure 4-2 A). During the treatment no

decrease of the cellular NAD+ content could be detected. 6.5 h after the beginning of the isoproterenol

treatment a reduction of the intracellular NAD+ content of about 30% could be observed. The cellular

NAD+ content was also quantified 24 h after the first isoproterenol dose, see Figure 4-2 B). As a

control, PBMCs were lysed direct at the beginning of the treatment without an isoproterenol dose (w/o

iso 0 min) and direct after the first isoproterenol dose (1x iso 0 min). As a positive control, PBMCs

were treated with 500 µM of H2O2 in PBS for 5 min at 37 °C, after the 24 h incubation time. No

significant difference of the NAD+ content between the isoproterenol treated cells (1x iso 0 min) and

the untreated cells (w/o iso 0 min) at the beginning of the treatment could be observed. Hence,

isoproterenol has no immediate effect on the cellular NAD+ content. Also no significant difference of

the cellular NAD+ content between the untreated cells at the beginning and after the 24 h incubation

time could be observed. Indicating, the incubation has no influence on the cellular NAD+ content. The

H2O2 treatment led to a significant decrease of the cellular NAD+ pools. Also the isoproterenol

treatment resulted in a significant decrease of the cellular NAD+

content of PBMCs. The strongest

decrease, about 30%, of the NAD+ content could be observed after the 8-fold isoproterenol treatment.

These results showed that the isoproterenol treatment induced the depletion of NAD+. Moreover, the

reduced NAD+ content could be the reason and the explanation for the reduced PAR formation in

some cell population of the treated PBMCs.

Results

64

NA

D+

[µ

M]/

2*1

06

ce

lls

w/o

iso

0 m

in

1x iso

0 m

in

w/o

iso

1x iso

4x iso

8x iso

H2O

2 [

500µM

]

0 .0 0

0 .0 1

0 .0 2

0 .0 3

0 .0 4

0 .0 5

*

*

B )

**

#

2 4 h rs

no

rm

eli

ze

d t

o

co

ntr

ol

in %

0 m

in

30 m

in

60 m

in

90 m

in

120 m

in

150 m

in

180 m

in

210 m

in

6,5

h

0

5 0

1 0 0

1 5 0

A )

*

Figure 4-2: Intracellular NAD+ content of PBMCs during and 24 h after the 8-fold isoproterenol treatment. PBMCs

were cultured in RPMI-1640 w/o FCS and were treated every 30 min with isoproterenol. A) NAD+ content during the 8-fold

isoproterenol treatment. At each indicated point in time PBMCs were lysed, immediately after the application of a 10 µM

isoproterenol dose. The NAD+ content was measured by the NAD+ cycling assay. The values were normalized to untreated

controls. Data represent means with SEM of seven experiments (four measurements for point in time 6.5 h). B) Intracellular

NAD+ content 24 hours after the first isoproterenol treatment. PBMCs were either treated only with cell culture medium or

treated with a single dose (1x iso) of isoproterenol or interval treated, 4-fold (4x iso) or 8-fold (8x iso), with isoproterenol. In

addition, controls were treated with a single dose of isoproterenol or treated with medium and lysed immediately after the

treatment. Hydrogen peroxide (500 µM) was used as a positive control to deplete the NAD+ content of PBMCs. Data

represent means with SEM of seven experiments. Statistical analysis was performed using RM one-way ANOVA (#),

# P<0.05 followed by a Dunnett multiple comparison test (*), * P<0.05, ** P<0.01.

PAR formation after isoproterenol treatment under NAD+ saturated 4.1.3

conditions

The pilot study showed that the repeated isoproterenol treatment induced DNA strand breaks in

PBMCs and the treatment reduced the capacity of cells to form PAR, see appendix Figure 12-2 and

Figure 12-4 [421]. In addition, a decrease of the PARP1 protein level, see appendix Figure 12-5 [421],

and a reduction of intracellular NAD+

content of PBMCs, see section 4.1.2, could be observed 24 h

after the isoproterenol treatment. Since both the PARP1 protein level and the intracellular NAD+

content appear to be influenced by repeated isoproterenol treatment, the question arises what caused

the reduced PARylation capacity. Therefore, PAR formation under NAD+ saturated conditions was

analyzed. PBMCs were treated with the isoproterenol interval treatment. 24 h after the first treatment,

cells were fixed with a method that kept PARP1 functional. PARP1 could be active by DNA strand

breaks and PAR formation could be induced. In order to stimulate PAR formation in cells, cells were

supplemented with NAD+ and an oligonucleotide which mimics DNA strand breaks. Basal PAR

formation was measured without supplementation. After the immunostaining of PAR, the fluorescence

signal was measured by FACS and the fluorescence signal was expressed as relative mean

Results

65

fluorescence intensity (mfi). Figure 4-3 shows the intracellular PAR content after PARP1 activation by

the oligonucleotide under NAD+ saturated condition in control cells as well as in isoproterenol treated

cells. The PAR content was significant higher after PARP1 activation under the NAD+ saturation

conditions in the control cells and all isoproterenol treated cells compared to cells without the

supplementation. Moreover, approximately the same amount of PAR was formed in all isoproterenol

treated and untreated PBMCs under NAD+ saturated condition. This indicates that the reduced PAR

formation capacity in PBMCs (increase of the number of cells which had a lower PAR formation) is

caused by the reduction of the cellular NAD+ content.

re

lati

ve

mfi

co

ntr

ol

co

ntr

ol + o

lig

1x iso

1x iso

+ o

lig

4x iso

4x iso

+ o

lig

8x iso

8x iso

+ o

lig

0 .0

0 .5

1 .0

1 .5

2 .0 **** **** *******

Figure 4-3: PAR formation capacity after the repeated isoproterenol treatment under NAD+ saturated conditions. The

capacity of PARP1 to synthesize PAR was measured 24 h after the last isoproterenol treatment. PBMCs were isolated and

treated with either a single dose (1x iso) of isoproterenol or with an interval treatment of four (4x iso) or eight (8x iso) doses

isoproterenol in RPMI-1640 cell culture medium w/o FCS. Samples were then incubated with (black bars) or without (white

bars) an oligonucleotide that mimics DNA strand breaks under NAD+ saturated conditions. PAR was stained with the 10H

antibody followed by a secondary antibody coupled with Alexa Fluor 488. The mean fluorescence intensity was measured by

FACS. (Figure in cooperation with Canesia Amarysti, trainee, measurement of cellular PAR content under NAD+ saturated

conditions). Data represent means with SEM of 10 experiments. Statistical analysis was performed using paired t-test (*), ***

P<0.001, **** P<0.0001.

Formation of intracellular ROS in PBMCs during the repeated 4.1.4

isoproterenol treatment

The degradation of the natural occurring catecholamines epinephrine, norepinephrine and dopamine is

accompanied by the generation of ROS. Studies also show the formation of ROS during signaling

processes of G protein-coupled receptors like the β2-A [422, 423]. ROS is also known to damage the

DNA, which can result in DNA strand breaks. DNA damage induced in lymphocytes by epinephrine

can be reduced by a treatment with antioxidants [399]. Therefore, it was hypothesized that the

observed DNA strand breaks were induced by ROS. This idea was also supported by the finding that

isoproterenol is oxidized to isoprenochrome during the treatment, see section 4.3. Thus, the formation

of ROS in PBMCs during the repeated isoproterenol treatment was measured. Different methods are

Results

66

available for the detection of intracellular ROS. The most commonly used methods are based on the

use of fluorescence probes that change their fluorescence upon oxidation by ROS. Two of the most

used fluorescence probes are DHE and DCFDA. Both are cell-permeable dyes, DHE is specific for the

detection of superoxide, whereas, DCFDA can be oxidized by a broad range of ROS species. Both

dyes can only be used in living cells and cannot be used with fixed cells. Therefore, all experiments

were performed as quickly as possible and the fluorescence was measured immediately after

completion of the assay. Freshly isolated PBMCs were treated with isoproterenol in one of the three

tested cell culture media (RPMI-1640 w/o FCS (black), RPMI-1640 with FCS (grey), TexMACS

(green)). Cells were treated with isoproterenol and stained with DHE (10 µM) or DCFDA (20 µM) as

described in section 3.2.5. The fluorescence signal was measured 4 h after the first treatment (30 min

after the last isoproterenol treatment). This allowed the detection of ROS formed during the

isoproterenol treatment. Unstained cells were used as a control for the staining. MEN and TBHP were

used as positive controls to induce the oxidation of DHE or DCFDA. An increase of the fluorescence

after the DCFDA staining without further treatment was observed in both RPMI-1640 cell culture

media, but not in the TexMACS cell culture medium, see Figure 4-4 A), C) and E). In all three cell

culture media an increase of the fluorescence could be seen after the treatment with TBHP (200 µM,

500 µM or 50 mM). Hence, DCFDA had to be present in these cells. The isoproterenol treatment did

not increase the fluorescence signal. This indicated that the isoproterenol treatment did not induce the

formation of ROS in PBMCs. Or at least this experimental setup was not sensitive enough to detect the

ROS formation. Interestingly, different doses of THBP were needed to induce ROS in PBMCs,

depending on the used cell culture medium. The staining with DHE without further treatment also led

to a significant increase of the fluorescence signal, see Figure 4-4 B), D) and F). This indicated the

intracellular presence of the dye. Also a dose-dependent increase of the fluorescence was seen after the

MEN treatment (10, 50, 200 and 500 µM). However, also with DHE staining no formation of

intracellular ROS could be detected. Again, different concentrations of MEN were needed, depending

on the used cell culture medium, to induce the oxidation of the fluorescence probe. Taken together, the

interval treatment of PBMCs with isoproterenol did not induce the formation of ROS.

Results

67

flu

ore

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in

ten

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[AU

]

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/o s

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A

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[200 µ

M]

w/o

DC

FD

A

w/o

iso

1x iso

4x iso

8x iso

TH

BP

[200 µ

M]

0 .0

0 .5

1 .0

1 .5

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2 .5

A )

***

**

flu

ore

sc

en

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in

ten

sit

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[AU

]

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ntr

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/o s

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N [

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M]

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E

w/o

iso

1x iso

4x iso

8x iso

ME

N [

10 µ

M]

ME

N [

200 µ

M]

w/o

DH

E

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

**

**

***

B )fl

uo

re

sc

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in

ten

sit

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[AU

]

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ntr

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/o s

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w/o

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FD

A

TH

BP

[500 µ

M]

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FD

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w/o

iso

1x iso

4x iso

8x iso

TH

BP

[500 µ

M]

0

1

2

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**

C )fl

uo

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in

ten

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[AU

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/o s

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E

w/o

iso

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4x iso

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ME

N [

50 µ

M]

ME

N [

500 µ

M]

0 .0

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1 .0

1 .5

D )

**

*

***

flu

ore

sc

en

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in

ten

sit

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[AU

]

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/o s

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BP

[50 m

M]

w/o

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FD

A

w/o

iso

1x iso

4x iso

8x iso

TH

BP

[50 m

M]

0 .0

0 .5

1 .0

1 .5

**

E )

flu

ore

sc

en

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in

ten

sit

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[AU

]

co

ntr

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/o s

tain

ing

8x iso

w/o

DH

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ME

N [

500 µ

M]

w/o

DH

E

w/o

iso

1x iso

4x iso

8x iso

ME

N [

50 µ

M]

ME

N [

500 µ

M]

0 .0

0 .5

1 .0

1 .5

2 .0

*

**

****

F )

Figure 4-4: The formation of intracellular ROS during the repeated isoproterenol treatment of PBMCs. Freshly

isolated PBMCs were resuspended in one of the three following cell culture media: RPMI-1640 w/o FCS (black) or RPMI-

Results

68

1640 with FCS (grey) or TexMACS (green). PBMCs were either treated with medium (w/o iso) or with a single dose of

isoproterenol (1x iso) or with four (4x iso) respectively eight doses (8x iso) of isoproterenol (red box). Each given dose had

an isoproterenol concentration of 10 µM. A, C and E) PBMCs were stained with 20 µM of DCFDA. PBMCs without

DCFDA staining were used as negative control (first three columns). TBHP was used as a positive control. The fluorescence

of oxidized DCFDA was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, 4 h after

the first isoproterenol administration. B, D and F) PBMCs were stained with 10 µM of DHE. PBMCs without staining were

used as negative control (first three columns). MEN served as positive control of the DHE oxidation. The fluorescence of

oxidized DHE was measured at an excitation wavelength of 520 nm and an emission wavelength of 610 nm, 4 h after the first

isoproterenol administration. Data represent means with SEM of five experiments. Statistical analysis was performed using

RM one-way ANOVA followed by a Dunnett multiple comparison test. For the isoproterenol treatments, no significant DHE

or DCFDA oxidation could be detected. Controls were compared by using a paired t-test (*), **** P<0.0001, *** P<0.001,

** P<0.01, * P<0.05.

Previous experiments have shown the maximum of DNA strand breaks evolved 6.5 h after

administration of the first isoproterenol dose. Therefore, the formation of ROS was also measured at

that point in time. The same experimental setup was used as for the measurements of ROS after 4h.

Again, an increase of the fluorescence signal could be detected after staining of the cells with DCFDA

as well as after the staining of cells with DHE, see Figure 4-5.

flu

ore

sc

en

ce

in

ten

sit

y

[AU

]

co

ntr

ol w

/o s

tain

ing

8x iso

w/o

DC

FD

A

TH

BP

[200 µ

M]

w/o

DC

FD

A

w/o

iso

1x iso

4x iso

8x iso

TH

BP

[200 µ

M]

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

A )

**

*

flu

ore

sc

en

ce

in

ten

sit

y

[AU

]

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ntr

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/o s

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ing

8x iso

w/o

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E

ME

N [

200 µ

M]

w/o

DH

E

w/o

iso

1x iso

4x iso

8x iso

ME

N [

10 µ

M]

ME

N [

200 µ

M]

0 .0

0 .1

0 .2

0 .3

0 .4*

*

B )

Figure 4-5: Formation of intracellular ROS during the first 6.5 hours after the first isoproterenol administration in

PBMCs. PBMCs were incubated in RPMI-1640 w/o FCS and either treated with medium (0x iso) or with a single dose of

isoproterenol (1x iso) or with four doses (4x iso) or with eight doses isoproterenol (red box). Each given dose had a

concentration of 10 µM isoproterenol. A) DCFDA (20µM) was used to detect the formation of ROS in PBMCs caused by the

isoproterenol administration. PBMCs without DCFDA were used as negative control and as control for the background

fluorescence of isoproterenol and TBHP (first three columns). TBHP was used as positive control to induce intracellular

oxidation of DCFDA. The fluorescence of oxidized DCFDA was measured at an excitation wavelength of 485 nm and an

emission wavelength of 535 nm, 6.5 h after the first isoproterenol administration. B) DHE (10 µM) was used to detect the

formation of intracellular superoxide. Cells without DHE staining were used as negative control (first three columns). MEN

was used to induce the oxidation of DHE in PBMCs. The fluorescence of oxidized DHE was measured at an excitation

wavelength of 520 nm and an emission wavelength of 610 nm, 6.5 h after the first isoproterenol administration. Data

represent means with SEM of five experiments for A) and seven experiments for B). Statistical analysis was performed using

RM one-way ANOVA followed by a Dunnett multiple comparison test for isoproterenol treatments, no significant DHE or

DCFDA oxidation could be detected. Paired test was used to compare the controls (*), ** P<0.01, * P<0.05.

The treatment with TBHP of DCFDA stained cells led to a further increase of the fluorescence. Also

the treatment with MEN of DHE stained cells induced a higher fluorescence signal. However, the

Results

69

interval treatment with isoproterenol did not induce the formation of ROS in PBMCs during the first

6.5 h. The experiments were performed only in RPMI-1640 cell culture medium w/o FCS, because the

strongest effect could be observed in cells cultured in this cell culture medium.

4.2 Repeated isoproterenol treatment induced senescence

like phenotype

Previous experiments with PBMCs of PTSD patients have shown that several genes involved in β-

adrenergic signaling, cell cycle regulation, DNA damage sensing and DNA damage repair were

transcriptionally dysregulated (Judy Salzwedel, personal communication). Moreover, ex vivo

experiments with PBMCs of healthy volunteers have shown that the repeated treatment with

isoproterenol induced: the formation of DNA strand breaks, the expression of senescence associated

beta-galactosidase, a change of the cell morphology to a senescence like phenotype and the ability of

PBMCs to proliferate after stimulation with PHE was inhibited (Palombo and Grath, manuscript in

preparation) [420]. It was hypothesized that isoproterenol may induce the expression of a senescence

like phenotype also in human PBMCs. This idea was supported by a recent published mice study

which demonstrated that the repeated isoproterenol infusion can induce a senescence like phenotype in

cardiomyocytes [416]. Therefore, further senescence markers were analyzed in PBMCs after the

isoproterenol treatment. Since the expression of senescence markers required some time the culturing

conditions of PBMCs had to be modified. Therefore, the RPMI-1640 cell culture medium was

replaced by the TexMACS cell culture medium. This medium was developed for the culturing of

immune cells. This increased the cell viability and allowed the culturing of PBMCs for several days,

see appendix Figure 12-8.

Gene expression in PBMCs after the repeated isoproterenol 4.2.1

treatment

Gene expression of genes involved in DNA damage signaling, DNA damage repair, cell cycle control,

oxidative stress and β-adrenergic signaling were analyzed by custom made real time quantitative PCR

(qPCR) arrays in PBMCs after the repeated isoproterenol treatment. These arrays were previously

used for the analysis of the gene expression in PBMCs of PTSD patients. The qPCR was performed to

identify genetic markers for a senescence phenotype in the isoproterenol treated PBMCs. PBMCs were

interval treated either with four or eight doses of isoproterenol, control cells were treated with cell

culture medium. 24 h after the first isoproterenol dose cells were lysed and mRNA was isolated and

transcribed into cDNA. The cDNA was used as template for the relative quantification of the gene

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70

expression. The expressions of the genes of interest were normalized to the expression of two

reference genes, GAPDH and HPRT1. Data were expressed as relative normalized expression (ΔΔCT).

Figure 4-6 shows the relative normalized gene expression of the 4-fold isoproterenol treated cells on

the left side and the relative normalized gene expression of the 8-fold treated cells on the right side.

The threshold for a down- or up-regulation was set to 2 (dotted lines). Meaning for an up-regulation,

the expression of a gene was two times higher in the treated sample compared to an untreated control.

For down-regulation, the expression of a gene of the treated sample was half of the expression of an

untreated control. Genes which are down-regulated are marked red and genes which are up-regulated

are marked green, unregulated genes are marked grey. Statistical analysis was performed for each gene

by using a two-tailed paired t-test, comparing the untreated control samples to the isoproterenol treated

samples. Several genes showed a significant difference in the expression. However, these differences

were small and the threshold for the regulation was not exceeded. Therefore, it was considered that the

expression of these genes was not regulated by the interval treatment with isoproterenol. After the

repeated four-fold isoproterenol treatment of cells, two genes, VCAN and BRAC2, showed an up-

regulation (6.5- and 2-fold increased expression compared to control cells). The up-regulation of the

BRAC2 gene was not significant. One gene, CCND1 (cyclin D1), was down-regulated by the 4-fold

isoproterenol treatment (2.17-fold decrease in isoproterenol treated cells compared to control cells).

Several other genes showed a significant difference in the expression between the control cells and the

four-fold isoproterenol treated cells such as SRC, GRK6 and ADRB2. An up-regulation of these genes

which was close to the threshold (1.8-, 1.86-, 1.73-fold increase compared to control cells) could be

observed. The eight-fold administration of isoproterenol induced up-regulation of the p16, BRCA2 and

VCAN genes (2.7- , 2.0- and 5.6-fold increase compared to control cells). However, the increase of the

p16 and BRCA2 gene expression was not significant. CCND1 was again down-regulated (3.33-fold

decrease compared to control cells). Some additionally genes were significantly regulated and close to

the threshold like NOS3, CDKN1C, B3GNT1 (1.9-, 1.78 and 1.83-fold incase compared to control

cells). The qPCR showed a down-regulation of the CCND1 gene expression. CCND1 is important for

the G1- to S-phase transition of the cell cycle. Since p16 (CDKN2A) and p21 proteins are key-

regulators of the cell cycle and only p21 was included in the original qPCR array the expression of

both was analyzed with new ordered primer pairs (second used p21 primers are indicated with

CDKN1A*). Therefore, the same cDNA stock was used as before for the qPCR arrays. Both CDKN1A

primer pairs gave the same results. The results of the gene expression with the results mentioned in

section 4.2 indicate that isoproterenol may induce a senescence like phenotype in PBMCs.

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71

Figure 4-6: Gene expression pattern of PBMCs after repeated isoproterenol treatment. PBMCs were repeatedly treated

either with four doses (4x iso) or eight doses (8x iso) of isoproterenol. 24 h after the first treatment, PBMCs were lysed and t

mRNA was isolated and transcribed into cDNA. The expression of 41 genes was analyzed by commercial available primer

pairs. Data were expressed as the relative normalized expression (ΔΔCT) (The gene expression of the control sample was set

to 1). Left side: Relative normalized expression of the genes after the four-fold isoproterenol (4x iso ΔΔCT) treatment. Right

side: Relative normalized expression of genes after the eight-fold isoproterenol (8x iso ΔΔCT) treatment. Dotted lines

indicate the threshold for the gene regulation which was set to 2. Grey bars marked genes that were not regulated. Grey bars

with green border marked genes with a regulation threshold above 1.8. Green bars marked genes that were up-regulated and

red bars marked genes that were down-regulated. CDKN1A was measured with two different primer pairs marked as

CDKN1A and CDKN1A*. Data represent means with SEM of five experiments, except for the CDKN2A gene which was

measured in 4 experiments. Statistical analysis was performed using paired t-test by comparing a treatment sample with

untreated control sample for each single gene (*), ** P<0.01, * P<0.05.

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72

p16 protein expression after repeated isoproterenol treatment 4.2.2

Besides the above described marker for senescence, the gene expression study provided further

evidence that isoproterenol may induce a senescence like phenotype in PBMCs. The significant down-

regulation of CCND1 after the isoproterenol treatment is a further hint for the induction of a

senescence like phenotype. Additional, CDKN2A expression exceeded the regulation threshold and

could be up-regulated by the 8-fold repeated isoproterenol treatment. CDKN2A is a biomarker of

aging, especially in T cells [424]. p16 inhibits the CDKs 4 and 6 and inhibits the progression from the

G1 phase to the S phase of the cell cycle. The result is a cell cycle arrest at the restriction point.

Therefore, p16 is also considered as a senescence marker. Expression of the p16 protein was measured

24 h and 48 h after the first isoproterenol treatment by a FACS assay, see Figure 4-7 A), B). The p16

protein level showed a tendency to increase 48 h after the first isoproterenol treatment. However, no

significant increase of p16 protein expression could be observed after the one-fold, four-fold or eight-

fold isoproterenol treatment at both points in time. Hence, the isoproterenol treatment did not induce

an up-regulation of the p16 protein. Taken together the results indicate that isoproterenol may induce a

senescence like phenotype in human PBMCs. However, we could not measure a strong indicator for

senescence such as an increased expression of p16 or p21.

w/o

iso

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Figure 4-7: p16 protein expression in PBMCs after the repeated isoproterenol treatment. Freshly isolated PBMCs were

treated with one dose isoproterenol (1x iso) (10 µM) or repeatedly treated with either four doses isoproterenol (4x iso) (each

dose 10 µM) or with eight doses isoproterenol (8x iso) (each dose 10 µM). Cells were incubated for 24 h A) or 48 h B). After

the incubation cells were fixed, permeabilized and stained with an anti-p16 antibody overnight. A secondary antibody

coupled with Alexa Fluor 488 was used for detection. The fluorescence signal was analyzed by FACS and quantified by the

mean fluorescence intensity (mfi). (Figure in cooperation with Canesia Amarysti, trainee, measurement of cellular p16

expression). Data represent means with SEM of eight experiments. Statistical analysis was performed using RM one-way

ANOVA followed by Tukey multiple comparison test.

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73

4.3 Degradation of isoproterenol under cell culture

conditions

Isoproterenol is a commonly used β-AR agonist. It is used in medicine for the treatment of

bradycardia, heart block and asthma. Moreover, it is also exploited for the investigation of the

signaling processes of ARs. However, the pharmacodynamics and pharmacokinetics of isoproterenol

have so far been investigated only in different animal models and in humans. These studies

demonstrated big differences in the absorption of isoproterenol depending on the administration route

[201, 205, 425]. The plasma half-life and metabolites of isoproterenol varies between different

administration routs and species. Cellular signaling processes are mostly investigated in cell culture

systems. Therefore, also data for uptake, clearance and breakdown of isoproterenol under cell culture

conditions are needed. For instance, it was shown that PBMCs can uptake, synthesis, metabolize and

store catecholamines [169]. One objective of this thesis was to investigate the stability and degradation

of isoproterenol in cell culture with PBMCs. A HPLC system was used to investigate the isoproterenol

concentration in cell culture medium during and after the interval treatment of PBMCs with

isoproterenol. We were interested in the isoproterenol concentration in cell culture medium, because

β-ARs are located at the cell membrane. Moreover, it is known that FCS and serum albumin binds

isoproterenol and increases the anti-oxidative capacity of the cell culture medium [426, 427]. This may

influence the oxidative degradation of isoproterenol. The ligand binding pocket of these receptors is

located on the extra cellular side [428]. Measurements of the intracellular isoproterenol concentrations

were not possible, because of the insufficient sensitivity of the available detectors. Therefore, we

investigated the stability of isoproterenol and its oxidation into isoprenochrome in basal cell culture

medium RPMI-1640 and also in the RPMI-1640 supplemented with 10% FCS. Moreover, the same

experiments were performed with TexMACS cell culture medium which is used for the culturing of

immune cells. TexMACS is FCS-free but contains serum albumin.

Detection of isoproterenol by absorbance detector 4.3.1

To investigate the fate of isoproterenol under cell culture conditions, the amount of isoproterenol was

measured by a HPLC. Isoproterenol was detected by its characteristic absorbance peak of 280 nm or

its fluorescence at an excitation wavelength of 280 nm and an emission wavelength of 316 nm.

Additionally, the amount of isoprenochrome was measured. Isoprenochrome is the aminochrome of

isoproterenol. It is a cytotoxic oxidation product and can be detected by its characteristic absorbance

peak at a wavelength of 490 nm. No further metabolites of isoproterenol in cell culture medium could

be measured, because the detectors were not sensitive enough. Samples were first centrifuged (5 min,

4 °C at 15000 g) to remove cells and cell debris, to avoid clogging of the HPLC. Samples were

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74

separated on a C18 column and relatively quantified. Previous studies showed a short plasma half-life

of isoproterenol of about 5 min [201, 208-210]. According to this plasma half-life, isoproterenol

should be degraded between two dosages during the repeated treatment. Since the time interval

between each dose was at least 30 min. To check this hypothesis, both detectors were calibrated and

optimized for the detection of isoproterenol and isoprenochrome. Therefore, a dilution series of

isoproterenol in cell culture medium was prepared on ice to avoid the degradation of isoproterenol.

Additionally, the isoproterenol stock was mixed with caffeine. Caffeine can be used for the calibration

of the diode array detector and is stable under the experimental conditions. Both substances could be

separated and detected, see Figure 4-8. Isoproterenol (X) was detected at a retention time of about

8 min. Caffeine (#) was detected at a retention time of about 21 min. Furthermore, unidentified

compounds of the cell culture medium (M) were detected in addition to isoproterenol and caffeine.

However, these compound peaks were separated from the isoproterenol and caffeine peaks. The peak

area of the isoproterenol peak and the peak area of the caffeine peak correlated linear with the

increasing concentrations of both substances, see Figure 4-8 I) and J). A tailing effect of the peaks

could be observed at higher concentration of isoproterenol and at higher concentrations of caffeine.

However, this effect could only be observed at concentrations above the experimental concentrations.

The linear correlation between the peak area and the concentration was still given.

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75

Figure 4-8: Dilution series of isoproterenol and caffeine in cell culture medium. Analysis was performed with the

diode array detector. Caffeine was dissolved in cell culture medium to a concentration of 1 mg/ml. Then isoproterenol was

dissolved in this caffeine solution. Samples were analyzed at an absorbance wavelength of 280 nm. A-H) Representative

chromatograms showing the dilution series of isoproterenol and caffeine: A) cell culture medium w/o isoproterenol or

caffeine (blank), B) 0.01 mM isoproterenol and 0.001 mg/ml caffeine, C) 0.05 mM isoproterenol and 0.005 mg/ml caffeine,

D) 0.1 mM isoproterenol and 0.01 mg/ml caffeine, E) 0.5 mM isoproterenol and 0.05 mg/ml caffeine, F) 1 mM isoproterenol

and 0.1 mg/ml caffeine, G) 5 mM isoproterenol and 0.5 mg/ml caffeine, H) 10 mM isoproterenol and 1 mg/ml caffeine. The

isoproterenol peak is marked by X and the caffeine peak by #. Peaks marked by M representing unknown cell culture

medium compounds. I-J) Quantification of the isoproterenol peaks (black) and caffeine peaks (green). Data represent means

with SEM of five experiments. Statistical analysis was performed using linear regression.

Besides the retention times of the peaks and the linear correlations of the peak areas with the

concentrations, peaks were also identified by the corresponding absorbance spectra, see Figure 4-9.

After the detection of a peak at 280 nm, the total absorbance spectrum from 200 nm till 900 nm was

recorded. This absorbance spectrum could be used for the identification of the compounds. Moreover,

the absorbance spectra could be used to control the purity of the peak. Impurities led to additional

absorbance peaks at different wavelengths. For example, Figure 4-9 D) and E) show the 3D plots of

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76

the isoproterenol peak and caffeine peak. No further absorbance peak could be observed. Also the

calculation of the peak purity showed no contaminations of the peaks with further substances. Hence,

the linear correlation of the peak area with the concentration, the retention times and the absorbance

spectra of each peak allowed the identification and relative quantification of isoproterenol by the diode

array detector.

Figure 4-9: Peak identification with the absorbance detector. Chromatographic peaks were identified according to their

retention time and their absorbance spectrum. Peaks were detected at a wavelength of 280 nm by the diode array detector.

After the detection of a peak, an absorbance spectrum from 200 nm till 900 nm was recorded for each peak. A)

Representative chromatogram of an isoproterenol (10 mM) and caffeine solution (1 mg/ml). B) 3D plot of the isoproterenol

peak, showing retention time (min) on the y-axis and absorbance intensity (mAU) on the z-axis. C) 3D plot of the caffeine

peak, showing retention time (min) on the y-axis and absorbance intensity (mAU) on the z-axis. D) 3D plot of the

isoproterenol peak, showing wavelength (nm) on the y-axis and absorbance intensity (mAU) on the z-axis. E) 3D plot of the

caffeine peak, showing wavelength (nm) on the y-axis and absorbance intensity (mAU) on the z-axis. D) and E) show no

additional absorbance peaks at higher wavelengths.

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77

Detection of isoproterenol by the fluorescence detector 4.3.2

The HPLC system was equipped with a second detector, a fluorescence detector. The advantage of the

fluorescence detector in comparison with the diode array detector is the 1000-fold higher sensitivity

and also the higher specificity. The fluorescence detector was calibrated according to the manual and

tested with a dilution series of isoproterenol in cell culture medium. Again, isoproterenol could be

detected at a retention time of about 8 min (X), see Figure 4-10.

Figure 4-10: Dilution series of isoproterenol in cell culture medium. Analysis was performed by the fluorescence

detector. Isoproterenol was dissolved in cell culture medium to a concentration of 10 mM. Afterwards, samples were directly

injected into the HPLC system. Isoproterenol was detected at an excitation wavelength of 280 nm and an emission

wavelength of 316 nm. A-G) Representative chromatograms of the isoproterenol dilution series: A) cell culture medium w/o

isoproterenol (blank), B) 1 µM isoproterenol, C) 2 µM isoproterenol, D) 5 µM isoproterenol, E) 10 µM isoproterenol, F) 50

µM isoproterenol and G) 100 µM isoproterenol. Isoproterenol peaks are marked by X. Peaks marked by M represent an

unknown compound of the cell culture medium. H) Quantification of the isoproterenol peaks (black). Data represent means

with SEM of nine experiments. Statistical analysis was performed using linear regression.

An unidentified compound of the cell culture medium was detected (M) at a retention time of about

5 min. However, the isoproterenol peak was separated from the peak of this compound. The

isoproterenol peaks were symmetrically and no tailing effect could be observed. The peak areas of the

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78

isoproterenol peaks correlated linear with the isoproterenol concentrations, see Figure 4-10 H). The

measured retention time of the isoproterenol peak was coincident with the retention time measured by

the diode array detector. However, it was not possible to calibrate the fluorescence detector for the

detection of isoprenochrome. The fluorescence detector did not provide further information’s on the

peak composition. Therefore, peaks had to be identified according to the retention times and their

relative retention times to the cell culture medium peak and if possible, by correlation with

information of the diode array detector.

Detection of isoprenochrome by the absorbance detector 4.3.3

Isoprenochrome is the aminochrome of isoproterenol. Aminochromes are oxidation products of

catecholamines and exhibit neuro- and cytotoxic properties. The formation of aminochromes involves

different oxidation steps with instable intermediates, including the formation of highly reactive

radicals. However, aminochromes are unstable and, therefore, must be synthesized in situ.

Catecholamines, including isoproterenol, can be chemically oxidized to the corresponding

aminochromes. The characteristic absorbance maxima at a wavelength of 490 nm distinguish them

from the catecholamines. To calibrate the diode array detector isoproterenol was oxidized with

different concentrations of sodium periodate (NaIO4), see Figure 4-11. On the one hand, increasing

concentrations of NaIO4 led to a decrease of the peak area of the isoproterenol peak (X), see Figure

4-11 left side (blue). On the other hand, increasing concentrations of NaIO4 led to an increase of the

peak area of the isoprenochrome peak, see Figure 4-11 left side (green).

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79

Figure 4-11: Chemical oxidation of isoproterenol to isoprenochrome by NaIO4. Isoproterenol was dissolved in oxidation

buffer yielding a starting concentration of 500 µM. NaIO4 was dissolved in oxidation buffer to a concentration of 50 mM.

Afterwards, the NaIO4 was titrated to the isoproterenol solution to the indicated concentration. A-F) Representative

chromatograms of the oxidation reaction with increasing NaIO4 concentrations. Blue chromatograms show the fluorescence

signal at an excitation wavelength of 280 nm and an emission wavelength of 316 nm (isoproterenol). Chromatograms in

green show the absorbance signal at a wavelength of 490 nm (isoprenochrome). A) 500 µM isoproterenol, B) 500 µM

isoproterenol with 10 µM NaIO4, C) 500 µM isoproterenol with 20 µM NaIO4, D) 500 µM isoproterenol with 30 µM NaIO4,

E) 500 µM isoproterenol with 50 µM NaIO4, F) 500 µM isoproterenol with 75 µM NaIO4.

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The area of the isoproterenol peak and the area of the isoprenochrome peak showed a linear

correlation with the NaIO4 concentration, see Figure 4-12 A). Figure 4-12 B) and C) show the

characteristic absorbance spectra of the isoproterenol peak (blue) and the isoprenochrome peak

(green).

Figure 4-12: Quantification of the isoproterenol and isoprenochrome peaks after the oxidation by NaIO4. A)

Quantification of the absorbance- and fluorescence-peaks of Figure 4-11. B) Absorbance spectrum of isoproterenol with the

characteristic absorbance maxima at 236 nm and 280 nm and an absorbance minimum at 248 nm. C) Absorbance spectrum of

isoprenochrome with the characteristic absorbance maxima at 240 nm, 308 nm and 490 nm and absorbance minima at

264 nm and 368 nm.

Isoproterenol stability in cell culture media at 4 °C 4.3.4

Isoproterenol was dissolved either in RPMI-1640 supplemented with FCS or in RPMI-1640 w/o FCS

or in TexMACS cell culture medium to a concentration of 10 mM. These stock solutions were

prepared fresh for each experiment and incubated on ice, protected from light. Previous experiments

have shown that isoproterenol is stable in saline solution at a temperature of 4 °C [200]. Therefore, the

stability of isoproterenol in cell culture media at 4 °C was analyzed. The isoproterenol stock solution

was aliquoted and incubated on ice protected from light. Every 30 min a sample was taken and the

concentration of isoproterenol and isoprenochrome was measured by the HPLC. Isoproterenol was not

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81

degraded in the test cell culture media during a 6 h incubation period, see Figure 4-13. Also no

formation of isoprenochrome could be detected.

t im e [m in ]

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Figure 4-13: Time course of the isoproterenol concentration during 6 h of incubation at 4 °C in three different cell

culture media. Isoproterenol was dissolved either in RPMI-1640 w/o FCS (black) or in RPMI-1640 with FCS (grey) or in

TexMACS (green) cell culture medium to a concentration of 10 mM. Afterwards, the solutions were incubated at 0 °C on ice

in the dark. Every 30 min a sample was analyzed and the absorbance was measured at 280 nm. The isoproterenol peaks were

relatively quantified. A degradation of isoproterenol could not be observed. Data represent means with SEM of four

experiments. Statistical analysis was performed using RM one-way ANOVA.

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82

Isoproterenol stability in cell culture media at 37 °C 4.3.5

The HPLC system was used to investigate the isoproterenol stability under cell culture conditions and

to analyze the oxidation of isoproterenol. It is known that isoproterenol degrades in saline solution at

37 °C [200]. To test whether isoproterenol degrades also in cell culture medium and isoprenochrome is

formed, isoproterenol was dissolved in three different cell culture media. These three culture media

were RPMI-1640 supplemented with 10% FCS, RPMI-1640 w/o FCS and TexMACS. RPMI-1640 is a

basal cell culture medium which is commonly used for the cultivation of PBMCs. However, for long-

term culturing of cells supplementation with FCS is required. This has the following disadvantages:

first, the exact chemical composition of FCS is unknown and may vary between different lots [419].

Second, the growth factors and hormones present in FCS can stimulate receptors and induce signaling

processes [418]. This may interfere with signaling processes induced by the stimulation of the β2-AR.

Third, it is known that isoproterenol is bound by serum albumin, which is also present in FCS [207].

Therefore, RPMI-1640 was used with and without (w/o) supplementation of FCS. TexMACS is a cell

culture medium with a defined chemical composition. It was evolved for the culturing of immune cells

in a serum-free cell culture medium. Isoproterenol was dissolved to a concentration of 500 µM in each

of these cell culture media and incubated at 37 °C. At the beginning of the incubation and every 30

min after the start of the incubation, samples were analyzed by HPLC. The isoproterenol content

showed a significant degradation during six hours of incubation in RPMI-1640 cell culture medium

with FCS as well as in RPMI-1640 cell culture medium w/o FCS, see Figure 4-14 A) and C). In

contrast, isoproterenol incubated in TexMACS cell culture medium was minimally degraded, see

Figure 4-14 E). Samples prepared in RPMI-1640 cell culture medium with FCS and RPMI-1640 cell

culture medium w/o FCS showed an increase of isoprenochrome during the incubation time, see

Figure 4-14 B) and D). At point of time 0 min, no isoprenochrome could be detected. After 30 min of

incubation, isoprenochrome could be detected. After additional 150 min of incubation, the formation

of isoprenochrome reached a maximum. Afterwards, the amount of isoprenochrome decreases. This

indicats a degradation of isoprenochrome. Although there was no significant decrease of isoproterenol

in the TexMACS cell culture medium during the incubation period, a small amount of isoprenochrome

was formed, see Figure 4-14 F). In contrast to the RPMI-1640 cell culture medium with and w/o FCS,

the amount of isoprenochrome was quite stable during the six hour incubation period. Taken together

the HPLC setup can be used for the detection, identification and relative quantification of

isoproterenol and isoprenochrome in cell culture media.

Results

83

t im e [m in ]

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Figure 4-14: Time course of the isoproterenol concentration and formation of isoprenochrome at 37 °C, in three

different cell culture media. Isoproterenol was dissolved either in RPMI-1640 w/o FCS (black) or in RPMI-1640 with FCS

(gray) or in TexMACS (green) cell culture medium to a concentration of 500 µM. Afterwards, the samples were incubated in

a water bath at 37 °C. Every 30 min samples were taken out and analyzed by HPLC. Isoproterenol was detected at an

absorbance wavelength of 280 nm (A, C and E). Isoprenochrome was detected at an absorbance wavelength of 490 nm (B, D

and F). Substances were quantified by the integration of the corresponding chromatographic peaks. Data represent means

with SEM of three experiments. Statistical analysis was performed using RM one-way ANOVA (*), *** P<0.001, ** P<0.01,

* P<0.05.

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84

Isoproterenol concentration after the single dose treatment of PBMCs 4.3.6

The isoproterenol concentration after the administration of single isoproterenol dose was measured

during an incubation time of 4.5 h. Therefore, freshly isolated PBMCs were treated with a single dose

of 10 µM isoproterenol and then incubated at 37 °C. At the beginning of the incubation and every 30

min during the incubation, a sample was taken and the cells were removed. The supernatant was

analyzed by the HPLC and the relative concentration of isoproterenol and isoprenochrome was

measured. In the two tested cell culture media TexMACS and RPMI-1640 w/o FCS, a statistical

significant linear decrease of the isoproterenol concentration could be observed during the incubation

time, see Figure 4-15 A).

t im e [h rs ]

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B )


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****

Figure 4-15: Time course of the isoproterenol concentration and isoprenochrome formation after the administration

of a single dose of isoproterenol to PBMCs. Freshly isolated PBMCs were resuspended either in RPMI-1640 w/o FCS or

TexMACS cell culture medium. Cells were treated with a single dose of 10 µM isoproterenol (red frame) and were incubated

at 37 °C. At the indicated points of time, samples were taken and analyzed by the HPLC. A) Isoproterenol was detected and

quantified by the fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of 316 nm. B)

Isoprenochrome was detected by the absorbance detector at an absorbance wavelength of 490 nm. Point of time 0 h was taken

immediately after the isoproterenol administration. Data represent means with SEM of four experiments. Statistical analysis

was performed using RM two-way ANOVA (*), **** P <0.0001, ** P<0.01.

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85

However, there was no significant decrease of the isoproterenol concentration after 30 min incubation.

The isoproterenol concentration in the RPMI-1640 cell culture medium w/o FCS showed the fastest

decrease. After 3.7 h, the isoproterenol concentration in RPMI-1640 cell culture medium w/o FCS

reached half of the starting concentration. In contrast, the degradation of isoproterenol in TexMACS

cell culture medium was slower. The half of the isoproterenol starting concentration was reached after

about 13 h. After 2.5 h incubation, the concentration of isoproterenol in TexMACS cell culture

medium is significantly higher compared to the RPMI-1640 cell culture medium. In both cell culture

media the formation of isoprenochrome could be observed after 30 min, see Figure 4-15 B). The most

isoprenochrome was formed in the RPMI-1640 cell culture medium w/o FCS. An increase of the

isoprenochrome concentration was observed during the first 3 h of incubation. Afterwards, there was

no further increase and the isoprenochrome concentration was stable in the observed time frame. In the

TexMACS cell culture medium, no further increase of the isoprenochrome concentration was detected

after an incubation period of 60 min. The isoprenochrome concentration was stable in the observed

time frame. Three hours after incubation start, the isoprenochrome concentration reached about a sixth

of the isoprenochrome concentration, measured in RPMI-1640 cell culture medium w/o FCS. The

results showed that the half-life of isoproterenol in cell culture studies and human or animal studies are

different and the results cannot be simply transferred. The hypothesis that isoproterenol is degraded in

the tested cell culture media in a timeframe of 30 min to 60 min must also be abandoned.

Isoproterenol concentration during and after the four-fold treatment 4.3.7

of PBMCs

During the interval treatment with isoproterenol, the PBMCs were treated with four doses of

isoproterenol. Animal and human studies showed that isoproterenol should be degraded after a time

frame of 60 min [201, 208-210]. However, the single dose isoproterenol treatment demonstrated that

degradation under cell culture conditions was slower compared to the animal and human studies.

Hence, an accumulation of isoproterenol in the cell culture medium during the repeated isoproterenol

treatment could be possible. On the other hand, a decrease of the isoproterenol concentration caused

by degradation processes (formation of isoprenochrome) and an increase of the isoproterenol

concentration caused by additional doses could interfere. To test this, freshly isolated PBMCs were

repeatedly treated with isoproterenol each dose had a concentration of 10 µM. The doses were applied

with an interval of 60 min and cells were incubated at 37 °C. Samples were taken every 30 min, direct

after the addition of one isoproterenol dose. At the beginning (0 min), no difference of the

isoproterenol concentration between the three cell culture media could be observed (tested by ordinary

one-way ANOVA followed by Tukey´s multiple comparisons test), see Figure 4-16 A).

Results

86

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n s**

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n s*

Figure 4-16: Time course of the isoproterenol concentration and isoprenochrome formation during and after the four-

fold isoproterenol treatment of PBMCs. Freshly isolated PBMCs were resuspended either in RPMI-1640 w/o FCS or in

RPMI-1640 with FCS or TexMACS cell culture medium. Cells received an interval treatment with isoproterenol. Therefore,

cells were treated at the beginning and then at one-hour intervals with isoproterenol, each dose 10 µM (red frame). PBMCs

were incubated at 37 °C during the treatment. At the indicated points of time samples were analyzed by the HPLC. A)

Isoproterenol was detected and quantified by the fluorescence detector at an excitation wavelength of 280 nm and an

emission wavelength of 316 nm. B) Isoprenochrome was detected by the absorbance detector at an absorbance wavelength of

490 nm. Data represent means with SEM of four experiments. Statistical analysis was performed using RM two-way

ANOVA (*), comparing two cell culture conditions with each other, **** P <0.0001, ** P<0.01, * P<0.05.

Results

87

An increase of the isoproterenol concentration could be observed after each isoproterenol

administration (at 1 h, 2 h and 3 h, red boxes) in all tested cell culture media. A statistically significant

decrease of the isoproterenol concentration between isoproterenol administrations (at points in time

0.5, 1.5 and 2.5 h) could not be observed in the TexMACS as well as in the RPMI-1640 cell culture

medium with FCS. In contrast, the isoproterenol concentration in RPMI-1640 cell culture medium w/o

FCS showed a tendency to decrease. However, this decrease was statistical not significant. The

maximum of the isoproterenol concentration was reached after the fourth dosage in all three cell

culture media. The increase of the isoproterenol concentration in RPMI-1640 cell culture medium w/o

FCS was significantly lower compared to the isoproterenol concentration in the two other cell culture

media. The isoproterenol concentration decreased in all three culture media after 3 h. The decrease of

the isoproterenol concentration was uniformly in the TexMACS as well as in the RPMI-1640 cell

culture media with FCS, in the observed timeframe. In the RPMI-1640 w/o FCS the decline was

reduced after 7 h. The isoproterenol stability in TexMACS and in RPMI-1640 cell culture medium

with FCS was significantly higher compared with the isoproterenol stability in RPMI-1640 without

FCS. The formation of isoprenochrome showed some difference between the three cell culture media,

see Figure 4-16 B). The formation of isoprenochrome could be detected 30 min after the first

isoproterenol dosage. Also an increase during the isoproterenol treatment could be observed in all

three cell culture media. The maximum of the isoprenochrome concentration was observed 4.5 h after

the first isoproterenol administration, 1.5 h after the last isoproterenol administration. The increase of

the isoprenochrome concentration in the RPMI-1640 cell culture medium w/o FCS was higher

compared to the two other cell culture media. 4.5 h after the first isoproterenol administration the

isoprenochrome concentration in RPMI-1640 cell culture medium w/o FCS was about three times

higher compared with the isoprenochrome concentration in the two other cell culture media. In RPMI-

1640 cell culture medium w/o FCS a decline of the isoprenochrome concentration could be observed

after 4.5 h. In TexMACS and in RPMI-1640 cell culture medium with FCS this was not the case. The

results showed that during the isoproterenol administration no significant breakdown of the

isoproterenol was detected. Therefore, isoproterenol accumulated in the cell culture media. The

accumulation of isoproterenol in the cell culture medium should cause a constant stimulation of the β2-

AR instead of a repeated stimulation of the receptor.

Isoproterenol concentration during and after the eight-fold 4.3.8

treatment of PBMCs

Additional to the four-time interval isoproterenol treatment, PBMCs were also repeatedly stimulated

with eight doses of isoproterenol and the isoproterenol concentration was analyzed, see Figure 4-17.

Results

88

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*****

Figure 4-17: Time course of the isoproterenol concentration and isoprenochrome formation during and after the

eight-fold isoproterenol treatment of PBMCs. Freshly isolated PBMCs were resuspended either in RPMI-1640 w/o FCS or

in RPMI-1640 with FCS or TexMACS cell culture medium. Cells were repeatedly treated with isoproterenol with an interval

of 30 min. Each dose had an isoproterenol concentration of 10 µM (red frame). PBMCs were incubated at 37 °C during the

treatment. At the indicated points of time samples were analyzed by HPLC. A) Isoproterenol was detected and quantified by

the fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of 316 nm. B) Isoprenochrome

was detected by the absorbance detector at an absorbance wavelength of 490 nm. Data represent means with SEM of four

experiments. Statistical analysis was performed using RM two-way ANOVA, comparing two cell culture conditions with

each other, **** P <0.0001, ** P<0.01, *** P<0.001, * P<0.05.

Results

89

The concentration of each single dose was 10 µM isoproterenol. The freshly isolated PBMCs were

treated at the beginning (0 min) and then every 30 min for the next 3.5 h. The samples were incubated

at 37 °C and every 30 min direct after the isoproterenol administration a sample was analyzed. All

three tested cell culture media showed an increase of the isoproterenol concentration during the

treatment, see Figure 4-17 A). The isoproterenol concentration showed a linear increase with each

further dose during the first 3.5 h of the incubation in the TexMACS and the RPMI-1640 with FCS

cell culture media. The maximum isoproterenol concentration was reached 3.5 h after the first

isoproterenol dose. In contrast, the isoproterenol concentration in RPMI-1640 cell culture medium w/o

FCS did not show the linear correlation with the addition of further isoproterenol doses. After about

2.5 h, the increase of the isoproterenol concentration reached a maximum which did not further

increase with the next two isoproterenol doses. This indicats that the degradation of isoproterenol

compensated the isoproterenol administration. After 3.5 h (last isoproterenol administration), a decline

of the isoproterenol concentration in all three cell culture media could be detected. In the RPMI-1640

cell culture medium w/o FCS the decline rate of the isoproterenol concentration slowed down during

time. In the TexMACS cell culture medium the isoproterenol was most stable and the decreasing

process showed a linear trend. The decline of the isoproterenol concentration in RPMI-1640 cell

culture medium with FCS showed also a linear trend. In all three tested cell culture media also the

formation of isoprenochrome could be detected, see Figure 4-17 B). 30 min after the first isoproterenol

dosage, isoprenochrome could be detected. The increase of the isoprenochrome concentration was the

highest in the RPMI-1640 cell culture medium w/o FCS. The maximum isoprenochrome concentration

was reached 4 h after the first isoproterenol administration (30 min after the last isoproterenol

administration). Afterwards, the isoprenochrome concentration constantly declined in the observed

timeframe. The isoprenochrome concentration in the RPMI-1640 cell culture medium with FCS

increased at a lower rate, in comparison to the RPMI-1640 cell culture medium w/o FCS. The

maximum isoprenochrome concentration was reached 5 h after the application of the first

isoproterenol dose. The maximum isoprenochrome concentration in RPMI-1640 cell culture medium

with FCS was about 1.9-fold lower compared with the maximum isoprenochrome concentration in

RPMI-1640 cell culture medium w/o FCS. After the maximum was reached, a constant decrease of the

isoprenochrome concentration in the RPMI-1640 culture medium with FCS could be observed. In

contrast, the increase of the isoprenochrome concentration in TexMACS cell culture medium was the

slowest. The maximum concentration was reached after about 5 h, and reached approximately a fifth

of the maximum isoprenochrome concentration in the RPMI-1640 cell culture medium w/o FCS.

Results

90

Isoproterenol concentration in cell culture media after a single 4.3.9

administration

Since PBMCs are able to take up, metabolize, store and syntheses catecholamines their influence on

the isoproterenol concentration and formation of isoprenochrome was analyzed. Therefore, the

experiments were repeated w/o PBMCs. 10 µM of isoproterenol were dissolved in the respective cell

culture medium and aliquoted like it was done before with the cell suspensions. The samples were

incubated at 37 °C, at indicated points in time samples were analyzed by the HPLC. In both cell

culture media a linear decrease of the isoproterenol concentration could be observed during the

incubation, see Figure 4-18.

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in T e x M A C S

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*

****

Figure 4-18: Time course of the isoproterenol concentration and isoprenochrome formation after the administration

of a single isoproterenol dose, in two different cell culture media RPMI-1640 w/o FCS and TexMACS. Isoproterenol

was dissolved either in RPMI-1640 w/o FCS or in TexMACS cell culture medium. A single dose of 10 µM isoproterenol (red

frame) was added to the respective cell culture medium and incubated at 37 °C. At indicated points of time samples were

taken and analyzed by the HPLC. A) Isoproterenol was detected and quantified by the fluorescence detector at an excitation

wavelength of 280 nm and an emission wavelength of 316 nm. B) Isoprenochrome was detected by the absorbance detector

at an absorbance wavelength of 490 nm. Point of time 0 h was taken immediately after the first isoproterenol administration.

Data represent means with SEM of four experiments. Statistical analysis was performed using RM two-way ANOVA (*),

comparing two cell culture conditions with each other, **** P <0.0001, ** P<0.01, * P<0.05.

Results

91

A comparison of the isoproterenol concentrations between culture conditions with cells and w/o cells

showed that there was no significant difference in the observed timeframe, see appendix Figure 12-10.

The formation of isoprenochrome could be detected in both cell culture media after an incubation

period of 30 min. The isoprenochrome concentration increased during the first 2 h of the incubation in

the TexMACS cell culture medium. Then the isoprenochrome concentration was stable during the rest

of the incubation. In RPMI-1640 cell culture medium w/o FCS the isoprenochrome concentration

increased during the first 4 h of the incubation. The maximum of the isoprenochrome concentration

was about 3.5-fold of the maximal isoprenochrome concentration in the TexMACS cell culture

medium.

Isoproterenol concentration in cell culture media during and after 4.3.10

the four-fold administration

The isoproterenol concentration and the formation of isoprenochrome during the four-fold

isoproterenol treatment were also investigated in three different culture media w/o cells. For this

purpose, isoproterenol was again dissolved in the respective cell culture medium. At the beginning and

then every hour for the next 3 h, 10 µM isoproterenol were added to the samples. The samples were

incubated at 37 °C and every 30 min a sample was analyzed by the HPLC. In all three tested cell

culture media an increase of the isoproterenol concentration could be detected, see Figure 4-19 A). No

significant difference of the isoproterenol concentration could be observed between the TexMACS and

the RPMI-1640 cell culture medium with FCS. After each additional isoproterenol dose

(1.5, 2 and 3 h) a stepwise increase of the isoproterenol concentration could be detected in both cell

culture media. The maximum of the isoproterenol concentration was reached after 3 h, with an

approximately four-fold increase of the starting concentration. There was no significant decline of the

isoproterenol concentration between the administrations (0.5, 1.5 and 2.5 h). In contrast, the

isoproterenol concentration in RPMI-1640 cell culture medium w/o FCS also increased stepwise

during the treatment. However, also a breakdown of the isoproterenol could be observed. Therefore,

after 3 h, the maximum isoproterenol concentration reached only about the three-fold of the starting

concentration. Between the isoproterenol administrations a decrease of the isoproterenol concentration

could be observed, which was significant at 2.5 h. After the last administration of isoproterenol, in all

three tested cell culture media a decline of the isoproterenol concentration, with a linear trend, could

be detected. The comparison of the isoproterenol concentration in cell culture media with cells to the

isoproterenol concentration in the respective cell culture media w/o cells showed no statistical

significant difference, see appendix Figure 12-11. The formation of isoprenochrome could be detected

in all three cell culture media after an incubation time of 30 min, see Figure 4-19 B).

Results

92

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Figure 4-19: Time course of the isoproterenol concentration and isoprenochrome formation during and after the four-

fold isoproterenol treatment, in three different cell culture media RPMI-1640 w/o FCS, RPMI-1640 with FCS and

TexMACS. Isoproterenol was dissolved either in RPMI-1640 w/o FCS, in RPMI-1640 with FCS or in TexMACS cell

culture medium. A single dose of 10 µM isoproterenol was added to respective cell culture medium and incubated at 37 °C.

For the next 3 h, every hour an additional isoproterenol dose was administered (red frame). At the indicated points of time

samples were taken and analyzed by the HPLC system. A) Isoproterenol was detected and quantified by the fluorescence

detector at an excitation wavelength of 280 nm and an emission wavelength of 316 nm. B) Isoprenochrome was detected by

the absorbance detector at an absorbance wavelength of 490 nm. Point of time 0 h was taken immediately after the

isoproterenol administration. Data represent means with SEM of four experiments. Statistical analysis was performed using

RM two-way ANOVA, comparing two cell culture conditions with each other (*), **** P <0.0001, ** P<0.01, * P<0.05.

Results

93

The isoprenochrome concentration in TexMACS cell culture medium increased until 5 h after the

beginning of the incubation. Then it was stable until the end of the measurement. In both RPMI-1640

cell culture media a more rapid increase of the isoprenochrome concentration could be detected. In

RPMI-1640 cell culture medium w/o FCS the maximum of the isoprenochrome concentration could be

detected 4.5 h after the beginning of the incubation. It was about 5.6-fold higher compared with the

maximal concentration of isoprenochrome in the TexMACS cell culture medium. The maximal

isoprenochrome concentration in RPMI-1640 cell culture medium with FCS was achieved 30 min

later, 5 h after the first isoproterenol dose. It was about 4-fold higher compared with the maximal

isoprenochrome concentration in the TexMACS cell culture medium.

Isoproterenol concentration in cell culture media during and after 4.3.11

the eight-fold administration

The eight-fold interval treatment was also repeated w/o PBMCs, to investigate the isoproterenol

concentration and the formation of isoprenochrome during the treatment. The isoproterenol was

dissolved in one of the three cell culture media. Afterwards, a 10 µM dose was added to the respective

medium and samples were incubated at 37 °C. Every 30 min an additional 10 µM isoproterenol dose

was applied until the last dose was given after 3.5 h. Samples were analyzed every 30 min after the

beginning, by HPLC. In all three cell culture media a linear increase of the isoproterenol concentration

during the treatment could be observed, see Figure 4-20 A). During the first 3.5 h of the isoproterenol

treatment, no significant difference of the isoproterenol concentration in the cell culture media

TexMACS and RPMI-1640 with FCS could be detected. The maximum concentration was reached

after the last treatment, after 3.5 h. The maximal concentration in both cell culture media reached

about the 8-fold of the starting concentration. In RPMI-1640 cell culture media w/o FCS the increase

of the isoproterenol concentration was slower. The maximal concentration was also reached after the

last isoproterenol dosage. The highest isoproterenol concentration was reached with a 6-fold increase

of the starting concentration. This indicated a significant degradation of isoproterenol during the

treatment. After 3.5 h, a linear decrease of the isoproterenol concentration could be detected in both

RPMI-1640 media. In the TexMACS cell culture medium a small decline could be observed. At later

points in time, the isoproterenol concentration was stable until the end of the measurements. For none

of the three cell culture media, a difference of the isoproterenol concentration could be detected in the

presence of PBMCs compared with the respective cell culture medium in the absence of PBMCs.

After an incubation time of 0.5 h, the formation of isoprenochrome could be observed in all three cell

culture media, see Figure 4-20 B).The isoprenochrome concentration increased linear and reach a

maximum after 4 h in the RPMI-1640 cell culture medium w/o FCS. Afterwards, a constantly decline

of the isoprenochrome concentration could be observed until the end of the measurement. In the

RPMI-1640 cell culture medium with FCS only a small increase of the isoprenochrome concentration

Results

94

could be observed for the first 3 h of incubation. Between 3.5 and 6 h an increase of the isoproterenol

concentration could be observed. No decline of the isoprenochrome concentration could be detected

until the end of the measurement. A slow increase of the isoprenochrome concentration in TexMACS

cell culture medium could be detected. The maximal isoprenochrome concentration reached about a

fifth of the maximal isoprenochrome concentration in RPMI-1640 cell culture medium w/o FCS. This

maximum was achieved after 4.5 h of incubation time. Afterwards, the isoprenochrome content was

stable until the end of the measurement. Taken together the HPLC measurements demonstrated that

the half-life of isoproterenol under cell culture conditions is higher compared to the reported half-life

in animal and human studies. This also causes an accumulation of the isoproterenol in the cell culture

media during the repeated treatment. The degradation of isoproterenol in cell culture media is

associated with the formation of isoprenochrome. During this oxidation process free radicals and ROS

can be formed. The consequences of the formation of isoprenochrome and the unexpected high half-

life of isoproterenol on the performed studies will be discussed in the next section.

Results

95

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Figure 4-20: Time course of the isoproterenol concentration and isoprenochrome formation during and after eight-

fold isoproterenol treatment, in the three different cell culture media RPMI-1640 w/o FCS, RPMI-1640 with FCS and

TexMACS. Isoproterenol was dissolved either in RPMI-1640 w/o FCS, in RPMI-1640 with FCS or TexMACS cell culture

medium. Isoproterenol doses with a concentration of 10 µM were administered every 30 min for the next 3.5 h. (red frame).

Solutions were incubated at 37 °C. At the indicated points of time samples were analyzed by HPLC. A) Isoproterenol was

detected and quantified by the fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of

316 nm. B) Isoprenochrome was detected by the absorbance detector at an absorbance wavelength of 490 nm. Data represent

means with SEM of four experiments. Statistical analysis was performed using RM two-way ANOVA, comparing two cell

culture conditions with each other, **** P <0.0001, *** P<0.001, ** P<0.01.

Discussion

96

5 Discussion

In the last decades, it became clear that psychological stress can influence many physiological

functions of the human body [4, 25, 152]. Especially, studies which investigated the influence of

chronic stress could demonstrate, at least partially, the adverse effect of stress on the human health

[71, 72, 83, 429]. In particular, the influence of chronic stress on the immune system is of special

interest. Although the connection between stress and the immune system is under investigation and

many lessons have been learned at the last decades, there are still open questions. The influence of

stress on the genomic stability becomes a new interesting research field. Several human-, animal- and

cell studies demonstrate the influence of stress on the genomic stability and its association with the

development and progression of cancer [27, 30, 32, 73, 379, 400, 401]. For instance, previous studies

suggest an accelerated aging of PTSD patients [82]. PBMCs isolated from PTSD patients have an

accumulation of DNA strand breaks [83]. Furthermore, dysregulation of DNA repair genes could be

observed in PBMCs of PTSD patients (Judy Salzwedel, personal communication). PTSD can be

considered as chronic stress model because PTSD patients have a chronically elevated level of stress

compared to healthy people. On a molecular level, an increase of catecholamine levels in the blood

and urine is observed in PTSD patients [57, 59-61]. Stress hormones, especially catecholamines, can

influence the genomic stability [399]. Catecholamines bind to ARs which are present on various cell

types, including immune cells. The subsequent downstream signaling influence physiological

processes which influence the cancer progression. Moreover, catecholamine can induce DNA damage

by various mechanisms, such as chemical or enzymatically degradation processes, but also by

signaling processes of the β2-AR [400, 401]. The combination of the following findings leads to the

hypothesis that “chronic stress induces DNA damage via the repeated stimulation of the β2-AR by

catecholamines”: PBMCs of PTSD patients have more DNA damage compared to healthy control

persons, animal- and human studies of chronic stress showed an increase of DNA damage in stressed

subjects, catecholamines can induce DNA damage by the formation of ROS and repeated stimulation

of the β2-AR by catecholamines can induce the accumulation of DNA strand breaks in human cancer

cell lines, repeated treatment of mice with isoproterenol could induce a senescence like phenotype in

myocardiac cells. To prove this hypothesis and to identify biomarkers of stress in PBMCs, an ex vivo

model for the repeated release of catecholamines was previously established by Schumacher and

Moreno-Villanueva [405]. Therefore, PBMCs of healthy volunteers were isolated and repeatedly

treated with isoproterenol, a synthetic sympathomimetic drug. PBMCs were chosen because of the

pervious findings in PTSD patients. Moreover, PBMCs are primary human immune cells which can

easily be obtained by a minimal invasive procedure. PBMCs of PTSD patients were not selected for

several reasons: PTSD patients have a high rate of comorbidities which can influence immune cells,

available cell material is limited, and diagnosis of PTSD is based on an assessment of the subject by a

Discussion

97

medical psychotherapist and not by the investigation of biomarkers. Moreover, PBMCs are primary

cells, therefore, they are closer related to the in vivo situation than cell lines. Cell lines often have a

different functional phenotype compared to their cognate primary cells [430]. However, the use of

primary cells also has some disadvantages, which must be taken into account. For example, the cell

material is more limited, the genomic variability between donors can influence the statistical power of

the experiments and the results. Additional, PBMCs are no homogenous cell population. They consist

out of different subpopulations, such as T cells, B cells, natural killer cells and monocytes. The exact

composition of the subpopulation can vary between donors and also between sample points of time.

PBMCs can be affected by diet, life style and medication of the donor and also by environmental

influences which could not be controlled in this study. The common used density centrifugation

method for isolation of PBMCs allows not the complete removal of other blood cells like erythrocytes

and thrombocytes. Hence, these cells can contaminate the isolated PBMCs. Isoproterenol was chosen

instead of epinephrine to stimulate the β2-AR, because it is often used in cell studies, unlike to

epinephrine, it selectively binds to β-ARs. Isolated PBMCs were cultured in a standard RPMI-1640

cell culture medium w/o the supplementation of fetal calf serum. Since FCS is a complex and

undefined mixture of various proteins, hormones, and growth factors which can affect the signaling

processes of the β2-AR. Investigation of the signaling pathways usually requires a serum starvation,

because many of the signal transducers, such as kinases, are shared by many different signaling

pathways. Hence, the simultaneously activation of signaling pathways by different stimuli can induce

an overlay of the different signal processes. This makes it difficult or impossible to analyze the signal

of interest. Additional, one of the main components of FCS is serum albumin which is known to bind

various small molecules, including isoproterenol [207, 431]. Moreover, FCS can stimulate the

cytokine production of PBMCs, especially the production of IL-2 was increased, and change their

immune response [418]. Withdrawal of FCS has also same adverse effect, because the cell tended to

adhere [405]. Therefore, the cells were incubated in a shaking water bath which reduced the observed

adherence. Only a small reduction of the cell viability of approximately 6% could be observed under

these culture conditions after 24 h of incubation, see appendix Figure 10Figure 12-1 [421]. Longer

incubation times of 48 h or 72 h, under serum starvation, reduced the cell viability (data not shown).

Therefore, experiments with longer incubation times than 24 h were not performed in RPMI-1640 cell

culture medium w/o FCS. In order to overcome this problem in experiments which required a longer

incubation period, PBMCs were cultured in TexMACS cell culture medium. TexMACS is a cell

culture medium that was developed for the culturing of immune cells without the supplementation of

FCS [432]. However, TexMACS cell culture medium contains human serum albumin. For some

comparative experiments (HPLC measurements) also RPMI-1640 cell culture medium supplemented

with FCS was used. Hara et al. used subcutaneously implanted osmotic pumps for the continuously

infusion of isoproterenol 30 mg kg-1

d-1

for 28 days in mice to simulate chronic stress and the repeated

release of catecholamines [400]. Cell lines were treated every 12 h with 10 µM isoproterenol for

Discussion

98

3 days. This treatment protocol was not feasible for the treatment of PBMCs, because of the long

incubation time. Therefore, a new treatment protocol was used. PBMCs were treated either with a

single dose of isoproterenol (10 µM), or with four doses of isoproterenol (10 µM each dose) with an

interval of 1 h between each dose, or with eight doses of isoproterenol (10 µM each dose) with an

interval of 30 min between each dose. Each sample was treated every 30 min until the last

isoproterenol dose was administered. At points in time when the samples were not treated with

isoproterenol, 1-fold and 4-fold treatments, samples got the solvent administered. An administration of

10 µM isoproterenol is a commonly used dose in cell culture experiments. Since this dose leads to a

maximum cAMP response in the cells, including PBMCs [433]. Using this ex vivo model of the

release of catecholamines during chronic stress, some interesting findings have been obtained which

prepared the basis of the presented thesis. The repeated 8-fold isoproterenol treatment induced the

formation of DNA strand breaks 6.5 h after the first isoproterenol administration, see appendix Figure

12-2 [421]. The treatment of PBMCs with a dose of 10 µM propranolol alone, a dose which is

sufficient to block the cAMP signaling of the β2-AR (see Figure 4-1), did not induce the formation of

DNA strand breaks. In contrast, the administration of 10 µM of propranolol 10 min before the start of

the repeated isoproterenol treatment could significantly reduce the amount of DNA strand breaks, see

appendix Figure 12-2 [421].

5.1 Isoproterenol mediated DNA damage

Pretreatment with propranolol could not completely inhibit the formation of DNA strand breaks. This

indicates that additional processes must be responsible for the formation of DNA strand breaks. Hara

et al. hypothesized the formation of DNA strand breaks is caused by the cAMP/PKA signaling

pathway which causes the generation of ROS [400]. According to this hypothesis, the ROS formation

and the resulting DNA damage should be completely blocked by the propranolol which is not the case

in our experiments. However, there are other mechanisms for the formation of ROS that could not be

inhibited by propranolol. Isoproterenol can be oxidized to isoprenochrome extracellular as well as

intracellular. In both cases the formation of free radicals and ROS could be observed, discussed in

section 5.1.1. Recent data indicate that isoproterenol may act as biased agonist of α-ARs [434].

Although the presence of α-ARs at the surface of immune cells is still under discussion, there is some

evidence for their presence. For example, the selective and potent α2-AR antagonist, yohimbine, binds

to human lymphocytes [435]. T cells express the mRNA of α-ARs [436]. Natural killer cells express

α1- as well as α2-adrenergic receptors [437]. Activation of β2-AR by agonists can induce the expression

of α-ARs at monocytes [438, 439]. However, ROS can induce DNA single as well as double-strand

breaks which are well known to activate PARP1. The binding to DNA strand breaks activates the

enzymatically activity of PARP1 and leads to the formation of PAR. PARP1 as well as PAR are

Discussion

99

important for the repair of the DNA strand breaks, because both recruit and modulate the activity of

further DNA repair factors. Therefore, the capacity of PARPs to form PAR was measured 24 h after

the first isoproterenol administration, see appendix Figure 12-4 [421]. The repeated isoproterenol

treatment affected the capacity of PARP1 to form PAR in some cells. The more isoproterenol doses

were administered, the more cells showed a lower capacity to form PAR. In addition to DNA strand

breaks, there are two main factors that influence the PAR formation. On the one hand the expression

of PARP1 as the main contributor to the PAR formation. On the other hand, the cellular NAD+

content, because NAD+ is the substrate for PAR formation [440]. Therefore, the intracellular NAD

+

content was measured during the 8-fold isoproterenol treatment and 24 h after administration of the

first isoproterenol dose, see Figure 4-2. The 8-fold repeated isoproterenol treatment induced a

reduction of the cellular NAD+

content, discussed in section 5.1.2. However, also the PARP1 protein

level decreased after the repeated isoproterenol treatment, see appendix Figure 12-5 [421]. PARP1 is

known to be constitutive expressed in cells. The expression of the PARP1 mRNA was not regulated

by the repeated isoproterenol. However, a donor dependent decrease of the PARP1 protein level could

be observed, see appendix Figure 12-6 [421]. The PARP1 protein level was repeatedly measured in

three different subjects at different days. For each donor the measurement was repeated at least three

times. The results of the measurements for each subject were repeatable. Demonstrating the

differences of the PARP1 protein expression was no side effect of the experimental conditions. Hence,

the observed reduced PARP1 protein level must be caused by a breakdown of the protein. PARP1 can

be degraded by various types of enzymes which produce a specific pattern of protein fragments [441,

442]. A time course analysis of the PARP1 protein and posttranslational modifications and eventual

evolving protein fragments could give further information about the decrease. Moreover, the

fragmentation patterns can be associated with different kinds of cell death. An intra-individual

variability of the poly(ADP-ribosyl)ation capacity was observed in other studies [414]. To determine

whether the cellular NAD+ content or the PARP1 protein level is the limiting factor of the poly(ADP-

ribosyl)ation capacity of PBMCs, the PAR formation was analyzed under NAD+ saturated conditions.

This assay was developed earlier in the group by Kunzmann et al. [414]. The assay allows the

detection of the maximum intracellular PAR capacity in permeabilized cells. Since an oligonucleotide

mimics DNA strand breaks under saturated NAD+ concentrations. Using these conditions, no

difference of the PAR formation between control cells and isoproterenol treated cells could be

observed, see Figure 4-3. Therefore, the cellular NAD+ pools seem to be the limiting factor and not the

PARP1 protein level. However, it is possible that the influence of slightly reduced PARP1 protein

level could not be detected. Or the reduction of the PARP1 protein is compensated by other PARPs.

PARP2 and PARP3 can also be activated by DNA damages. PARP1 and PAR play also an important

role in the induction of apoptosis. Hyperactivation of PARP1 by DNA strand breaks induces a

depletion of cellular NAD+ and ATP pools. This leads to an energy crisis and subsequently to cell

death. Therefore, during apoptosis PARP1 is cleaved by caspases to preserve the NAD+ and ATP

Discussion

100

pools. However, there was no detectable caspase 3 or caspase 7 activity (data not shown), suggesting a

caspase independent apoptosis induction. The repeated isoproterenol treatment induced apoptosis in

treated PBMCs, see appendix Figure 12-7 [421]. Interestingly, as for the PARP1 protein level, also for

the isoproterenol-mediated apoptosis, a strong inter-individual variability could be observed. The

PBMCs of some donors undergo apoptosis, while the PBMCs of other donors seem to be more

resistant. It was previously shown that dobutamine, a synthetic catecholamine, can induce apoptosis in

PBMCs in a dose-dependent manner after 24 h and 48 h incubation, which was partially mediated by

the β2-AR [443]. In this study it was not possible to measure all of the biomarkers: DNA strand breaks,

NAD+ and ATP levels, PAR formation capacity, and apoptosis within the same subject. Therefore, it is

not possible to correlate the PARP1 protein level with the PAR capacity or the induction of apoptosis.

Follow-up studies should be performed to do this. Moreover, the kinetics of these biomarkers should

be measured between the 3.5 h and 24 h after the first isoproterenol treatment, in order to obtain

information on the timing of the cellular events. Since different scenarios could explain the observed

results. The isoproterenol induced DNA strand breaks could stimulate the PARP1 activity over longer

time periods. Resulting in a NAD+ pool depletion and subsequent in an energy crisis and apoptosis

[444]. The formed PAR polymer is degraded by PARG and the free PAR polymers can induce the

release of apoptosis-inducing factor (AIF) from the mitochondria and subsequently induce cell death

(Parthanatos) [310, 445-447]. Therefore, also the temporal and spatial cellular localization of the AIF

should be measured. An investigation of the DNA fragmentation pattern, small-scale vs. large-scale

fragmentation, could give further information with regard to cell death. Investigations of the timing of

the cellular process could also help to pinpoint the cause of the DNA strand breaks, which were

detected 24 h after the first isoproterenol dose. Since the DNA strand breaks could be a result of

isoproterenol treatment itself or caused secondary by apoptosis. The results show indications for an

apoptotic cell death as well as for necrotic cell death (energy depletion). PBMCs are a mixture of

different cell types. It may be possible that a cell type undergoes necrosis while other cell types

undergo apoptosis in response of the isoproterenol treatment.

Formation of intracellular ROS 5.1.1

The levels of intracellular ROS were measured, because ROS could be responsible for the formation

of the observed DNA strand breaks. Catecholamines can induce the formation of ROS. However, the

most human and animal studies showed only an indirect correlation between catecholamines and ROS

mediated DNA damage, no direct measurement of ROS was performed [400, 401]. ROS should be

detected intracellular if it is responsible for the induction of DNA damage. The different origins of

ROS in the reported setup can be the chemical degradation of isoproterenol to isoprenochrome, the

intracellular breakdown of isoproterenol, the formation of ROS induced by the β2-adregernic signaling

or indirect secondary effects. The formation of intracellular ROS can be detected by various methods.

Discussion

101

The most common used methods in cell biology are fluorescence probes which interact with ROS. In

this study DCFDA and DHE were used in single staining’s for the detection of ROS during the

repeated isoproterenol treatment, 4 h and 6.5 h, after the first isoproterenol dose. The 6.5 h point in

time was selected, because at this point in time the formation of DNA strand breaks had peaked.

However, the results showed that the detection of ROS was not possible after 4 hours of incubation or

after 6.5 h of incubation. Since, the cell culture medium have an influence on the formation of ROS,

see section 4.3, the ROS measurements were performed in all three used cell culture media for an

incubation time of 4 h. The ROS measurement of the 6.5 h point in time was only performed in RPMI-

1640 cell culture medium w/o FCS. Since the results were negative and the strongest effects were

expected in RPMI-1640 cell culture medium w/o FCS. Although, the measurements of intracellular

ROS indicate no formation of ROS, see Figure 4-4, this should be critically considered for several

reasons. DCFDA is a generalized oxidative stress indicator which reacts with various ROS. However,

it can detect intracellular peroxides only efficiently after their decomposition into radicals [448].

DCFDA is a cell-permeable, non-fluorescent dye which must be deacetylated by intracellular

esterases. The hydrolysis of the two acetyl-groups creates 2´,7´-dichlordihydrofluorescein (H2DCF).

H2DCF is cell membrane impermeable and accumulates in the cell, it can be oxidized by ROS which

creates the highly fluorescent DCF compound. DCF can pass the cell membrane and can leak out of a

cell over the time [448, 449]. According to the supplier Abcam, DCF should be retained in cells for

the observed time period. However, a leakage could not be completely excluded, because no data were

available with regards of PBMCs. Therefore, control samples were included which were not stained

with DCFDA. In all measurement series, except for the measurements in TexMACS cell culture

medium, see Figure 4-4 E) a significant increase of the fluorescence could be observed after the

DCFDA staining. This indicates the presence of a fluorogenic compound in the cells. Moreover, the

fluorescence signal increased after the treatment with TBHP which is used as positive control. The

fluorescence of the cells was not influenced by isoproterenol or TBHP treatment alone. DHE is also a

cell membrane permeable dye which reacts specifically with the superoxide radical anion [450]. The

oxidation product is ethidium which intercalates into DNA. As for DCFDA measurements, an increase

of the fluorescence signal could be observed after the DHE staining compared with DHE unstained

cells. This demonstrates that a fluorogenic substance was taken up by the cells. The fluorescence

signal was not increased by the isoproterenol or MEN treatment alone. The positive control, MEN,

further increased the fluorescence signal of DHE stained cells. Hence, it should have been possible to

detect ROS during the 4 h as well as during the 6.5 h incubation period. An additional problem could

have been the sensitivity of the used experimental setup. The increase of the fluorescence signal after

treatment with the positive control was statistical significant but low, 2- to 3-fold for the DCFDA

staining and up to 4-fold for the DHE staining. This detection range might be too small and therefore

too insensitive to detect the ROS formation induced by isoproterenol in PBMCs. For example,

fluorescence microscopy or flow cytometry measurements showed an increase of the fluorescence

Discussion

102

signal of DCFDA induced by ROS to only about 1.5-fold after the treatment of HEK cells with

isoproterenol or other catecholamines [422, 423, 451]. Moreover, HEK cells are human embryonic

kidney cells which represent, unlike PBMCS, a homogenous cell population. Additional, the most

reactive oxygen species have a short half-life ranging from nanoseconds to milliseconds [452]. The

large time differences between the half-life of the reactive oxygen species and the observed time frame

which is several hours could be a limiting factor. As already mentioned, there are several sources for

the ROS formation in the used assay setup. The HPLC experiments showed the oxidation of

isoproterenol to isoprenochrome. This is an indirect indication that ROS or free radicals are formed

extracellular by chemical reactions. Some reactive oxygen species, like peroxides can diffuse into the

cells and damage cell components, including the DNA. Electron paramagnetic resonance spectrometry

(EPR) could be used for the detection of ROS and free radicals in the cell culture medium [453, 454].

Another source of ROS could be the intracellular degradation of isoproterenol. In contrast to

epinephrine and norepinephrine, isoproterenol is no substrate for MAO [201, 455]. The degradation of

catecholamines by MAOs induces the formation of hydrogen peroxide and is a main source of the

catecholamine induced ROS formation. However, other enzymes like oxidases could catalyze the

oxidation of isoproterenol and subsequent induce the formation of ROS. In cell lines, mainly HEK 293

cells, and animal models it was demonstrated that ROS play a role in cell signaling processes [451].

Also the signaling of the β2-AR was linked to the formation of ROS via the cAMP/PKA signaling

pathway [400, 401]. The NADPH oxidase is important in the receptor signaling induced ROS

formation. NADPH oxidase can be found in human PBMCs [456]. However, this signaling induced

ROS formation has not been demonstrated previously in PBMCs. Hara et al. demonstrated the

formation of ROS only indirectly by the formation of DNA strand breaks, analyzed by γ-H2AX foci

[400]. The partial inhibition of the DNA strand break formation by a pretreatment of the PBMCs with

propranolol also indicates that the ROS formation is not only mediated by the cAMP/PKA signaling

pathway. Since propranolol inhibits the cAMP formation. Other receptors like the α-AR could be

responsible for the formation of ROS. Norepinephrine induces the formation of ROS via α-ARs in

human PBMCs [457]. Additional, it was shown that persistent activation of the β2-AR protects

myocardiac cells from apoptosis which was induced by ROS [141, 143, 458]. Therefore, the

performed ROS measurements should be repeated using other strategies and methods. For example,

the measurement of a time series after each isoproterenol administration could improve the time

resolution and reduce the probability of a leakage of the fluorescence dyes from the cells. However,

this would require a lot of experiments, because both dyes cannot be used with fixed cells and both

dyes are not resistant against detergents [459]. Hence, the measurement and staining must be

performed for each point in time separately. Chemical improved versions of DCFDA and DHE are

available which can overcome these limitations. Moreover, some fluorescence probes are specific for

cellular compartments, allowing a higher spatial resolution of the ROS detection. Additionally, the use

of fluorescence microscopy for the detection of the fluorescence signals could improve the sensitivity

Discussion

103

for the ROS detection. EPR spectrometry could be used for the detection of intracellular ROS. Finally,

an increase of the oxidative stress could be measured indirectly. ROS can damage various cellular

macromolecules. These oxidized macromolecules can be used as a marker for oxidative stress. This

markers can be oxidized lipids (lipid peroxidation), oxidized proteins and oxidative DNA lesions

[452]. The most interesting marker with regards to the study topic would be the oxidative DNA

damage. For instance, the quantification of 8-oxoguanine which is one of the most common oxidative

DNA lesion could be used [460-462].

Intracellular cAMP and NAD+ content after repeated isoproterenol 5.1.2

treatment of PBMCs

The “classical” signaling pathway of the β2-AR is the cAMP/PKA signaling pathway. The formation

of the second messenger cAMP is an immediate and transient response of the cell induced by the

binding of an agonist to the β2-AR. The receptor must be present at the cell surface and must be

coupled to the Gs protein for the activation of the cAMP signaling. The cAMP content in PBMCs was

measured directly after the administration of the last isoproterenol dose. Forskolin was used as

positive control. Propranolol was used to block the β2-AR and as inhibitor of the cAMP formation.

Forskolin binds to the AC and increases the intracellular cAMP levels independently from the

receptor. The treatment of PBMCs with forskolin induces an approximately 8-fold increase of the

intracellular cAMP content, see Figure 4-2. This demonstrates that the PBMCs are able to induce the

formation of cAMP after the incubation. Interestingly, the forskolin treatment of PBMCs in the

TexMACS cell culture medium as well as in the RPMI-1640 cell culture medium w/o FCS showed no

differences of the cAMP induction. Treatment of cells with propranolol alone slight reduces the

intracellular cAMP levels, see Figure 4-2. Since propranolol is an inverse agonist, a reduction of the

cAMP level below the basal levels is expected [463, 464]. An increase of the intracellular cAMP

content could be observed after the isoproterenol treatment. This increase was inhibited by a

pretreatment with propranolol, with one exception, after the 8-fold isoproterenol treatment in the

RPMI-1640 cell culture medium. The increase of the cAMP content was the strongest after the 1-fold

isoproterenol administration. The observed increase of the intracellular cAMP content was

approximately 2-fold higher in the TexMACS cell culture medium compared to the RPMI-1640 cell

culture medium w/o FCS. Some substances of the TexMACS cell culture medium seem to synergize

the isoproterenol induced cAMP formation. Since the direct and maximal activation of the AC by

forskolin induced the formation of cAMP to the same quantity in both cell culture media. And the

TexMACS cell culture medium alone did not induce the stimulation of the β2-AR. During the

isoproterenol treatment, with the administration of further isoproterenol doses, the increase of the

cAMP content was lowered. Several mechanisms could explain these results. The repeated stimulation

of the receptor could induce its internalization and subsequently a down-regulation of the β2-AR.

Discussion

104

Moreover, repeated stimulation of the β2-AR changes the kinetics of the β-arrestin binding to the

receptor and leads to a faster internalization [123]. This reduces the isoproterenol binding sites at the

cell surface and the ability to activate the downstream cAMP signaling. For example, a long term

infusion of isoproterenol in man induced an up-regulation of β-ARs during the first hour of infusion.

After 4-6 h a decrease below the basal amount of β-ARs could be observed [465]. Also an uncoupling

of the Gs protein from the β2-AR which is observed after agonist binding could reduce the cAMP

dependent downstream signaling. Therefore, the β2-AR density at the cell surface should be measured

during and after the repeated isoproterenol treatment. Moreover, a time series of immunopurification

of the β2-AR could be used to investigate the quantity and the type of G proteins which are bound to

the receptor during and after interval treatment. A forskolin treatment directly after the 8-fold

isoproterenol treatment that increases the cAMP level to the same extant than the forskolin treatment

alone, would exclude a depletion of the cellular ATP pools. However, the results showed that the β2-

AR is stimulated and the downstream cAMP-signaling pathway is activated. Moreover, pretreatment

of cells with propranolol block the cAMP downstream signaling. The repeated treatment of PBMCs

with isoproterenol induced the formation of DNA strand breaks. This treatment also increased the

percentage of cells which showed a lower PAR content. Since the repair of DNA strand breaks and the

formation of PAR require energy, in the form of NAD+, the cellular NAD

+ content was measured

[249]. During the 8-fold isoproterenol treatment no reduction of the intracellular NAD+ content of

PBMCs could be detected, see Figure 4-2 A). This indicates that during this time period no significant

amount of DNA strand breaks occur, because DNA strand breaks activate PARP1. Activated PARP1

uses NAD+ as substrate for the PAR formation. This leads to the depletion of the cellular NAD

+

content. Poly(ADP-ribosyl)ation is the most important catabolic pathway of NAD+ in mammalian cells

[249]. However, during this early time period, during the isoproterenol treatment, no formation of

DNA strand breaks could be observed [466]. But 24 h after the first isoproterenol dosage a treatment

dependent decrease of the cellular NAD+ could be observed, see Figure 4-2 B). Cells lysed directly

after the isolation without incubation were used as a negative control. These cells showed a slightly

but not significant higher NAD+ content than PBMCs which were lysed after an incubation of 24 h.

The 8-fold isoproterenol treatment reduced the NAD+ content of PBMCs by approximately 30%.

Hence, in a time frame between 3.5 h and 24 h after the first isoproterenol administration, the cause for

the NAD+ depletion must occur. The FADU experiments showed 6 h after the first isoproterenol

treatment DNA strand breaks evolve. At this point in time also a reduction of the intracellular NAD+

content could be detected, see Figure 4-2 A). The observed DNA strand breaks explain the depletion

of the cellular NAD+

pools.

Discussion

105

5.2 Repeated isoproterenol treatment induced senescence

like phenotype

An emerging body of evidence demonstrates that psychological stress can affect the immune system.

Many results showed that age-associated changes of the immune system can be exacerbated by

psychological stress. Epidemiological studies have shown that stress increases the expression of

inflammatory markers (cytokines), the susceptibility to virus infections and reactivation rate of latent

virus infections [16, 403, 467-469]. Many of these detrimental effects could be correlated with a

senescence phenotype of immune cells. In particular T cells might have a senescence phenotype.

Senescence could be observed in human CD8 as well as in CD4 T cells [470]. T cells which have

reached replicative senescence in cell culture could still retain normal functions [471, 472].

Senescence of T cells can be induced by an inflammatory environment. For instance, the inflammatory

cytokines TNFα and IFNγ can induce senescence in CD8 T cells [473]. Also a shortening of telomeres

could be observed in T cells [470, 474]. Senescent T cells also loss the expression of the surface

protein CD28, a co-stimulatory protein essential for optimal T cell activation [475]. Beside T cells,

late memory B cells showed characteristics of cellular senescence, like the expression of SASP and

p16 [476, 477]. A senescence phenotype can also be induced in natural killer cells by DNA damage

response signaling. Induced by the activation of CD158d which is associated with the expression of

SASP [478]. The telomeres of PBMCs isolated from PTSD patients were shortened compared to

healthy age matched controls. Also some genes which are important in DNA damage repair and cell

cycle control were dysregulated in these PTSD patients (Judy Salzwedel, personal communication).

Telomere shortening or the accumulation of DNA strand breaks that induce a long-lasting DNA

damage response are cardinal markers for replicative senescence [479, 480]. Besides these correlations

between chronic stress and senescence, a recent mice study demonstrated that isoproterenol could

induce a senescence like phenotype in mouse cardiomyocytes [416]. Long-term (7 day) subcutaneous

injections of isoproterenol were used in a model for the induction of pathological induced cardiac

hypertrophy. The protein levels of p16, p21 and p53 were significantly higher in the cardiomyocytes

of isoproterenol treated animals compared to control animals. Moreover, cultured neonatal

cardiomyocytes treated for 48 h with isoproterenol showed a senescence like phenotype. The cells

were positive for SA-β-GAL and showed an accumulation of lipofuscin [416]. We observed the

infliction of DNA damage after the isoproterenol treatment of PBMCs. Activation of the DNA damage

response might contribute to the induction of senescence. The question arose whether isoproterenol

would induce biomarkers of senescence in PBMCs. Depending on the cell type and the trigger of the

senescence it takes several days to induce a senescence phenotype in cells [335]. As mentioned above,

the before used experimental setup was not appropriate for the incubation of PBMCs longer than 24 h.

Because the viability of PBMCs incubated longer than 24 h in FCS free medium was too low.

Discussion

106

Therefore, different cell culture media were tested to increase the cell viability. The highest cell

viability of PBMCs were observed in the TexMACS cell culture medium [417]. At the beginning of

the incubation, cell viability was approximately 85%, see appendix Figure 12-8 A). After 24 h of

incubation, cell viability slightly decreased to approximately 78%. Also no apoptotic PARP1 cleavage

could be detected, see appendix Figure 12-9. Afterwards, cell viability was stable and no further

significant cell loss could be observed until 120 h of incubation, see appendix Figure 12-8 A). Also no

significant difference of the cell viability between untreated and 8-fold isoproterenol treated PBMCs

during the incubation time of 120 h could be observed, see appendix Figure 12-8 B). Prior to the

presented study, several additional experiments in our group indicated a correlation between the

repeated isoproterenol treatment and the induction of cellular senescence. For example, the 8-fold

isoproterenol treatment induced the expression of SA-β-GAL in PBMCs, especially in T cells [417].

The cell morphology of PBMCs changed to a phenotype associated with cellular senescence, cells

were enlarged and flattened [417]. Finally, the isoproterenol pretreatment inhibited the proliferation of

lymphocytes after the stimulation with PHA (Palombo and Grath, manuscript in preparation) [420].

Based on these findings the expression of 41 genes was analyzed by quantitative real-time PCR.

Therefore, custom made real-time PCR arrays were used. The investigated genes can be clustered into

8 groups: adrenergic signaling, cell cycle control, immune response, N-glycosylation, oxidative stress,

DNA repair and telomeric regulation. The selected genes showed a dysregulation in a previous

performed transcriptome study with PBMCs of PTSD patients (Judy Salzwedel, personal

communication). The mRNA expression profile of treated PBMCs was normalized to the mRNA

expression profile of untreated PBMCs. In the view of the fact that PBMCs are a heterogeneous cell

population and the heterogeneity between different donors, the regulation threshold was set to a 2–fold

change. After the 4-fold isoproterenol treatment 3 genes, VCAN, CCND1 and BRCA2 exceeded the

regulation threshold and were significantly regulated, see Figure 4-6. Versican (VCAN) is a matrix

proteoglycan and belongs to the lectican protein family. It is involved in cellular adhesion, migration

and proliferation. The expression of VCAN positively correlates with the cellular inflammatory

response [481]. It is also important in the activation and adhesion of T cells [482]. The observed

cellular adhesion and the morphological changes after the isoproterenol treatment of the PBMCs are in

accordance with the VCAN up-regulation. Moreover, it was demonstrated that versican protects cells

from oxidative stress-induced apoptosis [483]. The expression of VCAN is regulated by various

cytokines, transcription factors and signaling pathways such as TGFβ, IL-1α, IL-1β, CREB, p53 and

the PI3K/PKB signaling pathway, which are also regulated by β-adrenergic signaling [484, 485].

However, the long-acting β2-adrenergic agonists, formoterol and salmeterol alone did not affect the

expression of VCAN [486, 487]. Besides VCAN also BRCA2 exceeded the regulation threshold but the

difference between the 4-fold isoproterenol treated PBMCs and the untreated control PBMCs was not

significant. BRCA2 is an important DNA repair protein involved the repair of DNA double-strand

breaks by homologous recombination [488, 489]. BRCA2 is a tumor suppressor gene with very high

Discussion

107

importance with regards to breast- or ovarian cancer [490]. The most interesting result with regards of

the cell cycle control is the down-regulation of CCNA1 (cyclin D1) after the 4-fold isoproterenol

treatment. Cyclin D1 is a key regulator of the cell cycle. It is a sensor and integrator of extra cellular

stimuli in the G1 phase of the cell cycle. Cyclin D1 is essential for the progression of the G1 phase

into the S phase, mediated by binding to CDKs, histone acetylase and histone deacetylase. PBMCs are

non-proliferating (quiescent) cells without an external stimulation. However, they express the cyclin

D1 protein and mRNA with approximately the same quantity as PHA-stimulated lymphocytes and

malignant lymphocytes [491, 492]. The cyclin D1/CDK4/6 complex, together with a complex of

cyclin E/CDK2, phosphorylates the RB protein which inhibits its function and induces the release of

E2F transcription factors. E2F transcription factors are essential for the transcription of genes needed

for entering the S phase of the cell cycle. Hence, a down-regulation of CCNA1 induces a cell cycle

arrest at the restriction point in the G1 phase. The down-regulation of the expression of CCNA1 by β2-

adrenergic receptor signaling was also shown for other cell types. The treatment of human airway

smooth muscle cells with the β2-AR agonist salbutamol reduces the expression cyclin D1 and blocks

the cell cycle at the restriction point [493]. The expression of cyclin D1 is also inhibited by cAMP in

fibroblast cell lines and induces a cell cycle arrest in the G1 phase [494]. The differences in the gene

expression pattern between the 4-fold and the 8-fold isoproterenol treatment are low, see Figure 4-6. In

both cases only VCAN and CCNA1 showed a significant regulation that exceeded the threshold.

Interestingly, after the 8-fold isoproterenol treatment an increase of the CDKN2A (p16) expression

could be observed. However, this up-regulation was not significant. p16 is an important cell CDK

inhibitor which specifically inhibits the progression from the G1 phase into the S phase. The p16

expression in peripheral blood T cells is a biomarker for aging and replicative senescence [424, 495,

496]. p16 binds to CDK4 as well as to CDK6 which blocks the interactions with cyclin D. Hence, RB

is not phosphorylated by the complex of cyclinD1/CDK4/6 and is retained in its active, transcription

repressing form. Taking together, the results of the gene expression analysis showed that the

expression of CCND1 is influenced by the repeated isoproterenol treatment and the expression of

CDKN2A might be influenced by the repeated isoproterenol treatment. The down-regulation of the

CCND1 expression and a higher expression of CDKN2A might indicate a cell cycle arrest at the G1

phase of the cell cycle restriction point. Besides the p16/RB pathway also the p53/p21 pathway is

important for the induction of senescence. The expression of p21 was measured by two different

primer pairs (CDKN1A and CDKN1A * (Biorad and Biomol)). However, the isoproterenol treatment

induced no changes in the CDKN1A expression pattern under this culture conditions, see Figure 4-6.

Since expression of CDKN2A might be up-regulated after the 8-fold isoproterenol treatment, the p16

protein level was measured in PBMCs treated with one, four or eight doses of isoproterenol after 24 h

and 48 h. The results showed no significant increase of the p16 protein levels, see Figure 4-7. The p16

protein levels were slightly higher after the isoproterenol treatment. In general, senescent cells cannot

be identified by a single marker instead of a combination of several senescence markers must be used

Discussion

108

for the identification of senescent cells. Today, many such senescence markers are known, see section

1.4. However, a given cell type will not express all of them, the detectable senescence markers are

dependent on the cell type, the cause of senescence and the time frame which has passed after the

senescence induction. Two major molecular senescence markers are the expression of p16 and p21.

Both are cell cycle inhibitors and induce a cell cycle rest, a cardinal feature of senescent cells. In the

case of immune cells, p16 is considered as possible biomarker of aging and senescence marker.

However, immune cells express different p16 protein levels. T cells are the cell type of PBMCs with

the highest expression of p16 compared to the other cell types B cells, monocytes and naturel killer

cells. They express only 30% or less of the p16 protein level of T cells [424]. A senescence like

phenotype is observed for T-helper cells as well as for cytotoxic T cells. The other cell types of

PBMCs natural killer cells, B cells, and monocytes, show no clear indication of a senescence like

phenotype. Either only a sub-cell population shows a senescence like phenotype, in the case of B cells

the late memory B cells [477]. Or only special stimuli induce a senescence like phenotype, like the

activation of CD158d in the case of natural killer cells [478]. Therefore, further studies should be

performed with purified sub-cell populations. The measurement of senescence markers in a specific

cell type should give clearer results compared to the measurements in a cell mixture such as PBMCs.

For example, the express of SA-β-GAL in T cells showed a clear treatment-dependency [417]. T cells

are the most interesting cell type in PBMCs with regards to senescence, because T cells are part of the

adaptive immune response. After T cell activation via the T cell receptor and co-stimulatory molecules

by the specific antigen, presented by antigen-presenting cells (APCs), T cells start to proliferate and

undergo clonal expansion [467]. Moreover, additional senescent markers which are specific for T cells

could be analyzed, like a down-regulation of CD28 and a lower expression of the heat shock protein

HSP70 [475, 497, 498]. Taken together, the results give the indications that the isoproterenol might

induce a senescence like phenotype in PBMCs, at least in T cells. Although an increase of the p16 or

p21 protein expression, two strong senescence markers, was not observed. The reason for that could be

the short incubation times before the measurements. Since the mouse study which used isoproterenol

to induce senescence in cardiomyocytes showed an increase of SA-β-GAL within 2 days of the

isoproterenol treatment. In contrast, the higher expression of p16 and p21 was observed 7 days after

the first isoproterenol infusion [416]. T cells could be used to investigate longer treatments with

isoproterenol, because normal human T cells can be cultured ex-vivo for at least 25 population

doublings using a constant expose to IL-2 [499-501]. A IL-2 treatment has the disadvantage that it

may influences the β2-AR density at the T cell surface [183]. In this study replicative senescence does

not play an important role. Because blood was draw from young subjects and cells were not stimulated

to proliferate during the incubation this could explain the lack of p16 upregulation in our experiments.

Discussion

109

5.3 Degradation of isoproterenol under cell culture

conditions

In our studies, a treatment protocol with repeated administrations of isoproterenol was used. This

treatment protocol was established as a model system to simulate the repeated release of stress

hormones during chronic stress. Isoproterenol is a synthetic catecholamine which is often used in

science because of its selectivity for β-ARs. The natural catecholamines epinephrine and

norepinephrine bind to α- and to β-ARs. Although isoproterenol is often used in cell culture models for

the investigation of signaling processes, its metabolism in cell lines in the cell culture is not known to

our knowledge. However, studies in animals and humans have shown that the isoproterenol

degradation depends strongly on the administration route [211-213]. Catecholamines circulating in the

blood system are metabolized mainly in the liver. Most studies measured a plasma half-life of natural

catecholamines of only several minutes in humans. However, the half-life which was found for

isoproterenol ranges from several minutes to hours [205, 208, 214]. The two most important enzymes

of the catecholamine metabolism are MAO and COMT [160]. Isoproterenol is no substrate for MAO

because of its isopropyl group at the nitrogen atom of the amine [201]. PBMCs express MAO, COMT,

uptake enzymes, and storage vesicles for catecholamines [169]. Therefore, they might be also able to

metabolize isoproterenol. Moreover, beside the enzymatic degradation of isoproterenol, it undergoes

chemical degradation processes [232-235]. Therefore, it was expected that the isoproterenol in the cell

culture medium was degraded within a 30 min. At least, a timeframe of 1 h should be enough for the

breakdown of the isoproterenol and the recycling of the β2-AR. As the β2-AR is stimulated by

extracellular ligands, the main interest was to determine the isoproterenol concentration in the cell

culture medium. Additional, we were interested in the formation of isoprenochrome an oxidation

product which is known to be cytotoxic [221, 222]. In the two studies that were performed in parallel,

we used two different cell culture media in otherwise identical conditions. The chemical composition

of cell culture media and supplements like FCS and serum albumin can influence signaling processes

and the stability of chemicals like isoproterenol [207, 418, 419, 427, 502, 503]. Therefore, the stability

of isoproterenol, the formation of isoprenochrome and the influence of the cell culture media were

investigated. Therefore, a HPLC protocol for the measurement of catecholamines and aminochromes

in blood was adopted. This allowed the detection and relative quantification of isoproterenol and

isoprenochrome in an easy way. Only the cells and suspended particles had to be removed by

centrifugation to avoid the clogging of the HPLC. No purification steps were needed that could

influence the recovery of the substances. Also the quick procedure avoids the degradation of

isoproterenol or isoprenochrome. Hence, it was possible to investigate the concentration of both

substances in “real time”. Samples were taken and analyzed every 30 min, although the retention times

were lower than 10 min, because to have enough time to complete the treatment and the sampling.

Discussion

110

And the cell culture media RPMI-1640 with FCS and the TexMACS contained substances that had

high retention times, about 20 till 25 min. These substances could cause ghost-peaks which could

overlay with the isoproterenol and isoprenochrome peaks of the next run. Therefore, the time was used

to elute these substances before the next run was started. It was possible to detect isoproterenol with

the diode array detector and with the fluorescence detector. A combination of both detectors increases

the reliability and the sensitivity of the measurements. The absorbance detector is less sensitive and

less specific than the fluorescence detector. However, the absorbance detector allows after the

detection of substance at a specific wavelength, in this case 280 nm for isoproterenol and 490 nm for

isoprenochrome, an absorbance scan from a wavelength of 200 nm till 900 nm of the substance peak.

The absorbance spectrum was used for the identification of the isoproterenol or isoprenochrome peak

in the chromatogram by comparing the absorbance spectra with spectra in literature [504]. Additional,

an absorbance spectrum of pure isoproterenol was recorded in PBS buffer and HPLC eluent by a

spectrometer and used for comparison. Moreover, the recording of the absorbance spectra of each peak

allowed the software (ChemStation, Agilent) to calculate the purity of a chromatographic peak and to

project a 3D plot. The calculated purity as well as the 3D plot showed that the chromatography peaks

of isoproterenol and isoprenochrome were pure. There was no additional substance peak with an

absorbance maxima of 280 nm or 490 nm and an equal or similar retention time that could overlay

either with the isoproterenol or isoprenochrome peak. The fluorescence detector has a higher

specificity and sensitivity compared with the absorbance detector. However, it was not possible to

obtain a fluorescence spectrum of isoprenochrome by attempting to calibrate the detector for

isoprenochrome according to the manufacturer´s handbook. The broad absorbance peak at 490 nm of

isoprenochrome could be an explanation. In the literature no fluorescence spectrum of isoprenochrome

could be found. Hence, isoprenochrome could only be detected by the diode array detector. For both

detectors a linear correlation between the peak area of the isoproterenol peaks and the dissolved

isoproterenol concentration could be observed, see Figure 4-8 and Figure 4-10. A tailing effect could

be observed at higher isoproterenol concentrations that is an indication for an overload of the column.

However, these concentrations were not reached during the experiments and there was no interference

with adjacent peaks. Therefore, this was accepted. Also a negative correlation of the isoproterenol

peak area and an increase of the NaIO4 concentration could be observed. And at the same time a

positive correlation between the isoprenochrome peak area and the NaIO4 concentration could be seen.

The dilution series of isoproterenol prepared either in RPMI-1640 cell culture medium w/o FCS,

RPMI-1640 cell culture medium with FCS or TexMACS showed the same slopes and y-interceptions.

It is well known that isoproterenol binds to plasma proteins [206, 207]. The identical slopes and y-

interceptions indicated no differences in the isoproterenol concentrations. However, this did not

exclude the binding of isoproterenol to proteins. Since the binding of isoproterenol to a protein could

be reversed by the HPLC eluent during the separation. If isoproterenol is bound under cell culture

conditions by proteins which are part of the FCS or the TexMACS cell culture medium should be

Discussion

111

analyzed in the future. The binding to proteins could influence the degradation and oxidation

processes of catecholamines [234]. Therefore, isoproterenol could be dissolved in the respective

medium and the proteins could be removed, for example by ultrafiltration before the HPLC run. If

isoproterenol is bound by proteins, the isoproterenol concentration should be lower in the cell culture

medium after filtration of proteins. Also the chemical composition of the solvent influences the

isoproterenol concentration. Isoproterenol, dissolved in phosphate buffer or water, is stable when it is

stored at 4 °C and protected from light [200]. However, different metal cations and enzymes can

catalyze the oxidation of catecholamines [232-235]. Since cell culture media contain such factors, the

stability of isoproterenol has been tested in cell culture media at 4 °C. At 4 °C, no degradation of

isoproterenol was observed in each tested cell culture medium during a time span of 6 h. Hence,

freshly prepared stock solutions could be used for the interval treatment. In contrast, at 37 °C, a

decrease of the isoproterenol concentration could be observed during a time span of 6 h in all culture

media. The formation of isoprenochrome could be observed in all three cell culture media. The

isoprenochrome concentration in the TexMACS cell culture medium was only approximately a sixth

compared with the isoprenochrome concentration in both RPMI-1640 cell culture media. Indicating

that isoproterenol is most stable in the TexMACS cell culture medium. However, the degradation of

isoproterenol under cell culture conditions seemed to be slower compared to the degradation in

humans and animals. This was confirmed by the measurements of the isoproterenol stability after a

single administration of 10 µM to PBMCs. In contrast to the initial hypothesis, there was nearly no

degradation of isoproterenol detectable after 30 min of incubation. Also after 60 min only a small

decrease of the isoproterenol concentration could be observed. However, in both tested cell culture

media a linear decrease of the isoproterenol concentration could be observed. Linear regressions were

used to determine the degradation rates of isoproterenol. The following degradation rates were

determined, in RPMI-1640 cell culture medium w/o FCS 72.69 ± 8,23 𝑚𝐿𝑈

ℎ (or 1.6 ± 0.18

µ𝑚𝑜𝑙

𝑙

ℎ ), in

TexMACS cell culture medium 40.83 ± 6.04 𝑚𝐿𝑈

ℎ (or 0.87 ± 0.13

µ𝑚𝑜𝑙

𝑙

ℎ ). TexMACS do not contain

FCS, but it contains human serum albumin. Albumin is a major component of FCS and the main

component of plasma proteins [419]. Albumin or a unknown other substance of TexMACS could

increase the isoproterenol stability. This could be tested by incubating isoproterenol in RPMI-1640

cell culture medium that contains different concentrations of albumin. In contrast to the chemical

composition of the cell culture medium, the presents of PBMCs in the cell culture media had no

significant influence of the isoproterenol degradation, see appendix Figure 12-10. Therefore, the

degradation of isoproterenol must be due to chemical processes. It is well known that catecholamines

undergo different oxidation processes. Different metal ions catalyze the oxidation of catecholamines in

aqueous buffers at intermediate pH 6-8 [234]. The products of these oxidation processes are

aminochromes. They are chemical unstable and undergo further oxidation processes. The degradation

of isoproterenol is accompanied in both cell culture media with PBMCs or w/o PBMCs by the

Discussion

112

formation of isoprenochrome. The formation of isoprenochrome is induced during the incubation

because at the point in time 0 min no isoprenochrome could be detected. After 30 min at 37 °C, the

formation of isoprenochrome could be observed. The highest degradation rate of isoproterenol was

measured in the RPMI-1640 cell culture medium w/o FCS. This suggested the formation of

isoprenochrome was the fastest in the RPMI-1640 cell culture medium w/o FCS and the lowest

TexMACS cell culture medium. Indeed, the lower formation rate of isoprenochrome could be seen in

TexMACS cell culture medium. During the first 2.5 to 3 h the increase of isoprenochrome seems to be

linear with the time, in the RPMI-1640 cell culture medium w/o FCS. Then the concentration of

isoprenochrome was constant, indicating that isoprenochrome undergoes further reactions. The slow

degradation rates of isoproterenol indicated that isoproterenol will accumulate in the cell culture

medium during the repeated treatment. In fact, the accumulation of isoproterenol could be observed

for the 4-fold as well as for the 8-fold isoproterenol treatment in all tested culture media. During the 4-

fold isoproterenol treatment a stepwise increase could be detected for the TexMACS and the RPMI-

1640 cell culture medium with FCS. In contrast, in the RPMI-1640 cell culture medium w/o FCS a

small decrease of the isoproterenol concentration could be observed. After 3 h, about 19% of the total

administered isoproterenol dose had been degraded. The highest isoproterenol concentration could be

observed after the last treatment in all three cell culture media. Afterwards, a liner decrease of the

isoproterenol concentration could be observed. Linear regressions were used to determine the

degradation rats. The following degradation rates were determined, in RPMI-1640 cell culture medium

w/o FCS 230.7 ± 31.28 𝑚𝐿𝑈

ℎ (or 5 ± 0.68

µ𝑚𝑜𝑙

𝑙

ℎ ), in TexMACS cell culture medium

192.6 ± 24.01 𝑚𝐿𝑈

ℎ (or 3.92 ± 0.46

µ𝑚𝑜𝑙

𝑙

ℎ ), RPMI-1640 cell culture medium with FCS

168.2 ± 29.36 𝑚𝐿𝑈

ℎ (or 3.72 ± 0.65

µ𝑚𝑜𝑙

𝑙

ℎ ).The degradation rates after the 4-fold isoproterenol treatment

were faster compared with the 1-fold treatment. Again the formation of isoprenochrome could be

observed after 30 min of incubation in all three cell culture media. In TexMACS and in RPMI-1640

cell culture medium with FCS a slow increase could be observed. In contrast, in RPMI-1640 cell

culture medium w/o FCS a faster formation of isoprenochrome could be observed. The peak of the

isoprenochrome concentration could be observed 1 h after the maximum isoproterenol concentration.

Isoprenochrome undergoes also degradation in RPMI-1640 cell culture medium w/o FCS, as the

concentration that could be detected decreased after 4.5 h. This decrease could not be observed for the

other two other cell culture media. During the 8-fold isoproterenol treatment, a stronger overlay

between degradation processes of isoproterenol and increase of isoproterenol caused by additional

doses could be observed. This could be observed in particular for the RPMI-1640 cell culture medium

w/o FCS. After 2.5 h (administration of six doses) till 3.5 h (administration of 8 doses) no further

increase of the isoproterenol concentration could be detected. Hence, the degradation rate was about

880 𝑚𝐿𝑈

ℎ (or 20

µ𝑚𝑜𝑙

𝑙

ℎ ). As the isoproterenol degradation slowed down during time, there is no linear

Discussion

113

correlation between the isoproterenol concentration and the incubation time. Hence, a linear regression

cannot be used for the approximation of the degradation rate between 4 h and 8 h. In the two other cell

culture media a linear increase of the isoproterenol concentrations with additional doses could be

observed. The degradation of isoproterenol also appeared to be linear in the both cell culture media.

Again a linear regression was used to determine the isoproterenol degradation rates. In RPMI-1640

cell culture medium with FCS the degradation rate was in RPMI-1640 cell culture medium with FCS

440.1 ± 39.23 𝑚𝐿𝑈

ℎ (or 8.64 ± 0.77

µ𝑚𝑜𝑙

𝑙

ℎ ) and in TexMACS cell culture medium 216.5 ± 46.21

𝑚𝐿𝑈

ℎ (or

4.02 ± 0.86

µ𝑚𝑜𝑙

𝑙

ℎ . The formation of isoprenochrome could be observed again after 30 min of

incubation. The increase of the isoprenochrome concentration was the strongest in the RPMI-1640 cell

culture medium w/o FCS. The maximal concentration was reached 30 min after the last treatment.

Afterwards, a decrease of the isoprenochrome concentration indicates that also the isoprenochrome

undergoes degradation processes. In contrast to the 4-fold isoproterenol treatment, during the 8-fold

treatment in RPMI-1640 cell culture medium with FCS a decrease of the isoprenochrome

concentration could be observed. The peak concentration was reached after about 5 h of incubation. In

contrast, the isoprenochrome concentration in TexMACS cell culture medium was constant after

reaching the maximum concentration. The 4-fold and the 8-fold isoproterenol treatments were also

repeated without PBMCs. Again no significant differences between the experiments with and w/o cells

were observed. In all measurement series the fastest decrease of isoproterenol could be observed in the

RPMI-1640 cell culture medium w/o FCS. The slowest decrease could be observed in the TexMACS

cell culture medium. No statistical significant influence of PBMCs with regards on the isoproterenol

degradation could be observed. Nevertheless, this cannot be excluded because these effects can be

small and isoproterenol was administered in excess for a maximal stimulation. Such small effects can

be obscured by other influences, for example the variation caused by multiple dose administration

could be bigger than the effects caused by PBMCs. Moreover, the detectors could be not sensitive

enough to detect such small differences. In all measurement series, an increase of the isoproterenol

degradation rate could be observed with increased isoproterenol concentrations. Also the formation of

isoprenochrome increased with the number of administrations. Isoprenochrome is formed by oxidation

processes of isoproterenol, supporting the idea of a chemical degradation. Moreover, the oxidation of

isoproterenol to isoprenochrome contains several intermediates and is associated with the generation

of free radicals and ROS [233]. These intermediates are an unstable and undergo further oxidation.

These oxidations may be caused by various chemical species like ROS or free radicals but also by

other reaction intermediates like ortho-quinones or semiquinones [229, 231]. This might explain an

increased oxidation rate of isoproterenol during the repeated treatment. Intermediates such as o-

quinones, semiquinones or radicals that were formed after the first isoproterenol dose can increase the

oxidation rate of the following isoproterenol doses. Also the further oxidation of isoprenochrome to

melanin-like products could shift the reaction equilibrium and increase the oxidation rate of

Discussion

114

isoproterenol [228]. In addition to oxidation reactions, isoprenochrome undergoes also rearrangements

and other redox reactions. This might also explain the different isoproterenol degradation rates in the

different cell culture media. The chemical composition of a cell culture medium has an influence on

the lifetime of chemical compounds, especially with regards to ROS [505]. For instance, high

concentrations of dopamine (> 250 µM) induce cell death. Cell death is caused by H2O2 which is

formed during oxidation processes of dopamine in the cell culture medium [506]. It is well know, that

cell culture medium contains anti-oxidative substances, FCS increases the antioxidant capacity of a

cell culture medium [507]. Also albumin which is a compound of the TexMACS cell culture medium

acts as an antioxidant [426]. Isoproterenol has a high affinity for serum proteins [206, 207]. The

binding of isoproterenol to such proteins could reduce its reactivity and reduce the oxidation rate of it.

Such proteins can also bind metal ions and prevent that they undergo redox-reactions [427]. This could

also slow down the oxidation of isoproterenol because metal ions can oxidize catecholamines [226,

233]. The main goal was to determine the concentration of isoproterenol during the repeated treatment,

especially if isoproterenol was degraded between two administrations. The measurements of the

isoproterenol concentration showed that the degradation of isoproterenol is slower as initially thought.

No significant degradation between two doses during the 8-fold and during the 4-fold treatment could

be observed, at least in the beginning. Instead an accumulation of isoproterenol could be observed in

all cell culture media. As PBMCs could take up catecholamines and are able to store them in vesicles,

it would be interesting to measure also the intracellular isoproterenol concentration during and after

the treatment. Also the measurement of intracellular concentrations of isoprenochrome and further

metabolites would be interesting. In principle, the same protocol could be used but more sensitive

detectors would be needed. After centrifugation an adequate cell number could be lysed by the

addition of perchloric acid. The supernatant could be analyzed by LC-MS/MS or an HPLC equipped

with an electrochemical detector. The intracellular isoprenochrome concentration and the intracellular

concentration of other metabolites could give interesting information with regard to intracellular ROS

or free radicals. If isoprenochrome could be detected intracellular, it would be plausible to find

intracellular ROS or free radicals as well. However, the question arises how an accumulation of

isoproterenol could influence the PBMCs and how could this explain the influence of the cell culture

medium? It is known that long-term stimulation of cells with a β2-AR agonist induces a down-

regulation of the β2-AR. The long-term infusion (6 h) of isoproterenol in humans had a biphasic effect

on the β2-AR density on the cell surface of leukocytes. After 30 min, an up-regulation of the receptor

density at the cell surface could be observed. Afterwards, the receptor density was down-regulated

[179, 465]. In TexMACS cell culture medium the highest accumulation of isoproterenol could be

observed. These might lead to a stronger down-regulation of the β2-AR compared to the other culture

media. The reduced amount of receptors would also reduce the activity of intracellular signaling

pathways and the associated formation of ROS. The reduced ROS formation could lower the effects of

catecholamine, such as the formation of DNA damage. The high concentrations of isoproterenol

Discussion

115

caused by the accumulation during the repeated treatment seemed not to be responsible for the

observed effects in the two other studies. Already a single dose of 10 µM induced the maximum

cAMP response. However, this might be not true for the other signaling pathways of the β2-AR.

Therefore, an investigation of the other signaling pathways of the β2-AR must be taken into account

for a better understanding of the observed outcomes. Summing up the results of all three studies the

most plausible explanation for the observed isoproterenol induced effects is caused by two

mechanisms. On the one hand, isoproterenol induces signaling of the β-ARs. On the other hand, the

oxidation of isoproterenol and the formation of isoprenochrome seemed also to be involved in the

observed effects.

Conclusions and outlook

116

6 Conclusions and outlook

Our studies have been designed with regards to findings that chronic stress on the one hand can impair

immune cells and on the other hand induce DNA damage. Several cell and animal studies of chronic

stress showed a decrease of the genomic instability in stressed subjects [399-401]. Especially, several

studies with PBMCs of chronically stressed PTSD patients showed an accelerated aging, an

accumulation of DNA strand breaks and an impairment of the DNA repair in these cells [82, 83].

These patients have also elevated blood plasma catecholamine concentrations [57-61]. Besides that,

catecholamines can induce the formation of ROS which induce DNA damage in mouse as well as in

human cell lines [399-401]. Therefore, the hypothesis was raised that repeated stimulation of the β2-

AR by catecholamines can induce DNA strand breaks in human PBMCs. To test this hypothesis an ex

vivo model was established [405]. PBMCs of healthy donors were isolated and repeatedly treated with

isoproterenol to mimic the increased and repeated secretion of catecholamines during chronic stress.

The experiments showed that repeated isoproterenol treatment induced the formation of β2-AR-

dependent and β2-AR-independent formation of DNA strand breaks [421]. These DNA strand breaks

were only partially repaired after 24 h. The responsiveness of the cAMP/PKA signaling pathway

decreased during the repeated stimulation of the β2-AR. Additional, the formation of the DNA strand

breaks could only be partially inhibited by the β-blocker propranolol. Both findings are indications

that additional processes must be involved in the induction of DNA strand breaks. Moreover, no

formation of intracellular ROS induced by the repeated isoproterenol treatment could be detected. The

formation of DNA strand breaks was accompanied by a decrease of intracellular NAD+ as well as ATP

pools. The repeated isoproterenol treatment reduced the PARP1 activity as well as the PARP1 protein

level and increased the number of apoptotic cells. The PARP1 protein expression as well as the

apoptosis rate seems to be subject-dependent. However, further studies are needed which measure all

parameters in the same subject at different points in time to order the effects and to determine the

mechanisms. Furthermore, the PBMCs are a heterogeneous cell population, consisting out of different

cell types. Since the different cell types respond differently to the isoproterenol treatment and the

composition of PBMCs varies between subjects, also the composition of the PBMCs could be

responsible for the observed variations. The data in literature indicated that half-life of isoproterenol

strongly depends on the administration route and has a plasma half-life of several minutes [205, 206,

208, 214]. Therefore, the initial hypothesis was that the isoproterenol was degraded between two

isoproterenol administrations and the cells had enough time for resensation of the receptor. In contrast

to that, measurements of the isoproterenol concentration in cell culture media revealed an

accumulation of the isoproterenol in the cell culture media. The degradation rate of isoproterenol

increased with further applications. Moreover, the degradation rate was highly influenced by the

composition of the cell culture media. The decrease of the isoproterenol content was accompanied by


117

the formation of isoprenochrome. Isoprenochrome is formed by the oxidation of isoproterenol. During

this oxidation processes ROS and free radicals are formed. If the oxidation of isoproterenol,

intracellular as well as extracellular, is the cause for the DNA strand breaks must be investigated by

further studies. Taken together the results indicate that isoproterenol under cell culture conditions is

mainly degraded by chemical processes and the degradation is mainly influence by the cell culture

medium. The data in literature also indicate the long-term treatment of myocardia cells with

isoproterenol induce a senescence like phenotype [416]. Our results, like the expression of SA-β-GAL,

higher mRNA expression of CCND1 and VCAN, morphological changes of the cells and the

impairment of cells to proliferate after PHA indicate that the repeated isoproterenol treatment can

induce a senescence like phenotype in human PBMCs. Moreover, the results indicated that mainly

T cells are responsible for the observed senescence phenotype. Further studies with purified cell

populations are required to confirm this and for further mechanistic studies. Our studies give some

indications that the repeatedly treatment of PBMCs with isoproterenol induces the expression of stress

biomarkers. But we couldn’t clarify the underlying mechanisms. For this purpose, further studies are

required which measure all parameters in a single subject for correlations. Moreover, a higher

temporal resolution is required, because also the additional signaling pathways of the β2-AR must be

taken into account. The phosphorylation processes responsible for the signal transduction are only

transient. Hence, the signaling cascades must be investigated with a high temporal resolution, at least

with 5 min steps after each isoproterenol treatment. Further, investigations of the cellular senescence

should focus on the T cell subset. Depending on the outcome of these studies, specific inhibitors for

the phosphorylation cascades of the β2-AR, treatments with antioxidants and β-blocker may be

considered as adjuvant therapeutics to reduce the adverse effect of chronic stress on the immune

system.

Introduction

118

CHAPTER II

7 Introduction

7.1 DNA damage and DNA damage repair

The deoxyribonucleic acid (DNA) is one of the most, if not the most important macromolecule of a

living cell. Six different molecules build up the DNA macromolecule. The backbone of a DNA strand

is formed by 2-deoxyribose and phosphate groups. The 2-deoxyribose molecules are linked together

by phosphate groups, forming phosphodiester bonds between the 5´ carbon atom of one sugar

molecule and the 3´ carbon atom of the next sugar molecule. At the 1´ carbon atom of each 2-

deoxyribose sugar one nucleobase (adenine, thymine, cytosine or guanine) is linked via a N-glycosidic

bond. Two single DNA strands with an antiparallel orientation build up the DNA double helix [508].

As the DNA is the carrier of genomic information, its structural integrity and stability is vital for each

cell. Any non-physiological modification of the DNA (DNA damage) is, therefore, a harmful threat.

These DNA lesions can cause mutations if they are not repaired or the repair is faulty. Every day cells

suffer a large amount of DNA damage, see Table 4.

Damage type Lesions per

day per cell

oxidative damage 10000 [509, 510]

depurinations 10000 [511, 512]

depyrimidinations 600 [513, 514]

single-strand breaks 55000 [515]

double-strand breaks 10 [516]

alkylations 3000 [517]

deaminations 500 [518] Table 4: DNA damage per cell, per day.

DNA lesions can be induced either by exogenous or endogenous sources. Exogenous sources are

environmental elements or factors and can be either physical or chemical agents like ionizing

irradiations, ultraviolet light, natural occurring radioactive compounds, medical treatment [514, 519,

520]. A big variety of chemical substance can damage the DNA like chemotherapeutics, toxins and

chemical warfare agents [517, 521, 522]. Life style can provide different kinds of sources for

exogenous DNA damages. For instance, cigarette smoke contains various DNA damaging substances.

The diet may contain DNA damaging agents like polycyclic aromatic hydrocarbons and nitrosamines

Introduction

119

[514]. Besides the exogenous sources, various endogenous sources are also responsible for the

occurring of DNA damage. Moreover, endogenous sources of DNA damage are the major cause of

mutations in human tissues [523, 524]. In aqueous solutions the DNA undergoes spontaneous

chemical reactions like hydrolysis of nucleobases, creating AP (apurinic/apyrimidinic) sites or

deamination reactions (mainly the deamination of cytosine to uracil) [511, 512, 525]. Also cell

metabolism itself produces DNA damaging agents. The most prominent are ROS and reactive nitrogen

species (RNS) which are generated during oxidative respiration, redox cycling reactions, or cell

signaling processes [422, 451, 461, 526]. Also the immune system is involved in the generation of

ROS and RNS during inflammation processes [169, 527-529]. Further, endogenous alkylating agents

induce DNA damage [523]. Additional, lipid peroxidation products, estrogen- and cholesterol-

metabolites and reactive carbonyl species are able to induce DNA damage [520, 523]. Therefore, cells

must cope with DNA damage and have evolved the DDR [530]. The DDR is a complex, highly

controlled network of DNA damage detectors, signal transduction pathways for these DNA damage

and multiple DNA damage repair pathways for the restoration of the DNA [530, 531]. Important

physiological processes are regulated by the DDR and determine the fate of a cell. This either results

in survival and DNA damage repair, replicative senescence or various types of cell death [347, 532,

533]. Important proteins of DDR are ATM, ATR, DNA-dependent protein kinase (DNA-PKcs) and

PARP1 and PARP2 [517, 534-538]. In response to a DNA damage over 900 phosphorylation sites in

over 700 proteins can be engaged by protein kinases ATM and ATR [539]. And over 10000 ATP

molecules are needed for the signaling of one DNA double-strand break [520]. The DNA damage

signaling provides the basis for the activation of the DNA damage repair pathways. The great variety

of chemical und physical impairments which are induced by DNA lesions must be counteracted. In the

view of the wide spectrum of these DNA lesions, a highly developed network of DNA repair pathways

has been evolved in cells. The most important DNA repair pathways are the direct lesion reversal,

MMR, base excision repair (BER), nucleotide excision repair (NER), non-homologous end-joining

(NHEJ) and homologous recombination (HR), see Figure 7-1 [530, 540, 541].

Introduction

120

Figure 7-1: Overview of DNA lesions and DNA damage repair pathways. Human cells have six main DNA repair

pathway: direct lesion reversal, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), non-

homologous end joining (NHEJ) and homologous recombination (HR). Each repair pathway is specialized in the removal of

specific DNA lesions which can be inflicted by various sources.

7.2 DNA strand break detection

The high abundance of DNA damage and its threat to health makes it a highly interesting research

area. In a clinical perspective, DNA lesions may be the starting point in carcinogenesis and, therefore,

life-threatening. Otherwise, the majority of therapies to treat cancer are genotoxic agents that kill

cancer cells by inducing DNA damage. Besides the medical aspect, there is a general interest of the

public, authorities and companies in the genotoxic properties of chemical and biological agents. The

exact knowledge of the genotoxic properties of a substance are important for a correct risk assessment,

a harmless production and placing on the market of the substance, but also for a correct use and

disposal of the substance. Therefore, a number of methods for the detection, analysis and

quantification of DNA damage and DNA damage repair have been developed. For some DNA lesions

such as oxidative DNA damage, there is a broad range of methods available for a direct or indirect

detection. However, a highly sensitive, robust, economical and easy-to-implement method for

detection of DNA single- and double-strand breaks is needed. The most common methods for

detection of DNA strand breaks are briefly described below. Based on the used technology the assays

can be divided in molecular and fluorescence methods [542].

Introduction

121

Molecular methods 7.2.1

Polymerase chain reaction (PCR) and agarose gel electrophoreses can be used for the detection of

DNA strand breaks. Strand breaks lead to a blockage of the polymerase at the DNA damage side and

inhibit further amplification of the DNA. The molecular weight of the amplified DNA strand with

DNA strand break is lower compared with the undamaged DNA strand. The result is a reduced

quantity of the DNA template and a fragmentation pattern on the agarose gel [543]. Besides the

normal PCR, PCR-based methods have been developed for the detection and quantification of DNA

damage. Quantitative PCR (qPCR) can be used for detection and quantification of DNA strand breaks

and adducts in a specific gene or gene section [544, 545]. Also the expression of important DNA

repair proteins or proteins involved in DNA damage signaling such as Ku and XRCC1 are used for an

indirect detection of DNA damage [542]. The one important protein with regards to DNA damage is

the histone γH2AX. γH2AX gets phosphorylated at serin-139 after the infliction of DNA double-

strand breaks. The amount of the phosphorylated γH2AX correlates with the amount of DNA double-

strand breaks [546].

Fluorescence methods 7.2.2

Many of the methods used for detection of DNA strand breaks are based on the measurement of a

fluorescence signal. The most common used fluorescence based methods are the Halo assay, Terminal

deoxribonucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Fluorescence in situ

hybridization (FISH), Comet assay and fluorometric detection of alkaline DNA unwinding (FADU)

assay [543]. The TUNEL assay was developed for detection of apoptotic DNA fragmentation [547].

But also DNA fragmentation induced by toxic substances can be detected. TUNEL staining uses the

ability of the enzyme terminal deoxynucleotidyl transferase to incorporate labeled 2´-deoxyuridine, 5´-

triphosphate (dUTP) into the free 3`-hydroxy termini of DNA strand breaks [548]. FISH is a molecular

cytogenetic assay which uses fluorescent probes that are complementary to a specific chromosomal

region. Chromosomal aberrations can be detected [549, 550]. The Halo assay is a semi quantitative

method for the detection of DNA single- as well as double-strand breaks on a single cell level [551,

552]. The Comet assay, also called single-cell gel electrophoresis, is the “gold standard” for the

detection of DNA single- or double-strand breaks [553, 554]. The assay principle is based on an

electrophoretic separation of DNA fragments in an agarose gel. The migration of DNA fragments, if

the rest of the electrophoretic parameters are constant, depends on the molecular size of the DNA

fragments. Test samples must be prepared as single cell suspension. Cells are embedded in low

melting agarose. Cells were directly lysed or incubated to allow DNA repair for a defined time and

lysed afterwards. The DNA is unwounded under alkaline conditions and an electrophoresis is

performed. DNA fragments migrate towards the anode, forming a comet-like image when viewed by

Introduction

122

fluorescence imaging after staining with a fluorescence dye. The head is formed by the nuclear region

and the tail by DNA fragments. Although the assay principle seems to be simple, various parameters

can influence the results of a Comet assay such as concentration of the agarose, the pH, temperature,

duration of alkaline DNA unwinding, pH of the unwinding buffer, temperature used for the unwinding

and the electrophoretic parameter (voltage, current direct at the sample not measured at the power

supply) [555, 556]. Besides the technical variations, there are also various methods for analyzing the

extant of DNA damage, such as visual scoring, percentage of DNA in the tail (%T), tail length, and

tail moment [557-560]. These variations make the interpretation of results of different laboratories

difficult. Today there are modified Comet assays for the detection of specific DNA lesions. The most

common is the adaption for the detection of oxidative DNA damage, but also assay modifications for

the detection of other DNA lesions are established [561-564]. Several inter-laboratory studies which

have been performed to validate the Comet assay for its reproducibility, showed a high variability

between these laboratories. The first inter-laboratory trail to attempt a standardization of the Comet

assay was performed by the European Standards Committee on Oxidative DNA Damage (ESCOOD).

The goal of one of their studies was to establish the background level of oxidative base damage in

human lymphocyte. Therefore, the Comet assay and HPLC combined with electrochemical detection

were used. The HPLC measured values for oxidative base lesions were about 6-12-fold higher

compared with the values detected by the Comet assay [562]. Moreover, only half of the laboratories

detected a dose response in standardized HeLa cell samples treated with a photosensitizer and light

[561]. The next attempt was an inter-laboratory validation study performed by the European Comet

Assay Validation Group (ECVAG). The study was conducted in 12 laboratories showed overt

differences in the reported DNA damage in standardized samples. All laboratories detected a dose-

response relationship, but the coefficient of variation of the reported DNA damage was 47%.

Normalization by a calibration curve prepared in each laboratory reduced the coefficient of variation

to 28% [558]. To solve such inter-laboratory variability Azqueta et al. and Ersson e. al. demonstrated

the importance of standardized protocols and equipment for the reproducibility of the Comet assay

[556, 565]. Based on these experiences, the Japanese Center for the Validation of Alternative Methods

(JaCVAM) started a new international validation study in 2006 [566]. The objectives of the study were

to demonstrate acceptable intra- and inter-laboratory reproducibility and to demonstrate the ability of

the Comet assay to identify reliable genotoxic chemicals in rodents. The ultimate goal was to establish

an Organization for Economic Co-operation and Development (OECD) guideline for the Comet assay

[566]. Moreover, the performance of the Comet assay was compared with the rat liver unscheduled

DNA synthesis (UDS) assay. Therefore, the genotoxic features of 40 reference chemicals were tested

in rats by the Comet assay [567]. In a pre-validation study a standardized protocol of the Comet assay

was created and reproducibility of the Comet assay was evaluated in five lead laboratories [568]. In

the main validation the 40 selected chemicals were tested in a blind study in 14 laboratories with the

standardized protocol. The results of the validation showed that the Comet assay fulfills the main task,

Introduction

123

the detection of genotoxic and carcinogenic chemicals that induce DNA damage. The results also

showed that the Comet assay could be at least equal to or maybe more sensitive in the detection of

genotoxic carcinogens than the UDS assay [569]. The JaCVAM study finally lead to the release of the

OECD guideline “guideline for the testing of chemicals In vivo mammalian alkaline comet assay”

(OECD/OCDE 489). However, the guideline also mentioned drawbacks of the current Comet assay

protocol such as: the Comet assay is not suitable for the detection of DNA damage in germ cells,

cross-link DNA damage cannot be detected, the protocol is only validated for rodent and only for liver

and stomach tissues, methodological aspects and experience of the experimenter have a critical impact

on reliability and reproducibility on the result, based on the present data, no cytotoxicity pre-test was

recommended [570]. Based on the major drawbacks of the Comet assay, the influence of the

experimenter on the results, the difficulties in intra- and inter-laboratory reproducibility and the time-

consumption of the Comet assay an automated version of the FADU assay was developed in our lab

[571].

7.3 Automated fluorometric detection of alkaline DNA

unwinding (FADU) assay

The FADU method was original described in 1981 by Birmboim and Jevcak [572]. The method is

based on the principle that the ends of DNA strands are starting points for the unwinding of the DNA

double helix into single stranded DNA. Such “ends” can either be natural, like chromosome ends,

replication forks or can be caused by DNA damaging agents, like X-rays or chemical agents. The

unwinding of the DNA is depending on the pH, temperature, and time. It is detected with the help of

fluorescence dyes that binds to double-stranded DNA. The more double-stranded DNA is present in a

sample, the higher the fluorescence signal is. DNA single- and double-strand breaks reduce the

fluorescence signal, because they are additional starting points for the unwinding of the DNA. The

amount of unwound DNA in a defined period of time depends on the amount of DNA strand breaks.

Hence, the fluorescence signal is inversely proportional to the amount of DNA strand breaks. Moreno-

Villanueva et al. introduced an automated form of the FADU assay, based on a liquid handling device

[571, 573]. This allows an accurate controlling of all important parameter and leads to an increase in

the reproducibility, sensitivity, and reduces the duration of the assay. The main advantages of the

automated FADU assay compared with the Comet assay are, all critical assay steps are automized and

the FADU assay takes only 174 min, while the Comet assay takes up to 715 min, see Figure 7-2 [574].

Furthermore, SYBR Green I is used for the detection of double-stranded DNA. In general, the

automated FADU assay consists of the following steps: first, cells are harvested and prepared for the

assay. Second, cells are treated with the test compound for a desired period of time. Optionally, the

chemical agent can be removed and cells can be cultured for a desired period of time. This allows cells

Introduction

124

to repair DNA damage. Third, cells are suspended in suspension buffer and transferred into the assay

plate. Fourth, cells are lysed with high concentrations of urea at 0 °C. This immediately inhibits all

DNA repair processes and denatures the chromatin. Fifth, lysates are treated with an alkaline solution,

which induces the unwinding of the DNA. This is a critical step, because the alkaline solution is added

on top of the lysate, in such a way that a second layer is formed. The alkaline solution only diffuses

into the lysate to prevent shearing forces that could induce artificial DNA strand breaks. Sixth, the

unwinding is stopped by the addition of neutralization buffer. Seventh, SYBR Green I is added and

mixed with the lysates. Eight, the fluorescence signal is measured at an excitation wavelength of

492 nm and an emission wavelength of 520 nm.

Figure 7-2: Workflow of the automated FADU assay. Steps in the red bracket are automated by the FADU robot.

For the interpretation of the data the following sample types are needed: T0, P0 and PX.

T0 samples: cells for T0 samples are untreated. These cells have only endogenous DNA strand breaks.

T0 samples are treated with neutralization buffer before the lysates are treated with alkaline solution.

Hence, the critical pH that is needed to induce the unwinding of the DNA is not reached. The DNA

double helix remains intact. Therefore, T0 samples represent the total amount of double-stranded DNA

of a cell type at the lysis.

P0 samples: cells for P0 samples are untreated. Hence, these cells have no artificial DNA strand breaks.

Unlike T0 samples, the cell lysates are treated with alkaline solution to induce unwinding of the DNA.

After this process, the lysates are treated with neutralization buffer to stop the unwinding. Hence, the

unwinding of the DNA double strands takes place at DNA sites that are accessible under physiological

conditions (chromosome ends, replication forks, transcription sides, etc.). The ratio between T0 and P0

represents the amount of single stranded DNA under physiological conditions.


125

PX samples: cells for PX samples are treated by a chemical test compound or by irradiation. Depending

on the test compound, this treatment can induce exogenous DNA strand breaks. The ratio between P0

and PX represents the extent of the induced DNA strand breaks. Based on the automated FADU assay

developed by the Bürkle laboratory, a commercially available version should be developed [571].

Moreover, the new FADU platform should be used in the parallel conducted studies with isoproterenol

to measure DNA strand breaks in human PBMCs.

8 Material and Methods

8.1 Material

Chemicals 8.1.1

Substance Supplier

4-nitrophenol Sigma-Aldrich, Steinheim, Germany

8-hydroxyquinoline Sigma-Aldrich, Steinheim, Germany

CasyClean OMNI Life Science GmbH, Bremen, Germany

CasyTon OMNI Life Science GmbH, Bremen, Germany

cyclohexyl-diaminetetraacetate Sigma-Aldrich, Steinheim, Germany

D-(+)-glucose-monohydrate Merck, Darmstadt, Germany

DMEM Gibco Life Technologies, Karlsruhe, Germany

DMSO Merck, Darmstadt, Germany

DTT Sigma-Aldrich, Steinheim, Germany

EDTA Sigma-Aldrich, Steinheim, Germany

ethanol pa VWR, Darmstadt, Germany

etoposide Sigma-Aldrich, Steinheim, Germany

eugenol Sigma-Aldrich, Steinheim, Germany

FCS Biochrome, Berlin, Germany

HCl 37% Riedel-de Haen, Seelze, Germany

menthol Sigma-Aldrich, Steinheim, Germany

MTT Sigma-Aldrich, Steinheim, Germany

myo-inositol Sigma-Aldrich, Steinheim, Germany

PBS Biochrome, Berlin, Germany

penicillin/streptomycin (5000 units/ml) Gibco Life Technologies, Karlsruhe, Germany

p-nitrophenol Sigma-Aldrich, Steinheim, Germany

potassium dihydrogen phosphate Riedel-de Haen, Seelze, Germany

RPMI-1640 Gibco Life Technologies, Karlsruhe, Germany

saccharin Sigma-Aldrich, Steinheim, Germany

SDS Sigma-Aldrich, Steinheim, Germany


126

sodium chloride Carl Roth, Karlsruhe, Germany

sodium hydroxide Merck, Darmstadt, Germany

sodium-deoxycholat Merck, Darmstadt, Germany

SYBER Green I Sigma-Aldrich, Steinheim, Germany

SYBR Green I Invitrogen, Karlsruhe, Germany

Tris-HCl Sigma-Aldrich, Steinheim, Germany

Trypan blue Sigma-Aldrich, Steinheim, Germany

trypsin-EDTA Gibco Life Technologies, Karlsruhe, Germany

urea Carl Roth, Karlsruhe, Germany

β-mercaptoethanol Merck, Darmstadt, Germany

Laboratory equipment 8.1.2

Object Type Supplier

384-Deep well microplates 384er Deep-well BRAND

96-Deep well microplates 96er Deep-well Greiner Bio-One

benchtop centrifuge Biofuge pico Heraeus Instruments

benchtop centrifuge Heraeus Fresco 17 Thermo Scientific,

Schwerte, Germany

benchtop centrifuge 5810 R Eppendorf

benchtop centrifuge Pico17 Hereaus

biological safety cabinet S2 HeraSafe Heraeus Instruments

biological safety cabinet S2 Lamin Air HB 2448 Heraeus

cell counter Casy CellCounter TT Innovatis

cell culture microscope Axiovert40C Zeiss

centrifuge 5810R Eppendorf

dosimeter UNIDOSE PTW

FADU-assay 96-well plate 96er Deep-well Greiner Bio-One

fluorescence reader FL600 Bio-TEK

fridge Premium Liebherr


hemocytometer Casy Innovartis

ice maker AF206 Scotsman

incubator Hera Cell 240 Heraeus Instruments

incubator Hera Cell Heraeus Instruments

magnetic stirrers IKAM Häberle Labortechnik

magnetic stirrers MR3001K Heidolph

micro clear platte 96-well for TOXXs Analyzer Greiner Bio-One

micro platte black bottom for TOXXs Analyzer Greiner Bio-One

micro scales CP2202S Sartorius

micro scales CP225D Sartorius

microscope Leitz DK IL Leica

MilliQ Reference A+ Millipore

minisaker Duomax 1030 Heidolph

minisaker MTS4 IKA


127

pipetboy Pipetboy Comfort IBS Integra Biosiences







pipetting head for

TOXXs Analyzer

R96/250 S/N CyBio AG

pipetting robot Genesis RSP 100 Tecan

pipetting robot Genesis RSP 150 Tecan

printer C3760dn Dell

scale AG 204 Delta Range Mettler

scale PM2000 Mettler

sonicater Sonorex Super RK102H Bandelin

sonicater TK52 Bandelin

thermostat Lauda cooler R204 Lauda cooler

thermostat for

TOXXs Analyzer

PelTherm 3T GmbH & Co. KG

thermostat Ecoline RE204 Lauda

TOXXs Analyzer

TOXXs Analyze

(based on the CyBio Felix)

Cetics GmbH/ Analytik

Jena AG

vortexer Vortex-Genie 2 Bender & Hobein AG

water bath 1083 GFL

water bath 1002 GFL

X-ray system XRAD 225IX PXI PRECISION X-

RAY

Consumables 8.1.3

Product Supplier

384-well microplate for cAMP assay Perkinelmer

Casy cup

OMNI Life Science GmbH, Bremen,

Germany

cell culture 96-well microplate Corning, Schiphol-Rijk, Netherlands




cryovials Corning, Schiphol-Rijk, Netherlands




gloves (Latex) MaiMed, Neuenkirchen, Germany

gloves (Nitril) VWR, Darmstadt, Germany




128




reaction vessel (SafeSeal 2 ml) Sarstedt, Nürnbrecht, Germany

Safety-Multifly-canula G21/0.8 mm Sarstedt, Nürnbrecht, Germany




tips long (200 µl) VWR, Darmstadt, Germany

Buffers and solutions 8.1.4

FADU suspension buffer

0.25 M myo-inositol

10 mM sodium phosphate


add MilliQ water

adjust pH (7.4)

FADU neutralization buffer

1 M D-(+)-glucose-monohydrate

14 mM β-mercapthoethanol

add MilliQ water

"modified" FADU neutralization buffer

1 M D-(+)-glucose-monohydrate

7 mM DTT

add MilliQ water

FADU lysis buffer

9 M urea

10 mM sodium hydroxide

2.5 mM cyclohexyl-diaminetetraacetate

0.1% (w/v) SDS

add MilliQ water

FADU alkaline unwinding buffer

42.5% FADU lysis buffer

0.2 M sodium hydroxide

add MilliQ water

PBS (pH 7.4)


2.7 mM potassium hydrogen phosphate

8.1 mM disodium hydrogen phosphate


129

1.8 mM potassium dihydrogen phosphate

add MilliQ water

MTT solution (MTT assay)

5 mg/ml MTT

add PBS

MTT solubilisation solution (MTT assay)

10% (w/v) SDS

10 mM HCl

Cell lines and cell culture reagents 8.1.5

Cell line Basal medium Supplements

Jurkat RPMI-1640

10% (v/v) FCS,


5 µg/ml streptomycin, 10 µg/ml neomycin

A549 RPMI-1641

10% (v/v) FCS,


5 µg/ml streptomycin,

10 µg/ml neomycin

8.2 Methods

Freezing of cells 8.2.1

For long time storage cells were frozen and kept in liquid nitrogen. Cells were passaged as described

below. The cell number was adjusted to 2*106 cells/ml. This cell suspension was mixed (1:1) with pre-

cooled (4 °C) two-fold freezing medium (cell culture medium, 20% DMSO and 30% FCS). The cell

suspension was aliquoted in cryovials, 1 ml per vial. The cryovials were put into a freezing container

filled with isopropanol and incubated at -80 °C overnight. On the next day the cryovials were

transferred into a cryotank filled with liquid nitrogen for long term storage.

Thawing of cells 8.2.2

Cell culture medium was pre-warmed to 37 °C in the water bath. For each cell aliquot, one 15 ml

conical tube was filled with 9 ml pre-warmed cell culture medium. The cryovials with cell aliquots

were taken from the liquid nitrogen storage (-197 °C) and cells were thawed in a water bath at 37 °C.

Immediately after the last ice crystals were melted, the cell suspension was transferred into a 15 ml


130

conical tube with the pre-warmed culture medium. Then cells were centrifuged at RT and 130 g, the

supernatant was removed, and cells were resuspended in cell culture medium. The cell suspension was

transferred into a cell culture flask with a cell density of 2.5*104 to 1*10

5 cells per ml. On the next day

the medium was exchanged by new culture medium to remove residue of the freezing medium.

Sub-culturing of suspension cells 8.2.3

Suspension cells (Jurkat cells) were sub-cultured at a cell density of about 3*106 cells/ml. For that, the

cell suspension was transferred into a 50 ml conical tube and a 10 µl cell aliquot was removed for cell

counting. The rest of the cell suspension was centrifuged at RT and 130 g for 5 min and the

supernatant was removed. Cells were resuspended in pre-warmed cell culture medium and transferred

into a cell culture flask with a density of 1*105 up to 1*10

6 cells per ml.

Sub-culturing of adherent cells 8.2.4

Cells were sub-cultured at a confluence of about 70-80% (all three to four days). Therefore, the cell

culture medium was removed and cells were washed two times with 20 ml pre-warmed PBS (37 °C).

Then 2 ml of a trypsin-EDTA solution were added per T-75 culture flask (3 ml of trypsin-EDTA

solution were added per T-175 culture flask) and cells were incubated for 5 min at 37 °C, to detach the

cells from the culture flask. To completely remove the cells from the culture flask and to inactivate the

trypsin, 10 ml of pre-warmed cell culture medium were rinsed over the bottom of the cell culture flask.

The detachment of the cells was controlled by a microscope observation. The resulting cell suspension

was transferred into 50 ml conical tubes and centrifuged at 130 g for 5 min. Supernatant was removed

and cells were resuspended in 10 ml culture medium. A 10 µl aliquot was used for the counting of the

cells. The cells were diluted with culture medium to the desired cell count. The cell suspension was

transferred into a cell culture flask and cells were cultured.

MTT assay 8.2.5

The MTT assay is a colorimetric assay that can be used to analyse cell viability [575]. The assay

principle is based on the fact that living cells are metabolic active. Hence, living cells can metabolize

MTT. The reduction of MTT leads to the formation of purple coloured formazan in the cell. Dead cells

are not able to reduce MTT. Therefore, the formation of formazan can be used to investigate the

cytotoxicity of chemical compounds. Cells were cultured as described in sections 8.2.3 and 8.2.4.

First, cells were seeded in 96-well microplates. Jurkat cells were seeded with a cell density of 70.000

cells per well, in 50 µl of RPMI-1640 cell culture medium without FCS. The MTT assay was


131

performed on the same day. A549 cells were harvested, as described in the section 8.2.4. A549 cells

were seeded at a cell density of 20.000 cells per well in 100 µl of cell RPMI-1640 culture medium.

The A549 cells were cultured for 24 h, to allow the attachment to the well bottom. The treatment with

the chemical compounds was performed as described in the following. For the treatment of the A549,

95 µl of the cell culture medium were removed and 45 µl of RPMI-1640 cell culture medium without

FCS were added. Both cell types were seeded with the following plate-layout: edges (grey wells) were

only filled with medium without FCS, see Table 5. These wells were used as blanks and as controls for

unspecific reactions of the test compounds with the cell culture medium or compounds of the MTT

assay.

1 2 3 4 5 6 7 8 9 10 11 12

A

B

cells cells cells cells cells cells cells cells cells cells

C

D

E

F

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H Table 5: Cell plate layout for MTT assay. White wells were filled with 50 µl of cell suspension (Jurkat cells 70.000 cells

per well, A549 cells 20.000 cells per well). Grey wells were filled with 50 µl of RPMI-1640 cell culture medium without

cells.

The following controls were used: untreated cells as metabolic control. As positive control to induce

cell death, cells were treated with different concentration of SDS. Jurkat cells were treated with 50 µM

and 25 µM of SDS in RPMI-1640 cell culture medium without FCS. A549 cells were treated with

800 µM and 200 µM of SDS in RPMI-1640 cell culture medium without FCS. The solvent control

contained the solvent of each test compound in the same concentration that was used in the treated

samples. The cells were treated with a concentration series of each compound to determine the toxic

concentrations that induce cytotoxicity, see Table 6.

Substance: Solvent:

Starting concentration

Jurkat cells [mM]

Starting concentration

A549 cells [mM]

eugenol RPMI-1640 7 7

8-hydoxyquinoline ethanol p.a. 5 5

4-nitrophenol RPMI-1640 10 10

sodium saccharin RPMI-1640 10 10

Table 6: Test compounds for the MTT assay.

Concentrations of the test compounds which induce cell death must be excluded in the FADU-assay,

because these concentrations induce artificial DNA strand breaks. For this purpose, a concentration

series of each test compound and the controls were prepared in a 96-well microplate in a two times

higher concentration as it was used in the assay. The compounds were diluted 1:1 with RPMI-1640

without FCS in each dilution step. For each compound six dilutions were prepared. The compound

plate had the following layout, see Table 7.


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Table 7: Plate layout with dilution series.

Then, 50 µl per well of the compound plate were transferred into the cell plate, yielding the following

plate layout for the cell treatment, see Table 8 and Table 9.

Table 8: Plate layout of the MTT assay. Dark grey wells are only filled with cell culture medium without cells and the

compound. Wells were used as blanks. Light gray wells were filled with the indicated chemical compound but without cells.

White wells were filled with cells and the indicated chemical compound.

Final concentration [mM]

Substance: c1 c2 c3 c4 c5 c6

eugenol 7 3.5 1.75 0.88 0.44 0.22

8-hydroxyquinoline 5 2.5 1.25 0.63 0.31 0.16

4-nitrophenol 10 5 2.5 1.25 0.63 0.31

Sodium saccharin 10 5 2.5 1.25 0.63 0.31 Table 9: Concentration of the test compounds used in the MTT assay.

Wells at the edges (dark grey) contained no cells, only medium without FCS was added. Light grey

indicate wells without cells but they contained the test compound or SDS. Afterwards, cells were

incubated at 37 °C for 30 min. Then, 10 µl of the MTT solution per well were added (final

concentration of MTT pro well 0.45 g/l) and the samples were incubated again at 37 °C for 3 h to

allow the cells to take up and metabolize the MTT. Next, cells were lysed and the insoluble precipitate

of formazan was solubilized by adding 100 µl of solubilisation solution per well. Samples were

incubated over night at 37 °C. Finally, the absorbance was measured at a wavelength of 590 nm and a

reference wavelength of 750 nm.

8.2.5.1 Evaluation of the MTT assay

First, the absorbance values measured at wavelength 750 nm were subtracted from the absorbance

values measured at 590 nm. The average of the blanks in each column was calculated and subtracted

from the sample value. Afterwards, the average of each sample was calculated out of the six replicates.

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133

The value of the negative control was set to 100% and all other values were expressed as percentage of

this value.

8.2.5.2 Pre-validation of the FADU assay

Jurkat cells were cultured as described in section 8.2.3. Jurkat cells were centrifuged for 5 min at 130 g

and RT. The supernatant was removed and cells were resuspended in RPMI-1640 without FCS to a

cell count of 1.44*106 cells/ml. Then 50 µl of the cell suspensions were transferred into each well of a

96-well assay plate (7.2*104 cells/well). Cells were incubated at 37 °C until the supplementation plate

was ready. The supplementation plate had the following layout, see Table 10.

Table 10: Supplementation plate for FADU assay.

The supplementation plate was prepared as following: T0 and P0 sample containing wells were only

filled with 200 µl of RPMI-1640 without FCS. The wells for the vehicle control were filled with

200 µl of RPMI-1640 without FCS containing the solvent. As positive control etoposide was dissolved

in DMSO to a stock concentration of 10 mM. This stock solution was further diluted with RPMI-1640

cell culture medium without FCS to a concentration of 20 µM and 10 µM etoposide and 200 µl per

well were transferred into the supplementation plate. As negative control mannitol was used, it was

dissolved in MilliQ to a stock concentration of 250 mM. This stock solution was also further diluted in

RPMI-1640 cell culture medium without FCS to a concentration of 500 µM and 200 µl were

transferred into the supplementation plate. Then the wells used for the dilution series (wells C2-C8)

were filled with 200 µl of RPMI-1640 cell culture medium without FCS. The C1 wells were not filled

with cell culture medium. The highest test compound concentration was prepared and 300 µl per well

were transferred into the C1 wells. Afterwards, 100 µl of the C1 solution were transferred into the C2

wells and mixed by pipetting. Again 100 µl of the resulting solution was transferred into the C3 wells

and mixed. This was repeated until the dilution was finished in C8. Then 50 µl per well of the

supplementation plate were transferred into the cell plate. The cells were incubated for 30 min at

37 °C. Afterwards, the cells were centrifuged for 5 min at 300 g and RT. The supernatant was

removed and the assay plate containing the treated cells was transferred into the FADU robot. The

following working steps were performed by a Tecan pipetting robot, in the dark. T0 and P0 values were

analyzed as twelve technical replicates, the other samples were analyzed as four technical replicates.

The sample plate was cooled down to 0 °C and 70 µl of ice-cold cell suspension were added per well.

Then 70 µl of lysis buffer at RT were added to each well and incubated for 12 min at 0 °C. This led to

1 2 3 4 5 6 7 8 9 10 11 12

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134

the lysis of the cells and the denaturation of proteins and the destruction of the chromatin. Next, the T0

samples were treated with 140 µl of neutralization buffer. Afterwards, 70 µl of ice-cold alkali solution

were added on top of each lysate. Samples were incubated for 15 min at 0 °C. Next, the sample plate

was warmed up to 30 °C and incubated for 60 min, allowing the unwinding of the DNA strands in P0

and PX samples. Then these samples received 140 µl of neutralization buffer to stop the unwinding of

the DNA and the sample plate was cooled down to 18 °C. Finally, 156 µl of SYBR Green I solution

(3:25000 in H2O) were added to each well and the samples were mixed one time by pipetting up and

down.

8.2.5.3 Fluorescence reading and data evaluation

Immediately after the SYBR Green I addition, the assay plate was taken out of the Tecan robot. The

fluorescence was measured with the help of a FL600 microplate fluorescence reader. The excitation

wavelength was set to 492 nm and the emission wavelength to 520 nm. The received data were

processed in Excel and GraphPad Prism. The averages of the technical replicates were formed. The

values for T0 samples were set to 100%, all other values were expressed in percentage of the T0 value.

8.2.5.4 Modification of FADU buffer

The original neutralization buffer contains β-mercaptoethanol. β-mercaptoethanol is important to

reduce disulfide bridges of proteins to make the denaturation irreversible. Moreover, it acts as an

antioxidant. However, it is toxic and highly volatile. Therefore, it was tested if β-mercaptoethanol can

be substituted by 1,4-dithiothreitol (DTT). DTT is less toxic and not volatile. For the comparison

Jurkat cells were harvested as in section 8.2.3. Cells were resuspended in RPMI-1640 cell culture

medium without FCS. Two different cell concentrations were used for the tests, 6*106

cells/ml and

3*106 cells/ml. Cell suspensions were aliquoted in 2 ml reaction vessels, 100 µl in each tube. The

reaction vessels were placed in a metal rack placed on ice. Cells were irradiated with different doses of

X-rays (0.69, 2.1, 3.7 and 6.2 Gy) to induce DNA strand breaks. Afterwards, the samples were

transferred into the Tecan robot. First, the cell suspension was diluted with 500 µl ice-cold suspension

buffer per reaction vessel. Then 70 µl of each sample were transferred as triplicate into a 96-well assay

plate. The following steps were performed as described above. With the adaption that one half of the

samples was treated with standard neutralization buffer (14 mM β-mercaptoethanol), whereas the

other half of the samples was treated with the modified neutralization buffer (7 mM DTT), see Table

11.


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Table 11: Sample layout for test of modified neutralization buffer.

Pre-validation of the TOXXs Analyzer 8.2.6

The TOXXs Analyzer is a modified liquid handling pipetting platform based on the CyBio Felix

platform produced by Analytik Jena AG, see Figure 8-1. The liquid handling pipetting platform

consist out of 2 main parts: the first part is the housing of the robot which contains the working space,

control units and a mounting system for a pipetting head. The mounting system can be connected to

different pipetting heads. The pipetting head is the second main part of the system. In this case, a

CyBio Felix Head R 96/250 µl was used, see Figure 8-3. This head uses 96 disposable pipetting tips.

There are some differences between the CyBio Felix based FADU platform and the Tecan based

system. The temperature of the assay plates is controlled by a liquid based temperature controlling

system, in the case of the Tecan system. A thermostat heats or cools the ethanol/water mixture which

is pumped to the assay plate- and buffer- holder. Hence, the temperature regulation of this system is

slow, especially between two FADU runs it takes several minutes until the system is cooled down for

the next FADU assay. Moreover, the temperature can only be set for the whole system and not for

single plate positions. The TOXXs Analyzer uses peltier-elements which are electrothermal

transducer, mounted on positions 1,2,3,4 and 8, see Figure 8-4. These peltier-elements are much faster

in the temperature regulation and each peltier-element can be regulated separate. This allows a faster

and more precise processing (no delay time) of assay plates, if more than one assay plate is used. The

second big advantage of the CyBio Felix based FADU platform is the use of a 96 pipetting tip head. In

contrast, the Tecan system uses a pipetting head with 8 tips. A 96 pipetting head allows the processing

of all samples at the same time. Hence, there is no time-gradient between the different columns of an

assay plate. Especially at the end of the FADU assay, after the addition of SYBR Green the time

differences can be a problem if more than one assay plate is used. Hence, the better cooling system

and the 96 tip pipetting head should increase the velocity and the accuracy of the FADU assay.

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Figure 8-1: Design of the CyBio Felix pipetting platform and pipetting head. A) Housing with three moveable decks

(decks A, B and C). Deck A is the lowest deck with the positions 1 till 6. Deck B and C are on the same level above deck A.

Deck B contains the positions 7 till 9 and deck C contains the positions 10 till 12. B) Schematic representation of the moving

parts of the robot. Left the pipetting head can move along the x- and the z-axis but not along the y-axis. All three decks can

only move along the y-axis. C) Left schematic representation of the CyBio Felix Head R 96/250 µl pipetting head. Right

magnification of the lower side of the pipetting head: 1 gripper for tip holder, 2 sealing pad, 3 frame for sealing pad. Pictures

are copies from the manual.


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8.2.6.1 Technical data of the housing and pipetting head of the TOXXs Analyzer

Figure 8-2: Original technical data sheet of the CyBio Felix pipetting platform in German.


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Figure 8-3: Original technical data sheet of the CyBio Felix Head R 96/250 µl in German.


139

Figure 8-4: Original technical data sheet of the deck positions of the TOXXs Analyzer in German. Position 1 to 3

holder for the assay plates (Modifications of the plate holder allowed the use of normal 96-well cell culture plates). Position 4

contains the cell suspension which was spread into the assay plates. Position 5 contains a plate for liquid waste. Position 6

contains pipetting tips for the T samples. Position 7 contains a plate for the storage of the lysis buffer (L4) and the SYBR

Green I solution. Position 8 contains a reservoir for the storage of the suspension buffer (L1) and the unwinding buffer (L3).

Position 9 contains a pipetting tip washing station. Position 10 contains pipetting tips for the P samples. Position 11 contains

the neutralization buffer (L2) and position 12 contains pipetting tips.


140

8.2.6.2 Precision test of the TOXXs Analyzer pipetting head

To test the pipetting precision of the pipetting head two test volumes were tested according to standard

operation procedure of Cetics, which uses different volumes compared to the standard operation

procedure from Analytik Jena AG. The variation coefficient (CV) was tested with a 96-well micro-

clear plate by the use of p-nitrophenol (dye solution). Afterwards, the absorbance was measured and

analyzed. First, the pipetting head was tested with a 70 µl test volume (simulating the spreading of the

cells). For that, a 12 mM p-nitrophenol stock solution in 0.1 M NaOH was prepared. For the test, this

stock solution was diluted 1:100 to a concentration of 120 µM p-nitrophenol. At position 4, 5 and 6,

three 96-well micro-clear plates were placed. At position 11 a deep-well plate filled with 500 µl/well

with the p-nitrophenol solution was placed. At position 12 new pipetting tips were placed. Then the p-

nitrophenol solution was mixed three times by the robot. This leads to a uniform wetting of the

pipetting tips. Afterwards, 70 µl were transferred from the deep-well plate into each well of the micro-

clear plates. Then the plates were centrifuged for 1 min at 1300 rpm and the absorbance was measured

at a wavelength of 405 nm. Second, the pipetting head was tested with a test volume of 156 µl. The

test-setup was the same as described above. Additionally, at position 7 a 384-well plate was placed.

The p-nitrophenol solution was mixed three times by the robot and 170 µl/well were transferred from

the deep well plate to the 384-well plate. Then 156 µl were transferred from the 384-well plate into

each well of the micro-clear plates. Again, the plates were centrifuged for 1 min at 1300 rpm and the

absorbance was measured at a wavelength of 405 nm. The data were evaluated by an Excel test sheet.

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9 Results

9.1 Pre-validation of the TOXXs Analyzer

The second project in this thesis was to support the further development of the automated FADU assay

as part of a cooperation project together with three partner institutions. These partners were the Cetics

Healthcare Technologies GmbH from Esslingen (Germany), the Eidgenössische Materialprüfungs-

und Forschungsanstalt (EMPA) from St. Gallen (Switzerland) and the University of Konstanz

(Germany). The project was financed by the Cetics GmbH, which had the leadership of the total

project. The other partners were part of the inter-laboratory validation of the automated FADU assay

device. Therefore, a completely new FADU platform was developed by Cetics GmbH in cooperation

with Dr. Maria Moreno-Villanueva in advance of this thesis. Three prototypes, called “TOXXs

Analyzer” were produced. Each partner obtained one prototype for the inter-laboratory validation. The

first step was to perform a pre-validation of these prototypes. The main objective of the pre-validation

was a technical test of the new FADU-platform. Depending on the results, technical adjustments

should be performed to increase the assay speed, reduce the assay volume and increase the assay

sensitivity. The technical support and development was performed by the Cetics GmbH. The final goal

of this project was the development of a market maturity version of the automated FADU assay.

Therefore, following workflow was used, see Figure 9-1.

Figure 9-1: Validation-workflow of the TOXXs Analyzer development.

For the validation of the TOXXs Analyzer two different human cell lines were chosen. On the one

hand, Jurkat cells (clone E6-1) and on the other hand A549 cells were selected. Both cells lines are

p53 and cytochrome P450 positive [576]. These proteins are important in the genotoxic response

[577]. Two cell lines were selected because the TOXXs Analyzer should be tested with an adherent

cell line, A549 cells, and with a suspension cell line, Jurkat cells. The automated FADU assay

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142

according to Moreno-Villanueva et al. required 96-deep well plates, because of a total assay volume of

506 µl/well [571]. This exceeds the maximum volume per well of normal 96-well cell culture plates

that can be used for the culturing of cells. The 96-deep well assay plates were not suitable for culturing

adherent cells because the surface is inadequate for the attachment of the cells. So far, adherent cells

had to be cultured in normal cell culture dishes and had to be trypsinized directly before the FADU

assay was performed. This method has several disadvantages: first, trypsin is a peptidase which

cleaves unspecific proteins on the cell surface including receptors, ion channels and transporters.

These surface proteins can be important for the investigation of genotoxic effects of chemicals.

Second, the trypsinization induces a detachment of the cells which changed the cell shape [578].

Third, the expression of important cellular proteins like p53 or p21 can be changed [579]. Therefore,

trypsinization could alter cytotoxic- and genotoxic-effects and impair their measurements. To avoid

these effects the assay volume of the TOXXs Analyzer should be halved to 253 µl per well. The

halved volume would allow the use of normal 96-well cell culture plates, which would allow the

culturing of adherent cells directly on the assay plate. Moreover, a reduced volume would also reduce

the needed cell material. Therefore, all technical adaptions for the reduction of the assay volume

should have been introduced during the performance of the Jurkat cells experiments. Next, the

chemical test compounds for the pre-validation of the FADU assay were selected from a list of

recommended chemicals for the development or improvement of genotoxic tests, published in 2008

from the EURL-ECVAM (European Union Reference Laboratory for alternatives to animal testing)

[580]. These chemicals can be categorized into three groups: The first group contains chemicals which

should be detected as positive in mammalian cell genotoxic tests. These chemicals are proofed

genotoxins in vivo (“true positives”). The second group of chemical shows no genotoxic effect in in

vitro mammalian cell genotoxic tests and also shows no effect in in vivo genotoxic tests (“true

negatives”). The third group of chemicals should give negative results in in vitro mammalian cell

genotoxicity tests. However, these chemicals have been reported to induce chromosomal aberrations

or mutations in the mouse lymphoma assay, at high concentrations (“false positives”). The following

14 test chemicals were selected, see Table 12.

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143

Compound: Group: Mode of action:

methylnitronitrosoguanidine (MNNG) 1 O6 and N

7 alkylator

4-nitroquinoline-1-oxide 1 aromatic amine

etoposide 1 topoisomerase inhibitor

camptothecin 1 topoisomerase inhibitor

D-mannitol 2 non-DNA-reactive

ethylenediaminetetraavetic acid trisodium salt

trihydrate 2 non-DNA-reactive

diethanolamine 2 non-DNA-reactive

D/L-menthol 3 non-DNA-reactive

phthalic anhydride 3 non-DNA-reactive

urea 3 non-DNA-reactive

sodium saccharin 3 non-DNA-reactive

4-nitrophenol 3 non-DNA-reactive

eugenol 3 non-DNA-reactive

8-hydroxyquinoline 3 non-DNA-reactive Table 12: Test chemicals for TOXXs Analyzer pre-validation.

Before the chemical substances could be used to test the TOXXs Analyzer, they had to be tested for

their cytotoxicity. Cytotoxic effects must be excluded, because cytotoxicity would impair the cell

viability and induce cell death, for example apoptosis or necrosis. Apoptosis as well as necrosis induce

a DNA breakdown, meaning additional DNA strand breaks would be evolved. The FADU assay is not

able to differentiate between DNA strand breaks induced by cytotoxic cell death and DNA strand

breaks induced by genotoxicity. Therefore, a cytotoxic compound could be identified falsely as a

genotoxin by the FADU assay. Hence, an MTT assay was used to determine a dose-range for each

chemical without cytotoxic effects. The MTT assay should be included as pre-test for the FADU assay

and, therefore, performed by the TOXX Analyzer. The pre-validation of the FADU assay should be

performed with all 14 test chemicals in each lab in three independent experiments. The first tests

should be performed with the Jurkat cell line. During this test series, all technical adjustments which

were necessary to reduce the assay volume to the half should be introduced by the Cetics GmbH.

Subsequently, all three prototypes should be adapted to these modifications and the test series with the

A549 cell lines should be performed. The resulting data of the TOXXs Analyzer pre-validation should

be used for the control of the specificity (detection of DNA strand breaks induced by the positive

controls), sensitivity (comparison with data in the literature and data of the old FADU system) and

most important for the technical pre-validation, the inter-laboratory and intra-laboratory variability

should be determined. However, this study could not be performed in the planned form due to

technical problems with the TOXXs Analyzer. These problems caused non-senses results of the

FADU assay, meaning there was no fluorescence signal detectable above the background fluorescence

signal (data not shown). Moreover, buffer residues and cell culture medium residues could be

observed on the sealing pad of the pipetting head after the run of a FADU assay. These residues were

an indication of a pipetting problem and could indicate a problem with the pipetting head. Therefore,

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the pipetting head was sent to the supplier for a revision. During the meantime the MTT assays were

performed without the TOXXs Analyzer manually. Moreover, the adjustments for the reduced assay

volume were introduced by the Cetics GmbH. These modifications should also solve the pipetting

problems of the TOXXs Analyzer. After the revision of the pipetting head and the introduction of the

modifications to reduce the assay volume, the results were still inacceptable. A precision test for the

pipetting head, designed by Cetics GmbH was performed to identify possible problems during the

FADU assay. Therefore, the spreading of the cells into the three assay plates and the addition of the

SYBR Green solution were simulated by the use of a p-nitrophenol solution. An absorbance

measurement was used as readout. The results of the pipetting precision test can be seen in Figure 9-2

and Figure 9-3. The pipetting precision test of the cell spreading showed that the values at plate

position 1 and plate position 3 were outside the acceptable limits. Only the values of plate position 2

were inside the limits. The pipetting precision test of the SYBR Green addition showed that the values

of all three plate positions were within acceptable limits. The results of the precision tests indicated

that the pipetting head was in principle functional. The cause for the insufficient performance of the

TOXXs Analyzer could so far not be determined. Hence, the pre-validation of the TOXXs Analyzer

could not be completed. The following sections display the achieved results during the pre-validation

in the course of the presented thesis at the University of Konstanz. All FADU assays were performed

with the old FADU system based on a TECAN handling device [571].

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Figure 9-2: Precision test of the TOXXs Analyzer pipetting head, simulating cell spreading. The TOXXs Analyzer

showed technical problems that make it impossible to use it as a platform for the FADU assay. Therefore, a trouble shooting

was started by the technical support (Cetics GmbH). Part of this trouble shooting was to test critical pipetting steps in the

FADU protocol, including a precision test of the pipetting head. Therefore, a 120 µM p-nitrophenol solution was pipetted

into the three assay plates 70 µl/well to mimic the cell spreading steps. An absorbance measurement was used to measure and

compare the accuracy of the pipetting volumes. A) 96-well assay plate at position 1, B) 96-well assay plate at position 2, C)

96-well assay plate at position 3, see deck positions Figure 8-4.

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Figure 9-3: Precision test of the TOXXs Analyzer pipetting head, simulating SYBR Green I pipetting step. The

TOXXs Analyzer showed technical problems that make it impossible to use it as a platform for the FADU assay. Therefore, a

trouble shooting was started by the technical support (Cetics GmbH). Part of this trouble shooting was to test critical

pipetting steps in the FADU protocol, including a precision test of the pipetting head. Therefore, 120 µM p-nitrophenol

solution was pipetted into the three assay plates 156 µl/well to mimic the SYBR Green I pipetting steps. An absorbance

measurement was used to measure and compare the accuracy of the pipetting volumes. A) 96-well assay plate at position 1,

B) 96-well assay plate at position 2, C) 96-well assay plate at position 3, see deck positions Figure 8-4.

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Determination of the cytotoxicity of the chemical test compounds 9.1.1

The cytotoxicity of the chemical test compounds was determined by an MTT assay. Therefore, test

chemicals were evenly distributed between the three laboratories. Each laboratory performed the

cytotoxic testing of the test chemicals in accordance with the standard operating procedure previously

defined by the three partners, see section 8.2.5. Therefore, the test chemicals were dissolved either in

the assay medium (RPMI-1640 cell culture medium without FCS) or in a vehicle solvent. For the pre-

validation, cells were treated with the compounds in FCS free cell culture medium for 30 min. The

following 5 test compounds were tested with Jurkat cells as well as with A549 cells: 4-nitrophenol,

sodium saccharin, 8-hydroxyquinolin, eugenol and propyl gallate. All compounds with the exception

of 8-hydoxyquinolin were dissolved in assay medium. 8-hydroxyquinolin was first dissolved in

ethanol and then further diluted in the assay medium (yielding a final concentration of 1% (v/v)

ethanol in the MTT assay). For all compounds, the maximum test concentration was 10 mM or the

concentration of the maximum solubility, if it was lower. However, the dose-range finding of propyl

gallate could not be performed with the MTT assay. Because propyl gallate reduced the MTT to

formazan in cell culture medium without cells, data not shown. Thus, it was removed from the test list

without substitution. Figure 9-4 shows the results of the MTT assay performed with Jurkat cells. In all

experiments SDS (50 and 25 µM) was used as positive control. A dose-dependent reduction of the cell

viability could be observed after the SDS treatment. 4-nitrophenol reduced the viability of Jurkat cells

significantly in a dose-dependent manner, see Figure 9-4 A). The highest test concentration, 10 mM of

4-nitrophenol, reduced the cell viability to about 33% of the viability of control cells. Already 1.25

mM of 4-nitrophenol reduced the viability of the Jurkat cells, but this was not significant. Sodium

saccharin also induced a significant dose-dependent reduction of the cell viability, see Figure 9-4 B).

2.5 mM of sodium saccharin reduced the cell viability to about 87% of the control cells. At the

maximal used saccharin concentration of 10 mM, a further reduction to approximately 75% could be

observed. A decline of the cell viability in a dose dependency could be seen after the 8-

hydroxyquinolin and the eugenol treatment, see Figure 9-4 C) and D). 8-hydoxyquinolin reduced the

cell viability significantly to about 71% of control cell at a concentration of 1.25 mM. The lowest cell

viability, about 45%, could be observed at a concentration of 5 mM 8-hydoxyquinolin. The highest

cytotoxicity was observed for eugenol. 0.88 mM of eugenol induced a decrease of the cell viability to

about 78%, but this was not statistical significant. 7 mM of eugenol, the highest usable concentration,

induced a statistical significant cytotoxic effect and reduced the cell viability to about 4%. Hence,

concentrations less than 0.63 mM of 4-nitrophenol and 8-hydroxyquinolin, 1.25 mM of sodium

saccharin and 0.44 mM of eugenol can be used for the genotoxic studies with the FADU assay, as at

these concentrations no cell death is induced under assay conditions.

Results

148

ce

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0.6

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1.2

5

2.5

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SD

S [5

0 µ

M]

SD

S [2

5 µ

M]

0

5 0

1 0 0

**

**

4 -n itro p h e n o l [m M ]

A )# #

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S [5

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SD

S [2

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SD

S [2

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S [5

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SD

S [2

5 µ

M]

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5 0

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**

***

e u g e n o l [m M ]

D )# # # #

Figure 9-4: Dose-range findings of Jurkat cells treated with 4-nitrophenol, sodium saccharin, 8-hydroxyquinolin or

eugenol. Jurkat cells were treated with a concentration series of 4-nitrophenol, sodium saccharin, 8-hydroxyquinolin or

eugenol in RPMI-1640 cell culture medium w/o FCS. Cell viability was determined with a MTT assay. A) Jurkat cells were

treated with a dilution series of 4-nitrophenol: 10, 5, 2.5, 1.25, 0.63, 0.31 and 0 mM. B) Jurkat cells were incubated with the

following dilution series: 10, 5, 2.5, 1.25, 0.63, 0.32 and 0 mM of sodium saccharin. C) Jurkat cells were treated with the

following doses of 8-hydroxyquinolin: 5, 2.5, 1.25, 0.63, 0.31, 0.16 and 0 mM. D) Jurkat cells were treated with the

following concentration series of eugenol: 7, 3.5, 1.75, 0.88, 0.44, 0.22 and 0 mM. As positive control, Jurkat cells were

treated with 50 µM and 25 µM of SDS. Cells were incubated for 30 min at 37 °C before the MTT solution (0,45g/l) was

added. After 3 h of incubation cells were lysed and the formazan was dissolved. Absorbance was measured at a wavelength

of 590 nm and a reference wavelength of 750 nm. Data were shown as percentage of untreated cells. Data represent means

with SEM of five experiments for A) and B) and seven experiments for C) and D). Data were shown as percent of untreated

cells. (Figure in cooperation with Monika Schulz, technical assistant, measurement of MTT assay). Statistical analysis was

performed using RM one-way ANOVA (#), #### P<0.0001, ## P<0.01, # P<0.05 followed by a Dunnett multiple

comparison test (*), **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05.

All observed DNA strand breaks observed at these concentrations of the test compounds should be

induced by genotoxic effects and not subsidiary by cytotoxic side effects, like cell death. A549 cells

were treated with the same concentrations of the test compounds as the Jurkat cells. As positive

control SDS was used, 800 µM and 200 µM. Also for the A549 a dose-dependent decrease of the cell

viability induced by SDS could be observed, see Figure 9-5). Treatment with 4-nitrophenol induced no

cytotoxicity in the tested concentration range. Rather an increase of the cell viability compared to the

Results

149

untreated control cells could be observed with increasing 4-nitrophenol concentration, see Figure 9-5

A). The treatments with either sodium saccharin or 8-hyxdroxyquinolin had no effect on the viability

of A549 cells, see Figure 9-5 B) and C). The highest tested 8-hydroxyquinolin concentration of 5 mM

induced a slight decrease to about 90% of the viability of control cells, but this was not statistical

significant. The only compound that induced cytotoxic effects in A549 cells was eugenol Figure 9-5

C). A concentration depended decline of the cell viability could be observed. A reduction of the cell

viability could be seen at concentrations higher than 1.75 mM of eugenol. Hence, only eugenol

concentrations higher than 1.78 mM must be excluded from the FADU assay

ce

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1 5 0

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2 5 0

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*


A )

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S [8

00 µ

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0

5 0

1 0 0

1 5 0

**


D )# #

Figure 9-5: Dose-range findings of A549 cells treated with 4-nitrophnol, sodium saccharin, 8-hydroxyquinolin or

eugenol. A549 cells were treated with a concentration series of 4-nitrophenol, sodium saccharin, 8-hydroxyquinolin or

eugenol in RPMI-1640 cell culture medium w/o FCS. Cell viability was determined with a MTT assay. A) A549 cells were

treated with a dilution series of 4-nitrophenol: 10, 5, 2.5, 1.25, 0.63, 0.31 and 0 mM. B) A549 cells were incubated with the

following dilution series: 10, 5, 2.5, 1.25, 0.63, 0.32 and 0 mM of saccharin. C) A549 cells were treated with the following

doses of 8-hydroxyquinolin: 5, 2.5, 1.25, 0.63, 0.31, 0.16 and 0 mM. D) A549 cells were treated with the following

concentration series of eugenol: 7, 3.5, 1.75, 0.88, 0.44, 0.22 and 0 mM. As positive control A549 cells were treated with 200

µM and 800 µM of SDS. Cells were incubated for 30 min at 37 °C before the MTT solution (0,45g/l) was added. After an

incubation of 3 h, cells were lysed and the formazan was dissolved. Absorbance was measured at a wavelength of 590 nm

and a reference wavelength of 750 nm. Data were shown as percent of untreated cells. (Figure in cooperation with Monika

Schulz, technical assistant, measurement of MTT assay). Data represent means with SEM of five experiments for A), C) and

D) and six experiments for B). Statistical analysis was performed using RM one-way ANOVA (#), ## P<0.01followed by a

Dunnett multiple comparison test, ** P<0.01, * P<0.05, ns = not significant.

Results

150

Modification of the neutralization buffer of the automated FADU 9.1.2

assay

In the course of the pre-validation, one object was to optimize the FADU assay in view of application-

friendliness. Thus, β-mercaptoethanol, a content of the neutralization buffer should be substituted

because of its toxicity, environmental treat and unpleasant odor. β-mercaptoethanol is used to reduce

disulfide bridges of proteins which makes a refolding of proteins impossible. This is important

because the DNA strand must be free of bound proteins for the unwinding process during the FADU

assay. Moreover, β-mercaptoethanol is used as an antioxidant for scavenging radicals. DTT and β-

mercaptoethanol are often interchangeable with each other. DTT is less toxic, less harmful and lower

concentrations are needed because DTT has two functional thiol groups in contrast to β-

mercaptoethanol. To test the substitution of β-mercaptoethanol by DTT, the FADU assay was

performed in parallel, either with standard neutralization buffer, containing 14 mM of β-

mercaptoethanol or with the modified neutralization buffer, containing 7 mM of DTT. Two

concentrations of Jurkat cells (3*106 cells/ml and 6*10

6 cells/ml) were used for the test. Cells were

either non-irradiated or irradiated with x-rays (0.7, 2.1, 3.7 and 6.2 Gy) to induce DNA strand breaks.

X-rays were used because no chemical carryovers were present after the treatment. The X-ray induced

DNA strand breaks led to a dose-dependent decline of the fluorescence signal, see Figure 9-6 A) and

B). No significant differences in the fluorescence signals between both assay setups could be seen.

Although, the assay setup which used DTT in the neutralization buffer showed minimal higher

fluorescence signals. However, this was statistically not significant. This allowed the conclusion that

β-mercaptoethanol can be substituted by DTT, at least in the tested assay setup.

Results

151

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D TT

****

ir ra d ia t io n d o s e [G y ]

B )

ns

Figure 9-6: Substitution of β-mercaptoethanol by DTT in the neutralization buffer. The substitution of β-

mercaptoethanol by DTT was tested with Jurkat cells. A) 3*106 cells/ml were irradiated with five different doses of X-rays

(0, 0.7, 2.1, 3.7 and 6.2 Gy) to induce DNA strand breaks. B) 6*106 cells/ml were irradiated with five different doses of X-

rays (0, 0.7, 2.1, 3.7 and 6.2 Gy) to induce DNA strand breaks. Afterwards, the FADU assay was performed and the

fluorescence was measured at an excitation wavelength of 492 nm and at an emission wavelength of 520 nm. Data represent

means with SEM of five experiments. Statistical analysis was performed using RM one-way ANOVA (*) followed by

multiple comparison Sidak´s test, **** P<0.0001, ns = not significant.

Genotoxicity of test compounds 9.1.3

After the cytotoxicity tests, two of the test compounds were tested for their ability to induce DNA

strand breaks by the FADU assay (old FADU platform). Jurkat cells were treated for 30 min with a

concentration series of either 4-nitrophenol or eugenol, see Figure 9-7. As positive control, to induce

DNA strand breaks etoposide (5 and 10 µM) was used. The fluorescence signal was normalized to the

total double-stranded DNA content (T0). The etoposide treatment of cells induced DNA strand breaks

Results

152

which reduced the fluorescence signal significantly. Concentrations lower than 10 mM of 4-

nitrophenol showed no induction of DNA strand breaks, because the fluorescence signal did not

decrease compared with the P0 value. A dose of 30 mM of 4-nitrophenol induced a significant amount

of DNA strand breaks which was even bigger than the amount of DNA strand breaks induced by

10 µM of etoposide. Eugenol showed also a dose-dependent induction of DNA strand breaks. Doses

higher than 0.77 mM of eugenol started to reduce the fluorescence signal. This indicated the formation

of DNA strand breaks. The highest dose tested at 7 mM induced approximately the same amount of

DNA strand breaks than 10 µM etoposide. However, the concentrations of eugenol as well as of 4-

nitrophenol needed to induce DNA strand breaks were higher than the concentrations needed to induce

cytotoxic effects (4-nitrophenol induced cytotoxic effect with concentrations higher than about

1.25 mM and eugenol induced cytotoxic effect with concentrations higher than about 0.88 mM).

Hence, the detected DNA strand breaks may be caused by secondary effects of the cytotoxicity.

flu

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T0

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30

10

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# # # #fl

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T0

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veh

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2.3

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0.7

7

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28

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09

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B )

*

#

Figure 9-7: DNA strand breaks induced by 4-nitrophenol and eugenol in Jurkat cells. The TOXXs Analyzer was due to

technical problems not ready for operation. Therefore, the pre-testing of chemical compounds was performed with the Tecan

robot. Jurkat cells were treated with etoposide (5 µM and 10 µM) as positive control. As negative control, D-mannitol was

used. Both test substances were diluted in assay medium and a dilution series (1:3) was performed getting the following

concentration series: 4-nitrophenol 30, 10, 3.33, 1.11, 0.37, 0.12, 0.04, and 0.013 mM and for eugenol 7, 2.33, 0.77, 0.26,

0.086, 0.028, 0.009 and 0.003 mM. Cells were incubated for 30 min at 37 °C. Afterwards, the supernatant with the test

compounds was removed and the automated FADU assay was performed. Fluorescence was measured at an excitation

wavelength of 492 nm and emission wavelength of 520 nm. Data represent means with SEM of five experiments for 4-

nitrophenol and three for eugenol. Statistical analysis was performed using RM one-way ANOVA (#), #### P<0.0001, #

P<0.05 followed by multiple comparison Dunnett test (*), ** P<0.01, * P<0.05.

Discussion

153

10 Discussion

Pre-validation of the TOXXs Analyzer 10.1.1

The TOXXs Analyzer represents the next generation of the automated FADU assay. In principle, the

TOXXs Analyzer is a fully automated pipetting platform which is commercially available (Analytik

Jena AG, CyBio Felix). The pipetting platform was modified by Cetics according to the requirements

of the FADU assay. This new platform has some technical advantages compared to the old automated

FADU system. This includes improvements in the software as well as in the hardware. The software of

the TOXXs Analyzer is more complex compared to the software of the old FADU system (Tecan).

However, it enables the parallel execution of processes independently of each other. The most

important improvements of the hardware are the reduction of the assay volume and in combination

with the software the reduction of the assay duration and an increase of the assay throughput. The old

FADU system allows only linear processes. This means the system can perform assay steps only in a

sequential order. A process must be completed for all assay plates before the next process can be

started. This limitation is caused by the software as well as by the hardware. The temperature system

can adjust and control the temperature of all assay plates only at once and not separately for each plate.

In contrast, the TOXXs Analyzer can perform parallel operations. Meaning, when one operating step

is completed for one assay plate, the next step can be started while the processing of the rest of the

assay plates is not completed. This required an autonomous regulation of the temperature for each

assay plate which was achieved by the use of peltier-elements. Moreover, these electrothermal

convertors are faster in the temperature regulation and more precise in the regulation of the

temperature then the water cooling system of the old FADU system. The pipetting steps are faster

compared to the old system, because of the use of a 96-tip pipetting head. In contrast, the old system

has an 8-tip pipetting head. The use of a 96-tip pipetting head also increases the accuracy of the

measurements, because all samples of an assay plate are processed at once. Therefore, there is no time

delay in the pipetting steps between different columns of an assay plate. This is important at the last

step of the FADU assay, the addition of the SYBR Green solution, because there is no time frame to

compensate the time delay of the previous pipetting steps. Finally, the reduction of the assay volume

allowed the use of normal 96-well plates. This has the advantage that adherent cells can grow directly

onto the assay plates, making trypsinization of the cells dispensable. Since trypsinization can affect

cellular processes by altering the cell shape, degradation of surface proteins and changing the

expression pattern of proteins, it can influence the outcome of a genotoxic analysis. The pre-validation

was designed to technically test the TOXXs Analyzer and verify it´s functionality and reliability. It

was not designed for the investigation of chemical compounds to generate new data about their

genotoxicity. Therefore, the test compounds are well known and had been tested before for their

Discussion

154

genotoxicity. These chemicals were classified and recommended by the “European Union Reference

Laboratory for alternatives to animal testing” for the validation of a genotoxic tests. For the pre-

validation one suspension cell line (Jurkat cells) and one adherent cell line (A549) were chosen. Both

cell lines are used in genotoxic tests. Since cell death would also induce DNA strand breaks the test

compounds were tested for their cytotoxicity. Therefore, a MTT assay was selected and both cell lines

were treated with dilution series of each compound to determine a dose response curve. Therefore, the

same incubation conditions were used as for the FADU assay. The maximum concentration used was

either 10 mM of the test compound or the concentration at the solubility maximum. However, due to

technical problems the TOXXs Analyzer was not ready to work. As the performance test of the

pipetting head showed, see Figure 9-2, the precision of the pipetting head was outside of an acceptable

range. However, this was not the case for all tested pipetting steps, see Figure 9-3. There were two

sources that could cause the pipetting problems. On the one hand, a hardware error can be responsible.

On the other hand, a software error can induce such problems. Since the error of the pipetting head

was not removed after maintenance by the producer (Analytik Jena AG) and was not occurring

constantly it would be likely that the error is caused by the software. However, Cetics was not able to

detect the error source and to remove it. Therefore, the pre-validation was stopped and only the MTT

assays and two measurement series with the old automated FADU could be performed. The chemical

compounds which were tested in the MTT assay were 4-nitrophenol, sodium saccharin, 8-

hydroxyquinolin, eugenol and propyl gallate. The remaining test substances were tested in the two

other labs. Propyl gallate could not be tested in the MTT assay, because it induced the reduction of

tetrazolium dye to formazan without the present of any cells (data not shown). Propyl gallate is a well-

known antioxidant which is used commercially in various products as additive to increase the

chemical stability [581]. Since propyl gallate undergoes oxidation in aqueous solutions it could reduce

the MTT to formazan, see Figure 10-1 [582, 583]. Therefore, propyl gallate was excluded from further

testing without substitution.

HO

HO

HO

O

O

+ 2 H+ + 2 e-O

HO

O

O

O

Figure 10-1: Oxidation of propyl gallate.

The remaining four substances were sufficient to generate enough data for the pre-validation. The

cytotoxicity of each substance was determined in Jurkat cells as well as in A549 cells. For 4-

nitrophenol, saccharin and 8-hydroxyquinolin a cytotoxic effect could be observed at concentrations

higher than 1 mM, after an incubation time of 30 min at 37 °C. The highest toxicity could be observed

Discussion

155

for eugenol which reduced the viability of Jurkat cells at lower concentrations. In contrast, a cytotoxic

effect in A549 cells could only be observed for eugenol at concentrations higher than 1.75 mM.

Saccharin and 8-hydroxyquinolin showed no cytotoxic effects in A549 cells. During the treatment

with 4-nitrophenol, a positive correlation between the increasing 4-nitrophenol concentration and an

increase of the reduction of MTT to formazan could be observed. This reduction was not caused by

redox reaction between the MTT and the 4-nitrophenol. Since blanks which contain both substances in

the same solvent and the same concentrations as the wells with cells showed no formation of

formazan. Hence, 4-nitrophenol had to influence cellular processes of A549 cells, which increased the

intracellular formation of formazan. The MTT assay is a metabolic assay which determines the

reduction of MTT by the mitochondrial electron transporting chain and by oxidoreductases. A cell

viability assay which is not based on the measurement of the metabolism could be used to confirm the

results [584, 585]. For example, the neutral red up-take assay or propidium iodide staining can be

used. In general, A549 cells seem to be more resistant against cytotoxic chemicals compared to Jurkat

cells. Since higher concentrations of SDS were needed to reduce the viability of A549 cells compared

to Jurkat cells. As the TOXXs Analyzer was defect, the old FADU system was used for the testing of

the chemicals and the treatment protocol. The old FADU system could only run with deep-well plates,

therefore, all experiments were performed with Jurkat cells. First, a modified neutralization buffer was

tested. The 14 µM of β-mercaptoethanol were substituted by 7 µM of DTT. This was done, because

DTT is more user-friendly than β-mercaptoethanol, it is less toxic and volatile. Two different cell

numbers and 5 doses of X-rays were used for a side by side comparison of the two neutralization

buffers. After normalization to the T0 values no statistical significant difference could be observed. All

four conditions showed the same dose response curve. Hence, DTT can be used as a substitute for β-

mercaptoethanol. The last experiment was a test of the protocol used for the pre-validation of the

TOXXs Analyzer. However, it was performed with the old FADU system. Etoposide was used as

positive control to induce DNA strand breaks via inhibition of topoisomerase 2. A dilution series of 4-

nitrophenol from 40 mM to 0.013 mM was used as well as a dilution series of eugenol from 7 to

0.003 mM. Both dilution series showed a dose response, increasing concentrations of 4-nitropehneol

as well as increasing concentrations of eugenol induced the formation of DNA strand breaks.

However, only the highest doses 30 mM of 4-nitrophenol and 7 mM of eugenol induced a statistical

significant formation of DNA strand breaks. These concentrations were higher than the concentration

used in the MTT assay that already reduced significantly the cell viability, 5 mM of 4-nitrophenol and

3.5 mM of eugenol. Hence, at these concentrations it is not possible to exclude a cytotoxic effect. The

detected DNA strand breaks could be caused by cell death which induces the breakdown of the DNA.

At lower concentrations which did not reduced the cell viability, no DNA strand breaks could be

observed. However, these findings confirm the literature, because 4-nitrophenol and eugenol are

classified as non-DNA reactive [580].


156

11 Conclusions and outlook

The pre-validation have shown that the TOXXs Analyzer had serious technical issues. Several

attempts by the Cetics GmbH were conducted to identify and to fix the malfunction. However, these

attempts were unsuccessful. Therefore, it must be concluded that at the current development stage the

TOXXs Analyzer is no reliable platform for the FADU assay. Nevertheless, the source of the

malfunction seemed to be caused by the software rather than by the hardware. Since all hardware parts

of the TOXXs Analyzer were fully functional and errors seemed to occur only at certain assay steps.

Also during a revision by the supplier of the robot (Analytik Jena AG) no hardware defects were

recognized. Therefore, the trouble shooting should be focused on the software of the TOXXs

Analyzer. The performed experiments showed that chosen chemical test substances, cell lines and

SOPs are in principal working and can be used for the test of the TOXXs Analyzer.

Appendix

157

12 Appendix

12.1 Supplementary figures

Figure 12-1: Cell recovery and cell viability after 24 h of incubation in RPMI-1640 cell culture medium w/o FCS. Left

side: at the beginning of the treatment the cell concentration was adjusted to 2*106cells/ml. No significant difference was

detected after 24 h of incubation. Right side: the cell viability after an incubation of 24 h. The fraction of living cells was

slightly decreased by about 5.85%. Data represent means with SEM of ten experiments. Statistical analysis was performed

using t-test (*), * P = 0.025. Figure was adjusted from Thomas and Palombo, experiment performed by J. Salzwedel [421].

Figure 12-2: DNA strand breaks in PBMCs induced by the repeated isoproterenol treatment. Left side: DNA strand

breaks induced by the 8-fold isoproterenol treatment (8x iso), 6 h after the first isoproterenol treatment. The amount of DNA

strand breaks was converted into an irradiation equivalent according to Junk et al. [573]. Propranolol (prop) was applied 10

min prior the isoproterenol treatment to block the β2-AR. Data represent means with SEM of ten experiments. Statistical

analysis was performed using Wilcoxon matched-pairs test (*), ** P=0.002. Right side: DNA strand breaks 24 h after the first

isoproterenol treatment. Cells were treated either with a single dose (1x iso) of isoproterenol or with a 4 doses (4x iso) or 8

doses (8x iso) of isoproterenol. Data represent means with SEM of twelve experiments. Statistical analysis was performed

using Friedman`s test (*), ** P=0.007. Figure was adjusted from Thomas and Palombo, experiment performed by V.

Bazylianska [421].

Appendix

158

Figure 12-3: Intracellular ATP content of PBMCs after the repeated isoproterenol treatment. The intracellular ATP

content of PBMCs was significantly reduced 24 h after the repeated isoproterenol treatment. H2O2 (10 mM) was used as

positive control. Data represent means with SEM of six experiments. Statistical analysis was performed using Friedman`s test

(*), **** P=0.0022. Multiple comparison test showed statistical significance for the 8-fold isoproterenol treatment when

compared to controls. Figure was adjusted from Thomas and Palombo, experiment performed by N. Schäfer [421].

Figure 12-4: PAR formation after the isoproterenol treatment. Left side: representative diagram of the PAR distribution

in PBMCs after the isoproterenol treatments. Increasing amounts of isoproterenol administrations induced a shift of the

fluorescence signal to the left side and lead to the formation of a cell population with lower PAR content. Right side:

isoproterenol untreated and PAR positive cell population was gated and cells shifted to the left were defined as cells with

lower PAR content and counted for each treatment. Data represent means with SEM of six experiments. Statistical analysis

was performed using Friedman`s test, *** P=0.0001. Figure was adjusted from Thomas and Palombo, experiment performed

by T. Schumacher [421].

Figure 12-5: PARP1 protein expression after the isoproterenol treatment. The relative PARP1 protein amount was

measured 24 h after the first isoproterenol treatment. The repeated isoproterenol treatment decreased the PARP1 protein

content in PBMCs. Data represent means with SEM of eleven experiments. Statistical analysis was performed using

Friedman`s test, ** P=0.0035. Figure was adjusted from Thomas and Palombo, experiment performed by T. Schumacher

[421].

Appendix

159

Figure 12-6: Intra-individual variability of the isoproterenol induced PARP1 protein decrease. The PARP1 protein

level was measured in three different subjects in at least three independent experiments. TOP: representative diagrams of the

PARP1 protein signal after the isoproterenol treatment. Bottom: relative quantification of the mean fluorescence intensity of

the PARP1 protein. The PARP1 protein level of Donor A showed no response to the isoproterenol treatment, whereas Donor

B and C showed a significant decrease of the PARP1 protein. Data represent means with SEM of 4 experiments for Donor A

and C and 3 experiments for Donor B. Statistical analysis was performed using Friedman`s test, * P=0.033, *** P=0.009.

Figure was adjusted from Thomas and Palombo, experiment performed by T. Schumacher [421].

Appendix

160

Figure 12-7: Isoproterenol induce apoptosis in PBMCs. Left side: PBMCs were treated with isoproterenol and incubated

for 24 h in RPMI-1640 cell culture medium. Apoptosis was analyzed by annexin V and propidium iodide staining. Apoptotic

cells were quantified by a FACS measurement. The amount of apoptotic cells correlates positively with the applied

isoproterenol doses. Data represent means with SEM of 16 experiments. Statistical analysis was performed using Friedman`s

test, **** P=0.033. Right side: 5 subjects showed a high rate of apoptotic cells, 61.8% of total cells, whereas 8 subjects

showed a lower rate of apoptotic cells, 27.7 % of total cells. Figure was adjusted from Thomas and Palombo, experiment

performed by T. Schumacher and G. von Scheven [421].

t im e [h rs ]

ce

ll v

iab

ilit

y [

%]

024

48

72

96

120

0

2 0

4 0

6 0

8 0

1 0 0

w /o is o

**

ns

A )

t im e [h rs ]

ce

ll v

iab

ilit

y [

%]

024

48

72

96

120

0

2 0

4 0

6 0

8 0

1 0 0

8 x iso

w /o is o

ns

B )

Figure 12-8: Cell viability of PBMCs during the incubation in TexMACS cell culture medium. A) PBMCs were

cultured in TexMACS cell culture medium for 120 h. The cell viability was measured every 24 h by the CASY cell counter.

The cell viability decreased about 7.5% during the first 24 h. During the following incubation period no further significant

decrease of the cell viability was observed. Data represent means with SEM of five experiments. Statistical analysis was

performed using RM one-way ANOVA (*), ** P=0.0072. B) Comparison of the cell viability between untreated PBMCs and

8-fold isoproterenol treated PBMCs during an incubation period of 120 h. Cell viability was measured every 24 h by the

Appendix

161

CASY cell counter. Data represent means with SEM of five experiments. Statistical analysis was performed using RM two-

way ANOVA. Figure was adjusted from, Master Thesis Anita Grath [417].

Figure 12-9: PARP1 apoptotic cleavage after the isoproterenol treatment in TexMACS cell culture medium. PBMCs

were incubated for 24 h after the first isoproterenol treatment in TexMACS cell culture medium. Cells were lysed and the

lysates were used for a Western blot analysis of the apoptotic PARP1 cleavage products. Left side: PARP1 was stained with

the anti-PARP1 antibody, clone FI23 which recognizes the apoptotic N-terminal PARP1 cleavage fragment of 24 kDa. As

control Jurkat cells were treated (J+) or untreated (J-) with 25 µM of etoposide for 5 h to induce apoptosis. Actin and histone

H1 were used as loading control. Right side: The same Western blot as showed on the left side. The PARP1 protein was

additional stained with the anti-PARP1 antibody, clone CII10 which recognizes the apoptotic C-terminal PARP1 cleavage

fragment of 89 kDa. Red box marks the apoptotic cleavage fragment which could be only observed in the positive control

(J+).

Appendix

162

h o u rs

pe

ak

ar

ea

(in

ten

sit

y m

LU

)

00.5 1

1.5 2

2.5 3

3.5 4

4.5

0

2 0 0

4 0 0

6 0 0

8 0 0is o p r o t e r e n o l

t r e a tm e n tR P M I-1 6 4 0 w /o F C S

w ith P B M C s

A )

ns

R P M I-1 6 4 0 w /o F C S

w /o P B M C s

h o u rs

pe

ak

ar

ea

(in

ten

sit

y m

LU

)

00.5 1

1.5 2

2.5 3

3.5 4

4.5

0

2 0 0

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6 0 0

8 0 0

T exM A C S

w ith P B M C s


t r e a tm e n t

B )

ns

T exM A C S

w /o P B M C s

Figure 12-10: Time course of the isoproterenol concentration after the administration of a single dose of isoproterenol

in RPMI-1640 w/o FCS and TexMACS in the presence or absence of PBMCs. Re-plot of the data from Figure 4-15 and

Figure 4-18 for comparison of the time course of the isoproterenol content in the presence and absence of PBMCs in A)

RPMI-1650 cell culture medium without FCS and B) TexMACS cell culture medium. Red boxes indicate points in times

were isoproterenol was administered.

Appendix

163

h o u rs

iso

pr

ote

re

no

l

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ea

(in

ten

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LU

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

0

1 0 0 0

2 0 0 0

3 0 0 0

R P M I-1 6 4 0 w /o F C S

w ith P B M C s

R P M I-1 6 4 0 w /o F C S

w /o P B M C s

is o p r o te r e n o l

t r e a tm e n t

ns

A )

h o u rs

iso

pr

ote

re

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ak

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ea

(in

ten

sit

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)

0 1 2 3 4 5 6 7 8

0

1 0 0 0

2 0 0 0

3 0 0 0

R P M I-1 6 4 0 w ith F C S

w ith P B M C s


w /o P B M C s


t r e a tm e n t

ns

B )

h o u rs

iso

pr

ote

re

no

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pe

ak

ar

ea

(in

ten

sit

y m

LU

)

0 1 2 3 4 5 6 7 8

0

1 0 0 0

2 0 0 0

3 0 0 0T exM A C S

w ith P B M C s

T exM A C S

w /o P B M C s


t r e a tm e n t

ns

C )

Figure 12-11: Time course of the isoproterenol concentration during and after the administration of four doses of

isoproterenol in RPMI-1640 w/o FCS, RPMI-1640 with FCS and TexMACS cell culture medium in the presence or

absence of PBMCs. Re-plot of the data from Figure 4-16 and Figure 4-19 for comparison of the time course of the

isoproterenol content in the presence and absence of PBMCs in A) RPMI-1650 cell culture medium without FCS, B) RPMI-

1650 cell culture medium with FCS and C) TexMACS cell culture medium. Red boxes indicate points in times were

isoproterenol was administered.

Appendix

164

h o u rs

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ea

(in

ten

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)

0 1 2 3 4 5 6 7 8

0

2 0 0 0

4 0 0 0

6 0 0 0

R P M I-1 6 4 0 w /o F C S

w ith P B M C s

R P M I-1 6 4 0 w /o F C S

w /o P B M C s

ns

A )is o p r o t e r e n o l

t r e a tm e n t

h o u rs

iso

pr

ote

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(in

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)

0 1 2 3 4 5 6 7 8

0

2 0 0 0

4 0 0 0

6 0 0 0


w ith P B M C s


w /o P B M C sn

s

B )is o p r o t e r e n o l

t r e a tm e n t

h o u rs

iso

pr

ote

re

no

l

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ak

ar

ea

(in

ten

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0 1 2 3 4 5 6 7 8

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4 0 0 0

6 0 0 0

T exM A C S

w ith P B M C s

T exM A C S

w /o P B M C s


t r e a tm e n t

ns

C )

Figure 12-12: Time course of the isoproterenol concentration after and during the administration of eight doses of

isoproterenol in RPMI-1640 w/o FCS, RPMI-1640 with FCS and TexMACS cell culture medium in the presence or

absence of PBMCs. Re-plot of the data from Figure 4-17 and Figure 4-20 for comparison of the time course of the

isoproterenol content in the presence and absence of PBMCs in A) RPMI-1650 cell culture medium without FCS, B) RPMI-

1650 cell culture medium with FCS and C) TexMACS cell culture medium. Red boxes indicate points in times were

isoproterenol was administered.

Appendix

165

12.2 Genes analyzed by qPCR

Gene: Name: Description:

ADRB2 β2-adrenergic receptor smooth muscle relaxation

AKT3 protein kinase B (gamma) inhibition of apoptosis and

stimulation of cell

proliferation

ARRB1 β1-arrestin mediates desenitization of β-

adrenergic

B3GALTL 1,3-galactosyltransferase-like transfers glucose towards

fructose with β-1,3-linkage

B3GNT1 UDP-GlcNAc: βGal β-1,3-N-

acetylglucosaminyltransferase 1

Essential for synthesis of poly-

N-acetyllactosamine

BLM Blood Sybdrome, RecQ Helicase-Like DNA repair, HR pathway

BRCA1 Breast Cancer 1, Early Onset HR pathway

BRCA2 Breast Cancer 2, Early Onset HR pathway

BRIP1 BRCA1 Interacting Protein C-Terminal Helicase 1 DNA repair, HR pathway

CAT Catalase antioxidant enzyme in defense

against oxidative stress

CCND1 Cyclin D1 cell cycle G1/S transition (cell

cycle progression)

CDKN1A Cyclin-Dependent Kinase Inhibitor 1A (P21, Cip1) inhibitor of cellular

proliferation in response to

DNA damage ( regulator of

cell cycle progression at G1)

CDKN1C Cyclin-Dependent Kinase Inhibitor 1C inhibitor of several cyclin/Cdk

complexes

CYGB Cytoglobin may be involved in

intracellular oxygen transfer

and protection during

oxidative stress

DHCR2 24-Dehydrocholesterol Reductase cholesterol biosynthesis and

protection during oxidative

stress

ERCC5 Excision Repair Cross-Complementing Rodent

Repair Deficiency, Complementation Group5

DNA repair, NER pathway

GALNT4 UDP-N-Acetyl-Alpha-D-Galactosamine:Polypeptide

N-Acetylgalactosaminyltransferase 4

catalyses initial reaction in

mucin-type O-linked

glycosilation in the Golgi

apparatus




mucin-type O-linked


apparatus

Appendix

166




mucin-type O-linked


apparatus

GRK5 G Protein-Coupled Receptor Kinase 5 deactivates G protein coupled

receptors

Lig4 Ligase IV NHEJ pathway and V(D)J

recombination

MRE11A Meiotic Recombination 11 Homolog A HR pathway, NHEJ pathway

NOS3 Nitric Oxide Synthase 3 (Endothelial Cell) neurotransmission and

antimicrobial

and antitumoral activities and

participates in vascular smooth

muscle relaxation

OGG1 8-Oxoguanine DNA Glycosylase An enzyme responsible for

excision of 8-oxoguanine

PARP1 Poly(ADP-ribose) Polymerase 1 DNA damage repair, BER

pathway, NER pathway, DNA

double-strand break repair and

genomic stability

PARP2 Poly(ADP-ribose) Polymerase 2 DNA damage repair

POLβ DNA-Polymerase β BER pathway

POMGNT1 Protein O-linked Mannose N-

Acetylglucosaminyltransferase 1

participates in O-mannosyl

glycosylation

PRKDC Protein Kinase, DNA-Activated, Catalytic Polypeptide NHEJ pathway, V(D)J

recombination and telomere

stabilization

RPA1 Protein A1, 70 kDa NER pathway, DNA

replication and DNA

recombination

S100AB S100 Calcium Binding Protein A8 regulation of inflammatory

processes and immune

response (pro-inflammatory,

antimicrobial, oxidant-

scavenging and apoptosis

inducing)

SOD2 Mitochondrial Superoxide Dismutase 2 degradation of superoxide,

anion radicals

SRC V-Src Avian Sarvoma may play a role in regulation

of embryonic development and

cell growth and lymphocytes

activation

TANK Traf Family Member- Associated NFκB Activator negative regulator of Toll-like

receptors and B-cell receptors

signaling

Appendix

167

TERF2 Telomeric Repeat Binding Factor 2 telomere maintenance and

protection against end-to-end

fusion of chromosomes

TP53 Tumor Protein p53 induces cell cycle arrest,

apoptosis, senescence, DNA

repair

VCAN Versican intracellular signaling and

connecting cell with ECM

WRN Werner Syndrome RecQ Helicase-Like NHEJ pathway

XCL1 Chemokine (C-Motif) Ligand 1 inflammatory and

immunologicalresponses,

is specifically chemotactic for

lymophocytes

XPC Xeroderma Pigmentosum Complementation Group C NER pathway

XRCC1 X-Ray Repair Complementing Defective Repair in

Chinese Hamster cells 1

BER pathway

12.3 Contribution

Section: Figure: Contributor: Contribution:

4.1.1 Figure 4-1 Anita Grath, master student,

University of Konstanz

Measurement of cAMP concentration in

PBMCs after repeated isoproterenol

treatment in TexMACS cell culture

medium

4.1.3 Figure 4-3 Canesia Amarysti, trainee,

Cardiff University

Measurement of cellular PAR content

under NAD+ saturated conditions

4.2.2 Figure 4-7 Canesia Amarysti, trainee,

Cardiff University

Measurement of cellular p16 expression

9.1.1 Figure 9-4

and Figure

9-5

Monika Schulz, technical

assistant,

University of Konstanz

Performing MTT assay

Appendix

168

12.4 Publications

1) Thomas M., Palombo P, Schumacher T, von Scheven G, Bazylianska V, Salzwedel J, Schäfer N,

Bürkle A. and Moreno-Villanueva M, Impaired PARP activity in response to the β-adrenergic receptor

agonist isoproterenol. Toxicology in Vitro, 2018. 50: p. 29-39.

2) Palombo P, Moreno-Villanueva M. and Mangerich A, Day and night variations in the repair of

ionizing-radiation-induced DNA damage in mouse splenocytes. DNA Repair, 2015. 28: p. 37-47.

3) Palombo P, Grath A, Laumann L,. Bürkle A. and Moreno-Villanuev M, Senescence-like Phenotype

after Chronic exposure to Isoproterenol in Primary Quiescent Immune Cells. (In preparation).

4) Palombo P, Salzwedel J, Thomas M. Bürkle A. and Moreno-Villanueva M, Isoproterenol stability

and formation of isoprenochrome in cell culture medium. (In preparation).

12.5 Oral presentations

10.12.2015 At the “Wissenschaft trifft Wirtschaft Forum”, Universität Konstanz, Titel

„Measurement of DNA damage and repair using the automated FADU assay:

Examples from in vitro and in vivi studies“.

15.02.2015 Scientific colloquium, Universität Konstanz, Titel „Investigation of the

molecular mechanism of traumatic stress and its influence on the genomic

stability“.

Appendix

169

12.6 Participation in courses within the teaching program

of the Konstanz Research School Chemical Biology

• Good Scientific Practice (Dr. Michael gommel)

• Practical Screening Data Analysis (Prof. Dr. Michael Berthold)

• Proteomics (Dr. Andreas Marquardt)

• Bioimaging ( Prof. Dr. Elisa May)

• Biomedicin (Dr. Stefanie Bürger, Dr. Florian Rohrbach, PD Dr. Suzanne Kadereit, Dr. Anette

Sommershof, PD Dr. Michael Basler)

• Einführung in die Sicherheitsproblematik der Gentechnik „Fortbildung nach

§ 15 der Gentechnik-Sicherheitsverordnung. (Dr. Norbert Kunze)

• Kurs zu Grundlagen der Versuchstierkunde nach FELASA-Richtlinien. (Dr. Gerald Mende)

Appendix

170

12.7 List of abbreviations

% percent

(v/v) volume per volume

°C degree celsius

µl microliter

µM micromole

µm micrometer

8-OH-dG 8-hydroxydeoxyguanosine

Å Angström

AC adenylate cyclase

ACTH adrenocorticotropic hormone

ADH alkoholdehydrogenase

AIDS acquired immune deficiency syndrome

AIF apoptosis inducing factor

AKAPs A-kinase anchor proteins

AKT protein kinase B

AMP adenosine monophosphate

ANS autonomic nervous system

AP site apurinic/ apyrimidinic sites

AP2 AP2 adaptor complex

APC antigen-presenting cells

APE apurinic endonuclease

ARF alternate reading frame

ART ADP-ribosyltransferase domain

ATM ataxia telangiectasia mutated

ATP adenosine triphosphate

ATR ataxia telangiectasia and Rad3-related protein

BCA bicinchoninic acid

BER base excision repair

bp base pairs

BRCA2 breast and ovarian cancer susceptibility gene

BRCT BRCA1 C terminus domain

BSA bovine serum albumin

ca. circa

cAMP cyclic adenosine monophosphate

CDK cyclin-dependent kinase

cDNA complementary DNA

CES-D center for epidemiological studies depression scale

CETN2 centrin 2

CNS central nervous system

CO2 carbon dioxide

COMT catechol-O-methyltransferase

COPD chronic obstructive pulmonary disease

CPDs cyclobutane pyrimidine dimers

Appendix

171

Cq value threshold for quantification (qPCR)

CREB cAMP responsive element binding protein

CRF corticotrophin releasing factor

CV variation coefficient

DANN deoxyribonucleic acid

DANN-PKs DANN-dependent protein kinases

DCF 2´,7´- dichlorofluorescein

DCFDA 2’,7’ –dichlorofluorescin diacetate

DDR DNA damage response

DHE dihydroethidium

DHEA dehydroepiandrosterone

DHPG dihydoxyphenylgycol

DMSO dimethyl sulfoxide

DNA-PK DNA-dependent protein kinases

DOPA 3,4-dihydroxyphenylalanine

DOPAC 3,4-dihydroxyphenylacetic acid

DSBR double-strand break repair

DSM Diagnostic and Statistical Manual of the Mental Disorders

DTT dithiothreitol

dUTP 2´-deoxyuridine, 5´-triphosphate

E2F E2F transcription factor

ECL enhanced chemiluminescence

ECVAG European Comet Assay Validation Group

EDTA ethylenediaminetetraacetic acid

EPAC exchange proteins activated by cAMP

EPR electron paramagnetic resonance spectrometry

ERCC1 excision repair cross-complementation group 1

ERK1/2 extracellular signal-regulated kinase 1/2

ESCOOD European Standards Committee on Oxidative DNA Damage

FACS flow cytometry

FADU fluorometric analysis of DNA unwinding

FCS fetal calf serum

FISH fluorescence in situ hybridization

fmol femtomole

g g-force

gDNA genomic DNA

GDP guanosine diphosphate

GGR global genomic repair

Gi inhibitory G protein

GPCRs G protein couple receptors

GRK G protein couple receptor kinases

Gs cAMP-depending pathway

GTP guanosintriphosphat

Gy gray

Gα alpha subunit of the G protein

Gβγ beta and gamma subunit of the G protein

h hour

Appendix

172

H2DCF 2´,7´-dichlordihydrofluorescein

H2O2 hydrogen peroxide

H-89 N-[2-(p-Bromocinnamylamino)ethyl]

-5-isoquinolinesulfonamide

dihydrochloride

HClO4 perchloric acid

HDACs histone deacetylases

HEK cells human embryonic kidney cells

HIV human immuno-deficiency virus

HPA hypothalamic-pituitary-adrenal axis

HPLC high performance liquid chromatography

HR homologous recombination

HSP heat shock protein

IBMX 3-isobutyl-1-methylxanthine

IDLs insertion/deletion loops

IFN interferon

IL interleukin

IMI imidazolidine

iso isoproterenol

JaCVAM Japanese Center for the Validation of Alternative Methods

JNK1 c-Jun N-terminal kinase 1

KClO4 potassium per chloride

L1 suspension buffer

L2 neutralization buffer

L3 unwinding buffer

L4 SYBER Grenn I solution

LPS lipopolysaccaride

M molar

mA milli ampere

MAO monoamine oxidase

MAPK mitogen-activated protein kinase

MDM2 mouse double minute 2 homolog

MEN menadion

mfi nuclear localization sequence

MGMT O6-methylguanin-DANN-methyltransferase

MilliQ

min minute

ml milliliter

mM millimole

MMR mismatch repair

mRNA messenger RNA

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

NAD+ nicotinamidadenindinucleotid

NADPH nicotinamide adenine dinucleotide phosphate

NaIO4 sodium periodate

NER nucleotide excision repair

Appendix

173

ng nanogram

NHEJ non-homologous end joining

NLS nuclear localization sequence

nm nanometer

OECD Organization for Economic Co-operation and Development

OIS oncogene-induced senescence

p14 ARF tumor suppressor

p16 cyclin dependent kinase inhibitor 2A

p21 cyclin dependent kinase inhibitor 1A

p38 P38 mitogen-activated protein kinases

PAR poly(ADP-ribose)

PARG Poly(ADP-ribose) glycohydrolase

PARP Poly(ADP-ribose) Polymerase

PARP1 Poly(ADP-ribose) Polymerase-1

PARP2 Poly(ADP-ribose) Polymerase-2

PBMCs peripheral blood mononuclear cells

PBS phosphate-buffered saline

PCR polymerase chain reaction

PDE phosphodiesterase

PES phenazine ethosulfate

PFA paraformaldehyd

pH pH-value

PHA phytohaemagglutinin

PI3K phosphatidylinositol-4,5-bisphosphate 3-kinase

PK1 positive control 1

PK2 positive control 2

PKA protein kinase A

PKC protein kinase C

pmol pikomole

PNI psychoneuroimmunology

PNKP polynucleotide kinase 3`-phosphatase

Polβ polymerase beta

POLδ polymerase delta

POMS the profile of mood states

PTSD posttraumatic stress disorder

PVN paraventricular nucleus

qPCR real time quantitative PCR

Rap1 ras-related protein 1

Ras rat sarcoma

RB retinoblastom protein

RIPA radioimmunoprecipitation assay

RM repeated measures

RNS reactive nitrogen species

ROS reactive oxygen species

rpm rounds per minute

RQ RNA quality assay

RT room temperature

Appendix

174

s second

SAHF senescence-associated heterochromatin foci

SAM sympathetic-adrenal-medullary axis

SASP senescence-associated secretory phenotype

SA-β-

galactosidase

senescence-associated beta-galactosidase

SDS sodium dodecyl sulfate

SDSA synthesis-dependent strand annealing

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEM standard error of the mean

SIPS stress-induced premature senescence

SNS sympathetic nervous system

Src proto-Oncogene Tyrosine-Protein Kinase Src

TBHP tert-butylhydroperoxid

TNF tumor necrosis factor

UV ultraviolet

UV-light ultraviolet light

w/o without

α-AR alpha-adrenergic receptor

β2-AR beta2-adrenergic receptor

β-AR beta-adrenergic receptor

References

175

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Danksagung

201

Danksagung

Zuerst möchte ich mich bei meinem Doktorvater Alexander Bürkle bedanken für die Möglichkeit bei

ihm zu promovieren und mir dabei die außergewöhnliche Möglichkeit bot angewandte Forschung und

Grundlagenforschung zu kombinieren. Dabei stand er mir immer mir Rat und Tat zur Seite.

Zudem danke ich Prof. Dr. Markus Christmann herzlichst für die Erstellung des Zweitgutachtens.

Ganz besonders möchte ich mich bei Dr. Maria Moreno-Villanueva bedanken für die Möglichkeit an

den interessanten Projekten zu arbeiten und die vielen „out of the Box“ Ideen. Außerdem für die vielen

und langen wissenschaftlichen Diskussionen und Gespräche, die großartige Betreuung und

Motivation, inklusive Standleitung nach Houston.

Zudem gebührt mein Dank meinem Thesis Komitee, bestehend aus Prof. Dr. Alexander Bürkle, Prof.

Dr. Valentin Wittmann und Prof. Dr. Christof Hauck.

Bedanken möchte ich mich auch aufs insbesondere bei der Cetics GmbH für die Finanzierung des

Promotionsprojektes. Bei unseren Kooperationspartner bei der Cetics GmbH Dr. Marcel Pilartz, Dr.

Inka Pfitzner und Dr. Karin Engelhart. Ebenso möchte ich mich bei unseren Kooperationspartner am

EMPA bedanken Dr. Peter Wick und Dr. Cordula Hirsch.

Einen ganz besonderen Dank schulde ich Anita, Mara, Tamara und Isabell die Ihre Masterarbeit am

selben Projekt durchgeführt haben und für den ein oder anderen Blödsinn zwischen durch zu haben

waren.

Ganz besonderen Dank schulde ich Monika, Gudrun, Thilo, Walli und Claudia für das Blutabnehmen,

die Aufreinigung von Litern von Blut, technischer Unterstützung und Hilfe bei administrativen

Dingen. Und für den ein oder anderen Blödsinn im Labor oder beim Essen.

Für die vielen wissenschaftlichen und nicht wissenschaftlichen Diskussionen bedanke ich mich bei

Matze, Jan, Sebastian, Benny, Judy, Tapes, Annika, Party Arty, Julia und Aswin. Mein herzlichen

Dank geht auch an Matze, Jan, Sebastian, Party Arty, Julia, Tapes, Benny, Judy, Magda, Jenny, Lisa,

Irmela, Mariam und Andy für die vielen Aktivitäten außerhalb des Labors.

Bedanken möchte ich mich auch bei den vielen Studenten die an dem Projekt mitgearbeitet haben oder

den ein oder anderen Quatsch während ihrer Zeit im Labor mitgemacht haben Daisy, Alex, Martin,

Matze, Steffen, Schorsch, Xiau, Canesia und Victoria.

Andy und Claudia für ihr Feedback beim Schreiben der Thesis.

Der größte Dank gilt jedoch meiner Familie für die Unterstützung in jeglicher Hinsicht.

Investigation of stress biomarkers in human peripheral blood ...

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