Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

Lack of molecular relationships between lipid peroxidation and

mitochondrial DNA single strand breaks in isolated rat hepatocytes

and mitochondria

Claudia Scottia,*,1, Luisa Iamele1a, Andrea Alessandrinib, Vanio Vanninia,Ornella Cazzalinia, Maria C. Lazzea, Raffaele Mellia, Monica Savioa, Roberto Pizzalaa,

Lucia A. Stivalaa, Silvia Biglieria, Aldo Tomasic, Livia Bianchia

aDipartimento di Medicina Sperimentale, Sezione di Patologia Generale, Universita di Pavia, Piazza Botta 10, 27100 Pavia, ItalybINFM e Dipartimento di Fisica, Universita di Bologna, Via Irnerio 46, 40126, Bologna, Italy

cDipartimento di Scienze Biomediche, Sezione di Patologia Generale, Universita di Modena e Reggio Emilia, Via Campi 287,

41100 Modena, Italy

Received 13 August 2002; received in revised form 4 December 2002; accepted 16 December 2002

Abstract

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand

breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that

mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free

radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible

molecular targets for the prevention of this kind of damage in vivo.

q 2003 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved.

Keywords: Mitochondrial DNA; Tert-butylhydroperoxide; Single strand breaks; Atomic force microscopy; Lipid peroxidation

1. Introduction

Mitochondria are the main site for the production

of reactive oxygen species (ROS) and up to 5% of the

total molecular oxygen utilized by mammalian

mitochondria is converted into ROS in the respiratory

chain of the inner mitochondrial membrane (Boveris

and Chance, 1973). Though continuously formed,

ROS occur physiologically at a controlled rate. Under

oxidative conditions their production is dramatically

increased and this induces alterations of cellular

biomolecules, like membrane lipids, proteins and

nucleic acids (Halliwell, 1994). Oxidative damage of

membranes, proteins and DNA has been associated to

several chronic degenerative diseases and aging

(Ames, 1983; Cerutti, 1985; Hertog et al., 1993).

Particularly, mitochondrial biomolecules are known

to be a main target of oxidative alterations. When cells

or isolated mitochondria are exposed to oxidant

1567-7249/03/$30.00 q 2003 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved.

doi:10.1016/S1567-7249(03)00004-7

Mitochondrion 2 (2003) 361–373

www.elsevier.com/locate/mito

1 These two authors gave equal contributions to the project.

* Corresponding author. Tel.: þ39-382-506-338; fax: þ39-382-

303-673.

E-mail address: [email protected] (C. Scotti).

http://www.elsevier.com/locate/mito

agents, mitochondrial DNA (mtDNA), that was

reported to be more sensitive to oxidative damage

than nuclear DNA (nDNA) (Richter et al., 1988;

Yakes and Van Houten, 1997; Bandy and Davison,

1990), undergoes a high frequency of base damage

(Richter et al., 1988; Yakes and Van Houten, 1997),

single-strand breaks (Kaneko and Inoue, 1998) and a

loss of its normal electrophoretic banding pattern

(Hruszkewycz and Bergtold, 1990). Oxidative

mtDNA lesions can interfere with mtDNA metab-

olism and processing, and might lead to the typical

alterations of mtDNA (point mutations, fragmenta-

tion, deletions, and duplications) that have been

implicated etiologically in human diseases (Johns,

1995) and aging (Shigenaga et al., 1994).

The detailed interrelationships between the mol-

ecular processes that lead to cell damage following an

increase in ROS are still unclear, but a close

association has been demonstrated between mem-

brane oxidation and DNA damage. Oxidation of lipids

has been shown to induce specific DNA damage

(Vaca et al., 1988a,b; Ames and Gold, 1991),

predominantly in the form of single strand breaks,

both in chemical model systems (Inouye, 1984; Ueda

et al., 1985) and in isolated cells (Nakayama et al.,

1986) or nuclei (Vaca et al., 1988a; Vaca and

Harms-Ringdahl, 1989a,b; Friedberg et al., 1995).

Yet, the details of the reaction mechanisms involved

are still poorly understood. Particularly, there is

considerable controversy over which intermediate

free radicals or secondary oxidation products are

responsible for DNA damage, while these data would

be extremely valuable to establish proper targets for

prevention and therapy.

Tert-butylhydroperoxide (TBH) is a short chain

analogue of a lipid hydroperoxide, and it is widely

used as a model compound in order to study oxidative

conditions involving membrane lipid impairment.

Beside the predominant transformation into the

metabolically inert tert-butanol by gluthation peroxi-

dase (Rush et al., 1985), TBH decomposes in the

presence of transition metals yielding tert-butoxyl or

tert-butylperoxyl free radicals, short-chain analogues

of lipid peroxidation intermediates (Kennedy et al.,

1992). They can trigger a number of cellular

alterations including lipid peroxidation and mitochon-

drial damage (Rush et al., 1985; Bellomo et al., 1984;

Guidarelli et al., 1997b; Castilho et al., 1995;

Nieminen et al., 1995), while single-strand breaks

(ssb) are the most relevant lesion caused at the level of

nDNA (Coleman et al., 1989; Sandstrom and

Marklund, 1990; Baker and He, 1991; Latour et al.,

1995; Guidarelli et al., 1995, 1996, 1997a). It has been

widely reported that cleavage of nDNA by TBH is

prevented by iron chelators and insensitive to chain-

breaking antioxidants (Coleman et al., 1989; Latour

et al., 1995; Guidarelli et al., 1995). This supports the

independence of nDNA ssb from a lipid peroxidation

mechanism and suggests the involvement of other

chemical species. It was shown that at least some of

these chemical species are actually formed in the

mitochondrion (Guidarelli et al., 1997b).

In this view, and considering its peculiar sensitivity

and location, mtDNA turns out to be an interesting

target of investigation. The aim of this work was first

of all to verify whether TBH could induce both lipid

peroxidation and mtDNA ssb in well characterized

experimental sets (cells and mitochondria isolated

from rat liver). We then gave a precise quantification

of the mtDNA ssb observed, defined the relationship

between mtDNA ssb and lipid peroxidation, and,

finally, determined the possible reactive chemical

species involved.

2. Materials and methods

2.1. Chemicals

A 70% (v/v) solution of TBH in water, bovine

serum albumin (fraction V), a-tocoferol (TF), defer-

oxamine mesilate (DFO), low melting point agarose

and all the chemicals for hepatocytes and mitochon-

dria isolation were purchased from Sigma (St. Louis,

MO, USA). 2,6-Di-tert-butyl-4-methylphenol (BHT)

was from Merck (Darmstadt, Germany), Seakem

Gold agarose from FMC BioProducts (Rockland,

ME, USA). Reagents for digoxigenin-labelling of

probes, agarase, Xho I, Pvu II, and plasmid pBR322

were from Boehringer (Mannheim, Germany). Ultra-

hyb was purchased from Ambion (Austin, TX, USA).

Solutions of nitroblue tetrazolium chloride and 5-

bromo-4-chloro-3-indolyl-phosphate, 4 toluidine salt

(BCIP) in dimethylformamide, and CDP-stare were

purchased from Roche Diagnostics (Milan, Italy).

Expand long polymerase chain reaction (PCR) kit was

C. Scotti et al. / Mitochondrion 2 (2003) 361–373362

purchased from Boehringer (Mannheim, Germany),

and AmpliTaqw DNA polymerase and other reagents

for PCR from Perkin Elmer Applied Biosystems

(Milan, Italy). 5-Diethoxyphospho-5-methyl-1-pyrro-

line n-oxide (DEPMPO) was from Oxis (Portland,

OR, USA).

2.2. Hepatocytes and mitochondria isolation

Male Wistar rats (200–250 g) were supplied by

Harlan Italy (Correzzana, Italy). The animals received

a standard diet and water ad libitum and were fasted

overnight before experiments. Hepatocytes were

isolated following the procedure described by Mol-

deus et al. (1978). Cells were then suspended at a final

concentration of 5 £ 106/ml in a modified Krebs

incubation buffer (pH 7.4) containing 86 mM NaCl,

20 mM KCl, 50 mM HEPES, 1 mM CaCl2, 2 mM

MgSO4, 1 mM Na2HPO4, 5.5 mM glucose. Cells

viability at the time of isolation ranged between 88

and 90%. For mitochondria isolation, rats were killed

by decapitation. Livers were removed, homogenized

in 0.25 M sucrose and subjected to differential

centrifugation (Darley-Usmar et al., 1987). The

mitochondrial pellet was then resuspended in 50

mM Tris–HCl pH 7.4, 150 mM KCl at 3 mg/ml final

protein concentration.

2.3. Incubations and assays

TBH was added to the samples from concentrated

stock solutions prepared in the appropriate incubation

buffers and cells or mitochondria were incubated at

378C in air in a thermoregulated shaking water bath.

BHT (1 mM final concentration) was used to stop the

oxidation reaction and, when required, 15 min pre-

incubations with 20 mM DFO or 10 mM TF were

performed. DFO was pre-dissolved in water and TF in

ethanol. TF was added to cells and mitochondria from

concentrated stock solutions, such that the final

ethanol concentrations were ,0.3% (v/v) and

,1.6% (v/v), respectively. Under these conditions,

ethanol did not produce any detectable effects on

thiobarbituric acid reactive substances (TBARS),

hepatocytes viability or mtDNA integrity (data not

shown). Protein content was determined by the

Lowry’s assay (Lowry et al., 1951) and cell viability

by the Trypan blue exclusion method. Following

protein precipitation with trichloroacetic acid, lipid

peroxidation was assessed spectrophotometrically as

TBARS (Sinnhuber and Yu, 1957) and expressed as

nmoles/mg proteins.

TBH free radicals were trapped with 50 mM

DEPMPO and spectra measured with a Bruker 300

electron spin resonance (ESR) spectrometer, operat-

ing at 9.5 GHz. Measurements were made at 378C,

using a standard flow dewar for temperature regu-

lation. The samples were placed into the ESR cavity

in a 100 ml flat cell. The spectrometer settings for the

samples containing DEPMPO were: incident micro-

wave power, 20 mW; modulation amplitude, 0.1 mT;

time constant, 160 ms; scan time, 3.20 s; scan range,

11.0 mT. Spectra simulations were performed with

the Bruker Symphonia Simulation package.

2.4. Analysis of mtDNA damage

2.4.1. DNA extraction

Plasmid pBR322 was cut with HinfI (pBR322-H)

and 500 ng were added to 1 £ 107 freshly isolated

hepatocytes. The same amount of plasmid linearized

with Pvu II (pBR322-P) was mixed with an aliquot of

mitochondria equivalent to 50 mg proteins. DNA was

then purified according to Driggers et al. (1997). After

an incubation with RNAse A, DNA was purified with

one volume chlorophorm:isoamyl alcohol (24:1) and

resuspended in Tris 10 mM, EDTA 1 mM, pH 8.0.

2.4.2. Depletion analysis

Southern blots were performed on total DNA from

isolated hepatocytes using the 18 Svedberg (18S)

nuclear gene as internal standard (Masini et al., 1999).

The 1870 bp of the rat 18S gene were PCR-amplified

in standard conditions using eukariote-specific pri-

mers A (sense primer: 50: AAC CTG GTT GAT CCT

GCC AGT: 30, nt 1–21) and B (antisense primer: 50:

GAT CCT TCT GCA GGT TCA CCT AC: 30, nt

1870–1847), while Buffer 3 from Expand Long PCR

kit, and primers P3 (sense primer: 50: TGG AGG TAA

GAT TAC ACA TG: 30, nt 98–117) and P4 (antisense

primer: 50: GAG GGT AGG CAA GTA AAG AGG

G: 30, nt 16,278–16,267) were used for rat mtDNA

amplification (16,005 out of 16,300 bp) following the

manufacturer instructions. These PCR products were

labelled with digoxigenin by random priming and

then used as probes.

C. Scotti et al. / Mitochondrion 2 (2003) 361–373 363

DNA extracted from isolated mitochondria was run

on a 0.6% Seakem Gold agarose gel with ethidium

bromide 5 mg/ml in 90 mM Tris base, 90 mM boric

acid, 1 mM EDTA at 0.8 V/cm for 20 h. The gel was

photographed on a UV transilluminator by a Polaroid

Camera (Fotodyne, New Berlin, WI, USA) on a 667

Polaroid film (St. Albans, UK).

The images of the gels and the filters were digitized

by a UMAX Power Look II scanner (UMAX Data

System, Taipei, Taiwan) and analysed using the

public domain NIH Image program (U.S. National

Institutes of Health, available on the Internet at http://

rsb.info.nih.gov/nih-image/) with the aid of the

bundled gel plotting macros II. For each sample, the

ratios between total mtDNA (equivalent to forms

I þ II þ III for isolated mitochondria) and the appro-

priate standard were calculated. Following similar

procedures, calibration curves were built.

2.4.3. Single-strand breaks analysis

For isolated hepatocytes, mtDNA ssb were quanti-

fied using denaturing gel electrophoresis as described

by Kaneko and Inoue (1998). Briefly, 12 mg DNA

were digested with Xho I to linearize mtDNA, mixed

with 6 £ loading buffer (300 mM NaOH, 6 mM

EDTA, 18% Ficoll, 0.25% Xylene cyanole) and

loaded on an 0.8% agarose gel prepared in running

buffer (30 mM NaOH, 0.1 M EDTA, pH 8.0). The run

was performed at 3 Volts/cm for 20 h. After the

capillary transfer and the first hybridization with the

mtDNA probe, the filters were stripped and hybri-

dized with a probe for the 1631 bp fragment of

pBR322-H. Ultrahyb was used as pre-hybridization

and hybridization solution following the manufacturer

instructions, and CDP-stare as substrate of the

alkaline-phosphatase conjugated anti-digoxigenin

antibody. Film digitization and analysis were per-

formed as described above and mtDNA/pBR322-H

was calculated. The average number of ssb (ssb) per

mtDNA molecule was estimated assuming a Poisson

distribution (Kaneko and Inoue, 1998):

ssb ¼ 2ln P0;

where P0 is the fraction of mtDNA/pBR322-H

remaining after each treatment.

For isolated mitochondria, mtDNA ssb were

quantified according to Epe and Hegler (1994), after

atomic force microscopy (AFM) (see below) had

confirmed the nature of each band. Agarose gel

electrophoresis and image analysis were performed in

the same conditions described above for depletion

analysis. A Poisson formula was used, assuming that

only the first single strand break in each molecule

induced the relaxation:

ssb ¼ 2ln½1:4sc=ð1:4sc þ ocÞ�;

where ssb is the average number of ssb per DNA

molecule, sc and oc the integrated pixel intensities of

forms I and II (35), respectively, and 1.4 an empirical

factor used to account for the relatively lower

fluorescence of ethidium bromide in supercoiled

compared to the relaxed form of DNA (Epe and

Hegler, 1994).

2.4.4. Atomic force microscope

AFM was performed with a Nanoscope III (Digital

Instruments, Santa Barbara, CA, USA) equipped with

a Multimode head, a 12-nm scanner (E-scanner) and a

tapping mode liquid cell. Silicon nitride triangular

cantilevers with integrated tips and a nominal force

constant of 0.38 N/m were used. Electron beam

deposited tips were grown on the pyramidal tips

(Keller and Chou, 1992). The substrates were

prepared according to the method reported by

Schabert and Engel (1994).

After the separation of the mtDNA forms by

agarose gel electrophoresis, the three bands of each

lane from control and TBH-treated samples were

excised and the DNA electrophoretically transferred

to an 0.8% low-melting point agarose gel. Slices

containing the DNA were cut and the gel dissolved

with b-agarase (1 U/200 mg agarose) at 458C

overnight. Agarose was precipitated with 0.1 volume

of 3 M sodium acetate (pH 5.5) on ice for 15 min. The

DNA was recovered from the supernatant with ice-

cold absolute ethanol and resuspended in a 10 mM

HEPES, 5 mM NiCl2 buffer. A drop of the DNA

suspension was placed on the freshly cleaved disc of

untreated mica and after 2 min the specimen was

rinsed and overlaid with double-distilled water with-

out a drying step. Such non-trapping condition would

leave mtDNA molecules free to progressively lose

their original three-dimensional conformation in

favour of a stable two-dimensional configuration.

The deposition time of 2 min, however, is not enough

for such long molecules to completely equilibrate


http://rsb.info.nih.gov/nih-image/

http://rsb.info.nih.gov/nih-image/

(Rivetti et al., 1996), and the addition of double-

distilled water ‘freezes’ them in their conformation.

This allows reliable information to be drawn from

AFM data. AFM images were acquired in tapping

mode (Zhong et al., 1993) at a cantilever oscillation

frequency of about 8.8 ^ 0.3 kHz with 512 £ 512

pixels. The scan rate was about one to two lines/s.

Pictures scans were acquired and analysed by the

installed Nanoscope software and by Image SXM 162

software developed by Steve Barret and available on

the Internet at http://reg.ssci.liv.ac.uk. For the images

shown, only a polynomial best-fit (with an order from

1 to 3 depending on the scan size of the image) line-

by-line subtraction was applied to eliminate scanner

artifacts.

2.5. Statistical analysis

Results are expressed as mean ^ SD, with n $ 3.

Statistical inference was performed using SPSS 10.0.6

for Windows. Quantitative data analysis for multiple

independent samples was performed by the Kruskal–

Wallis test followed by single comparisons calculated

with the Mann–Whitney non-parametric test. The

level of statistical significance was taken at P , 0:05.

3. Results

3.1. Effects of TF and DFO on TBH-induced lipid

peroxidation and free radicals production

In order to investigate the effects of TBH on the

mtDNA of living cells, the sublethal conditions (TBH

concentration and time of treatment) able to trigger

the highest lipid peroxidation in isolated hepatocytes

were determined. Several concentrations of TBH

(0.25–1.5 mM) and incubation times (15, 30 and 60

min) were tested. Statistical analysis of the results

(Fig. 1) showed that the treatment of hepatocytes with

1 mM TBH for 60 min induced the strongest lipid

peroxidation compared to the control (P , 0:05)

without affecting cell viability (P . 0:05). These

conditions were selected for use in subsequent assays.

Pre-treatment of the cell suspensions with anti-

oxidants (Fig. 2) showed that 10 mM TF and 20 mM

DFO were able to reduce TBH-induced lipid peroxi-

dation by 27% (P , 0:05) and by .100% (P , 0:05),

respectively. When associated to TBH, both com-

pounds decreased cell viability compared to the

control (P , 0:05 for both antioxidants) and TF also

compared to TBH-treated cells (P , 0:05).

In isolated mitochondria, TBARS level was

significantly increased by 1 mM TBH versus control

(P , 0:05, Fig. 2) and this effect was totally

prevented by both 20 mM DFO (P , 0:05 versus

TBH, P . 0:05 versus control) and 10 mM TF

(P , 0:05 versus TBH, P . 0:05 versus control).

Despite the high concentration of TF used in our

Fig. 1. Percentage cell viability (B: 15 min, : 30 min, W: 60 min)

and lipid peroxidation : 15 min, : 30 min, : 60 min) of

hepatocytes exposed to increasing concentrations of TBH. Cell

viability was assessed using the Trypan blue exclusion assay and

lipid peroxidation measured as TBARS. Results are expressed as

mean ^ SD of n ¼ 3 independent experiments. *Significantly

different from control, P , 0:05.

Fig. 2. Effect of a-TF and DFO on hepatocytes viability ( ) and lipid

peroxidation of cells ( ) and mitochondria ( ) treated with TBH.

Cells and mitochondria were pre-incubated with 10 mM TF or 20

mM DFO for 15 min. Following incubation, cells were exposed to 1

mM TBH for 1 h. Cell viability was assessed using the Trypan blue

exclusion assay and lipid peroxidation as TBARS. Results are

expressed as mean ^ SD of n ¼ 4 independent experiments.

*Significantly different from control, P , 0:05.


http://reg.ssci.liv.ac.uk

experimental system (20 mM), lipid peroxidation was

not brought down to the control levels in isolated

hepatocytes in contrast to what happened in isolated

mitochondria, where TF was more efficient at

reducing TBARS. It has often been questioned

whether exogenously added TF will, during short-

term incubations, insert into cell membranes in a

physiologic (and, therefore, functional) state. The

different efficiency of TF observed in the two systems

could be related to its different solubility in the

membranes of isolated mitochondria, whose compo-

sition is peculiar compared to the one of most other

cellular membranes. However, in both systems the

protection exerted by TF from TBARS accumulation

was statistically significant.

Two free radical adducts were detected following

incubation of isolated mitochondria with TBH and the

spin trap DEPMPO (Fig. 3).

Similar results, but with a much lower signal

intensity, were obtained incubating isolated hepato-

cytes (data not shown). Computer simulations of the

individual DEPMPO free radical adducts are shown in

Fig. 3 and are identified as: (F), the hydroxyl radical

adduct to DEPMPO (a N ¼ 13.9G, a H ¼ 13.9 G,

a P ¼ 46.6 G, (40)); (G), the nitroxyl resulting form

the trapping of an alkyl free radical by DEPMPO,

which is identified mainly from the typically large

proton coupling (a N ¼ 15.1G, a H ¼ 22.3 G,

a P ¼ 47.4 G, (40)). A computer simulation of the

combined identified free radical adducts (Fig. 3E)

compares well with the adducts formed in the

organelles in the presence of TBH (Fig. 3B).

Preincubation of TBH-treated samples with TF did

not significantly alter the spectrum (Fig. 3D), while no

signals were detected in control samples (Fig. 3A) and

in those pre-exposed to DFO (Fig. 3C). The

observation of the DEPMPO/alkyl free radical

adducts when TF is added to the system is explained

in terms of hydrogen abstraction by hydroxyl free

radicals at the TF aliphatic chain. Such reaction can

occur in competition with the hydrogen abstraction at

the hydroxyl groups because of the low selectivity of

the hydroxyl radicals and the large number of the

secondary and tertiary CZH bonds available; in the

presence of oxygen such radicals can initiate hydro-

peroxide chains.

3.2. Analysis of mtDNA damage

3.2.1. Depletion

In isolated hepatocytes, mtDNA depletion was

analysed by Southern blot, referring the amount of

mtDNA to a standard. Since the amount of 18S gene

was not affected by TBH when compared to pBR322-

Fig. 3. ESR spectra of DEPMPO free radical adducts formed on

incubation of TBH (1 mM final concentration), a-TF and DFO with

1 ml of isolated mitochondria (3 mg protein). (A) ESR spectrum of

control mitochondria after 5 min following incubation with

DEPMPO; (B) following an exposure to TBH 1 mM for 5 min;

(C) after a preincubation with 20 mM DFO for 15 min plus an

exposure to TBH 1 mM for 5 min; and (D) after a preincubation

with 10 mM TF for 15 min plus an exposure to TBH 1 mM for 5

min. (E) Computer simulation obtained from the combination of

spectra (F) and (G). (F) Simulated spectrum of an alkyl free radical

adduct to DEPMPO. (G) Simulated spectrum of the DEPMPO/hy-

droxyl free radical adduct. Instrumental conditions were:

power ¼ 20 mW, modulation amplitude ¼ 0.1 mT, time

constant ¼ 160 ms, scan time ¼ 3.20 s, scan range ¼ 11.0 mT.


H (data not shown), mtDNA/18S ratio can be

considered a good indicator of possible variations of

the mtDNA copy number. No difference was detected

in the mtDNA/18S ratio of TBH-treated hepatocytes

compared to the control (P . 0:05, Fig. 4A).

A typical agarose gel electrophoresis of mtDNA

purified from isolated mitochondria is shown in Fig.

4B, where the presence of the internal standard

(pBR322-P) excludes any illusory reduction in the

absolute amount of mtDNA from the TBH treated

sample. In fact, no variations in the mtDNA content

(evaluated from the ratio between the total of the three

forms and pBR322-P) were detected following TBH

treatment (P . 0:05).

3.2.2. Effects of TF and DFO on mtDNA ssb induced

by TBH

In isolated hepatocytes, the number of ssb was

determined using the denaturing gel electrophoresis-

Southern blot method described by Kaneko and Inoue

(Kaneko and Inoue, 1998) implemented with the use

of an external standard. Fig. 5 shows the estimated

frequency of ssb determined by TBH in mtDNA

relative to the control (P , 0:05). This damage was

unaffected by TF (P . 0:05 versus TBH), but totally

Fig. 4. Analysis of mtDNA depletion in isolated hepatocytes (A); and mitochondria (B) exposed to 1 mM TBH for 1 h. C: control samples. The

amounts of mtDNA detected in hepatocytes using Southern blot (A, top), or gel electrophoresis (B, top) were normalized by the respective

standard (A, bottom: 18S, 18 svedberg gene, pBR322-H, 1631 bp fragment of pBR322 cut with HinfI; B, bottom: pBR322-P, pBR322 plasmid

linearized with Pvu II). Results are expressed as mean ^ SD of n ¼ 4 independent experiments.

Fig. 5. Effect of 10 mM a-TF and 20 mM DFO on mtDNA ssb in

hepatocytes ( ) and isolated mitochondria ( ) exposed to TBH for

1 h. For hepatocytes, mtDNA ssb were evaluated with Southern

blot, and for isolated mitochondria with agarose gel electrophor-

esis. Values are expressed as mean ^ SD of n ¼ 4 independent

experiments. *Significantly different from control, P , 0:05.


prevented by DFO (P , 0:05 versus TBH, P . 0:05

versus control).

In isolated mitochondria, ssb were calculated

according to Epe and Hegler (1994) exploiting the

AFM results, which clarified the location of the

supercoiled and open circular forms (see below).

Results are shown following subtraction of the

corresponding control values (Fig. 5). The estimated

number of mtDNA ssb showed a definite increase

following TBH treatment (P , 0:05). Removal of

lipid peroxidation by means of TF was paralleled by

an increase in ssb (P , 0:05 versus TBH), while

complete protection from mtDNA breakage was

achieved using DFO (P , 0:05 versus TBH, P .

0:05 versus control).

3.2.3. Atomic force microscope

AFM images of mtDNA molecules from control

and TBH-treated mitochondria are shown in Fig. 6.

The sequence from Figs. 6A–C refers to the three

bands detected in the control sample, the sequence

from Figs. 6D–F to the corresponding bands of TBH-

treated samples. Fig. 6A shows a typical conformation

of mtDNA from the slowest band of agarose gel

electrophoresis. Complex quaternary structures, made

of several mtDNA molecules in different conden-

sation states, were observed. They can possibly be

referred to catenanes and/or intermediate forms of

replication. In Fig. 6D a single molecule from the

corresponding band of the TBH-treated sample

illustrates the important modifications induced by

TBH. Here, mtDNA consisted exclusively in scat-

tered, individual molecules in a relaxed display and it

was never possible to find mtDNA aggregates.

Samples from the second band displayed a linear

conformation of whole molecules both in the control

(Fig. 6B) and the treated samples (Fig. 6E). Figs. 6C,F

show representative images of the third band from the

control and TBH-treated samples, respectively. Both

present mtDNA molecules in a supercoiled confor-

mation, either with the looped structures of toroidal

forms (Fig. 6F) or the tight interwinding and knots of

plectonemic ones (Fig. 6C). Contour length measure-

ments performed on single molecules from the control

and the treated samples resulted in an average length

of 6.00 ^ 0.32 mm.

4. Discussion

The organic hydroperoxide TBH is widely used as

a model compound to simulate conditions of oxidative

stress. Particularly, it can mimick the action of long-

chain lipid hydroperoxides, triggering the peroxi-

dation of cellular membranes via a free-radical

mediated process. In rat hepatocytes and several

other cell types (Coleman et al., 1989; Sandstrom and

Marklund, 1990; Baker and He, 1991; Latour et al.,

1995; Guidarelli et al., 1996; Fraga and Tappel, 1988)

TBH is also known to cause peroxidation-independent

nDNA ssb, probably due to some chemical species

produced in the mitochondrion (Guidarelli et al.,

1996). Thus far, any proof of mtDNA ssb was

however missing. In this study, we show that TBH

can in fact cause this kind of damage and give

evidence of the main chemical species implicated in

the process.

Two essential features of our experimental system

were preliminarily defined. First, in order to exclude

the interference of events due to cell death, we defined

the sublethal concentration of TBH able to induce the

highest lipid peroxidation in isolated hepatocytes.

Second, using an internal standard method, we

demonstrated that, in these conditions, TBH could

not significantly modify the total mtDNA content of

both hepatocytes and isolated mitochondria. This

ruled out possible artefacts (due, for example, to the

loss of mtDNA molecules covalently linked to

proteins during oxidation) that could influence the

subsequent observations.

The first step consisted in the morphological

analysis of mtDNA molecules extracted from isolated

mitochondria. AFM, associated to the variations in

integrated pixel intensities of the different bands,

Fig. 6. Tapping-mode AFM images of mtDNA molecules from each band of agarose gel electrophoresis. The mtDNA is deposited on the mica

in a 10 mM HEPES, 5 mM NiCl2 buffer. Images from (A) to (C) refer to forms II, III, I, respectively, of the control sample (C), whereas images

from (D) to (F) refer to the corresponding forms of the TBH treated sample. The effect of TBH on the mtDNA conformation is most striking on

form II, where the molecules from the treated sample are in a less compact form and with no aggregates.



provided evidence, at a single molecule resolution, of

the transition of supercoiled mtDNA into its open

circular conformation in isolated mitochondria treated

with TBH. The structure of the molecules visualized

in each band of our gels confirmed the pattern

disposition adopted by Richter et al. (1995) and

allowed the calculation of ssb according to Epe and

Hegler (1994). It also showed that the slowest band

consisted only in open circular forms after TBH

treatment, and that the complex quaternary structures

of the control had been completely disrupted.

In the mtDNA of both hepatocytes and isolated

mitochondria exposed to the action of the hydroper-

oxide, we measured a significative increase in ssb and,

at the same time, an accumulation in lipid peroxi-

dation detected as TBARS. Interestingly, the results

obtained following the exposure of isolated mito-

chondria to TBH overlapped with what observed in

isolated hepatocytes. This demonstrates that mito-

chondria are in fact a self-sufficient source of TBH-

derived species able to interact with DNA and that no

extra-mitochondrial factors are essential in generating

mtDNA ssb.

In order to analyse the relationship between

mtDNA ssb and lipid peroxidation, we employed a

chain-breaking (TF) and a preventive (DFO)

antioxidant. A pre-load with TF lowered the

TBARS levels, but did not prevent mtDNA ssb

either in hepatocytes or in isolated mitochondria.

This demonstrated that mtDNA ssb are completely

independent of the propagation and termination

reactions of lipid peroxidation, in agreement with

what reported for nDNA ssb (Coleman et al., 1989;

Latour et al., 1995; Guidarelli et al., 1995).

Two observations can be made at this point. The

first is that mtDNA ssb, like nDNA ssb (Coleman

et al., 1989), appear to be totally independent of

cell killing. In fact, mtDNA ssb increased in the

TBH treated sample, where no related alteration of

cell viability was observed.

The second regards two distinct effects observed

when hepatocytes and isolated mitochondria were

treated with TBH after a preincubation with the

antioxidant TF. A peculiar reduction in cell viability

appeared in isolated hepatocytes, while an unexpected

increase in mtDNA ssb occurred in isolated mito-

chondria. Both these events were apparently elicited

by the interaction of TBH with TF, since they were

not observed when the molecules were separately

given. Masaki et al. (1989) showed that hepatocytes

killing induced by TBH occurred by two distinct

mechanisms, one dependent on and one independent

of lipid peroxidation. The TBH toxicity we observed

in hepatocytes and isolated mitochondria in the

presence of TF could certainly be ascribed to this

latter mechanism, since lipid peroxidation was

significantly reduced compared to the TBH treated

sample. Several factors can be indicated as possible

culprits. First of all, TF can reduce ferric iron, thus

acting as a conspirator in the activation of TBH

(Yamamoto and Niki, 1988). Secondly, the chain-

breaking action of TF is mainly due to its high

reactivity with peroxyl free radicals. In principle, TF

can not only interrupt the lipid peroxidation chain,

through the transfer of a hydrogen to the lipid

hydroperoxyl radicals, but also regenerate TBH

from its peroxyl radical, TBOOz. It would follow a

persistence of TBH activation with an effective

enhancement of the hydroxyl free radicals yield:

TBOOH þ Fe2þ $ Fe3þ þ TBO2 þ zOH

Finally, the relatively high amount of TF present in

the system (20:1 molar ratio versus TBH) could

inhibit the lipid peroxidation pathway, and deviate

active chemical species towards the alternative, iron-

dependent, pathway of TBH activation. The important

role of iron in determining cell toxicity is confirmed

by the fact that a preincubation with DFO was not

related to an increase in cell killing. In isolated

mitochondria, where TF has a higher chain-breaking

effect compared to isolated hepatocytes and where

extra-mitochondrial antioxidants cannot act, the

production of non-lipid free radical species would

be further enhanced and may thus explain the

observed increase in mtDNA ssb.

We then addressed the issue of defining which free-

radical mediators were involved in the induction of

mtDNA ssb during lipid peroxidation by ESR analysis

of mitochondrial suspensions. Computer simulation

of the experimental ESR spectra, detected in the

presence of TBH, clearly showed the presence of

alkyl and hydroxyl free radical adducts of DEPMPO.

Alkyl and hydroxyl free radicals can directly lead to

DNA modifications (Halliwell, 1994; Hix et al.,


1995). Particularly, hydroxyl free radicals can nick

the DNA backbone (Dizdaroglu, 1993).

Both alkyl and hydroxyl free radicals were still

detected by ESR after a pre-incubation of mitochon-

drial suspensions with TF. This supports the fact that

TF could not prevent the accumulation of mtDNA ssb

because unable to effectively scavenge the free

radicals responsible for mtDNA damage.

The likely involvement of hydroxyl free radicals in

mtDNA ssb during lipid peroxidation is also

supported by the iron-dependence of mtDNA ssb in

our model systems. In fact, DFO completely pre-

vented mtDNA ssb and our computer simulations of

ESR spectra confirmed the disappearance of hydroxyl

free radicals in TBH-treated samples pre-incubated

with DFO.

Iron is involved in TBH decomposition and,

though the binding of iron to DNA in vivo still

remains a matter of speculation, it was reported that

DNA complexes can form with iron ions (FeIII) in in

vitro systems (Kasyanenko et al., 1998; Tsitskishvili,

1983). Single strand breaks are produced in plasmid

DNA (Toyokuni and Sagripanti, 1999) and in mtDNA

when rat liver mitochondria are incubated in the

presence of Fe(III) gluconate (Yaffee et al., 1996).

Yaffee et al. (1996) suggested that the possible

mitochondrial reduction of Fe(III) to Fe(II), followed

by a reaction with H2O2, generates hydroxyl free

radicals that could nick the DNA strands. Because of

the short diffusion distance of hydroxyl free radicals

in cells (about 2 nm) (Ward et al., 1985), DNA

modifications are expected to occur when such

species, or ferryl radicals that then give zOH radicals,

are formed by a Fenton-like reaction in close

proximity to DNA (Chevion, 1988). DFO removal

of the hypothetical mtDNA-bound iron could thus

explain the prevention of ssb accumulation in mtDNA

we observed. In order to reach the highest chelating

efficiency and rate of membrane penetration (Kpart

0.01), and to push its well-known aspecific radical

scavenging activity, we employed DFO at a high

concentration (20 mM) (Coleman et al., 1989;

Caraceni et al., 1995; Kyle et al., 1990), which also

excluded any possible prooxidant effect. We opted not

to use a ferrioxamine control (DFO:iron in a 1:1 ratio),

since it was shown that this complex is toxic for

isolated hepatocytes (Bergamini et al., 1999).

In conclusion, our study gives evidence for the first

time of ssb induced by TBH on mtDNA. Taken

together, the data contribute to the definition of the

mechanism of action of the short-chain lipid-hydro-

peroxide analogue TBH, and help to clarify the

relationship between two orders of events likely to be

involved in human pathology: lipid peroxidation and

mtDNA ssb. They also suggest that hydroxyl anions

and iron, and not lipid free radicals, can be the main

molecular targets for the prevention of both nDNA

and mtDNA ssb.

Acknowledgements

We acknowledge Prof. Laura Zonta for her leads

about statistical analysis and Prof. Antonio Faucitano

for his important contributions to the discussions on

computer simulations of ESR spectra. We thank Dr

Patrizia Sommi, Dr Paola Perucca, and Dr Paola

Mignosi for their generous and constant support.

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Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

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