Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats

Cardiovascular and lung inflammatory effects induced by systemically

administered diesel exhaust particles in rats

Abderrahim NEMMAR1*, Sultan Al-Maskari2, Badreldin H. ALI3 and Issa S. Al-Amri4

1Department of Physiology, 2Dean’s Office, 3Department of Pharmacology,

4Department of Pathology, Electron Microscopy Unit, College of Medicine & Health

Sciences, Sultan Qaboos University, P O Box 35, Muscat 123, Al-Khod, Sultanate of

Oman

*Correspondence address to: Dr A. NEMMAR

Sultan Qaboos University

College of Medicine & Health Sciences

Department of Physiology

P O Box 35, Muscat 123,

Al-Khod

Sultanate of Oman.

Tel: 00968-24143435

Fax: 00968-24143514

Email: [email protected]

Page 1 of 30Articles in PresS. Am J Physiol Lung Cell Mol Physiol (November 3, 2006). doi:10.1152/ajplung.00240.2006

Copyright © 2006 by the American Physiological Society.

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Abstract

Pollution by particulates has consistently been associated with increased

cardiorespiratory morbidity and mortality. It has been suggested that ultrafine

particles, of which diesel exhaust particles (DEP) are significant contributors, are able

to translocate from the airways into the bloodstream in vivo. In the present study, we

assessed the effect of systemic administration of DEP on cardiovascular and

respiratory parameters. DEP were administered into the tail vein of rats and heart

rate, blood pressure, blood platelet activation, and lung inflammation were studied, 24

h later. Doses of 0.02, 0.1 or 0.5 mg DEP/kg (8, 42 or 212 µg DEP/rat) induced a

significant decrease of heart rate and blood pressure, compared to saline treated

rats. While the number of platelets was not affected, all the doses of DEP caused a

shortening of the bleeding time. Similarly, in addition to triggering lung oedema, the

bronchoalvealar lavage analysis revealed the presence of neutrophil influx in DEP-

treated rats, in a dose-dependent manner. We conclude that the presence of DEP

particles in the systemic circulation leads not only to cardiovascular and haemostatic

changes but it also triggers pulmonary inflammation.

Keywords: air pollution, diesel exhaust particles, Lung inflammation, heart.

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Introduction

Numerous epidemiological studies reported consistent associations between

exposures to particulate air pollution with a diameter ≤ 10 µm (PM10) and

cardiorespiratory mortality and morbidity (3; 40; 41). These studies found

associations between particulate matter and hospital admissions for various

cardiovascular diseases, including congestive heart failure (41; 42) and coronary

heart disease (37). Also, an increased risk for acute myocardial infarction (31; 32) and

cardiorespiratory symptoms (19) have been reported in association with particulate air

pollution.

The strongest associations were found for fine particles with a diameter <2.5

µm (PM2.5), and that have an important role in triggering pathophysiological changes

(31; 40). These particles, and particularly the ultrafine fraction (<100 nm), of which the

combustion-derived particulates of diesel exhaust are an important component,

penetrate deeply into the respiratory tract; and can carry large amounts of toxic

compounds, such as hydrocarbons and metals, on their surfaces (7).

Currently, different lines of particle-related research are being pursued (2; 25;

29; 46). It has been suggested that inhaled particles may lead to pulmonary

inflammation and subsequent release of soluble mediators that may influence blood

coagulation parameters (8). The autonomic nervous system may also be a target for

the adverse effects of air pollution (10). We (23; 28) and others (9; 18; 30; 44) have

reported extrapulmonary translocation of UFPs after intratracheal instillation or

inhalation, suggesting an alternative and/or a complementary explanation for the

cardiovascular effects of particles. However, the mechanisms related to the

cardiorespiratory effects of translocated particles are not well known.

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We recently reported in hamsters that DEP lead to a significant prothrombotic

tendency, activation of circulating blood platelets, as well as lung inflammation as

early as 1 h and persisting up to 24 h (22; 24; 27). Pulmonary inflammation and

peripheral thrombosis were correlated at 6 and 24 h, but the prothrombotic tendency

observed 1 h after DEP exposure did not appear to correlate with pulmonary

inflammation (27). The latter is compatible with direct platelet activation by DEP,

having presumably penetrated into the circulation (9; 18; 30; 44).

To circumvent the effects related to pulmonary accumulation of particles and

release of inflammatory mediators, several studies adopted a pharmacodynamic

approach consisting of administering precise amount of particles intravascularly. It

has been shown that within 1-2 h after their systemic administration, UFP cause

prothrombotic effects in the femoral vein of hamsters (26), ear vein of rats (43) and

the hepatic microvasculature of mice (16). However, the direct effect of particles on

cardiovascular endpoints and pulmonary inflammation is not known.

Therefore, the aim of this study was to investigate, in vivo, the acute (24 h)

effects of systemic administration of DEP on heart rate, blood pressure and

haemostasis, and to assess whether and to which extent these effects are associated

with the development of pulmonary inflammation.

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Material and Methods

Particles

We used diesel exhaust particles (DEP; SRM 2975) from the National Institute

of Standards and Technology (NIST, Gaithersburg, MD, USA). DEP were suspended

in sterile saline (NaCl 0.9 %) containing Tween 80 (0.1 %). To minimize aggregation,

particle suspensions were always sonicated (Clifton Ultrasonic Bath, Clifton, New

Jersey, USA) for 15 min and vortexed before their dilution and prior to intravenous

administration. Control animals received saline containing Tween 80 (0.1 %).

For electron microscopy, droplets (10 µL) of a suspension of 1 mg of DEP in

500 µL were placed on matured formvar/carbon film for 30 seconds. The samples

were then drained and inverted onto droplets of ultrapure water for 1 hour before

being drained, dried, and examined in a JEOL (JEM 1230) electron microscope.

Systemic administration of particles

This project was reviewed and approved by the Institutional Review Board of the

Sultan Qaboos University and experiments were performed in accordance with

protocols approved by the Institutional Animal Care and Research Advisory

Committee.

Sixteen-week-old Male Wistar Kyoto (WKY) rats (Taconic Farms Inc.,

Germantown, New York, USA), weighing 424 ± 8 g were placed in restrainers. The

tail was desinfected with ethanol, and 150 µl of vehicle or doses of 0.02, 0.1 or 0.5

mg DEP/kg corresponding to about 8 µg, 42 µg or 212 µg DEP/rat were injected into

the tail vein.

Experiments could not be completed on all animals the same day. However, at

least one relevant control animal was always included on each experimental day.

Twenty-four h after the systemic administration of DEP, the animals were subjected

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to heart rate and blood pressure measurements, tail bleeding time experiments, lung

wet-to-dry weight ratio and the analysis of bronchoalveolar (BAL) fluid.

Blood pressure and heart rate measurements

Twenty-four hours following the systemic administration of DEP, heart rate and

blood pressure were measured in the conscious restrained rats using a computerized

tail-cuff system (Harvard apparatus; Columbus Instruments) (4; 47; 50).

Bleeding time measurements

To determine the consequences of enhanced platelet function in DEP-treated

rats, bleeding time measurements were performed using a tail-cut model (17), which

was previously shown to be platelet dependent (13; 33; 45). Rats were anesthetized

by i.p. administration of a combination of ketamine (60 mg/kg) and xylazine (5

mg/kg). Then the tail was transected about 0.5 cm from the tip using a disposable

surgical blade. The tail was placed in 25 ml isotonic saline (pH 7.4, 37 °C)

immediately after being cut, and the bleeding time was measured from the moment of

transection until bleeding stopped completely.

Blood collection, BAL fluid analysis and lung wet-to-dry weight ratio.

In the same animals, immediately after measuring the bleeding time, blood

was drawn from the inferior vena cava in EDTA (4 %). A sample was used for

platelets, white blood cells (WBC) and red blood cells (RBC) counts using an ABX

Micros 60 counter (ABX Diagnostics, Montpellier, France). The remaining blood was

centrifuged during 15 min at 3,500 rpm, and the plasma samples were stored at -

20°C.

The rats were then killed with an overdose of ketamine. BAL was then

performed by cannulating the trachea, the left bronchus was clamped. The bronchi

and right lung were lavaged three times with 5 ml sterile 0.9% NaCl. The BAL fluid

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was pooled in a plastic tube on ice. No difference in the amount of recovered fluid

was observed between the different groups. BAL fluid was centrifuged (1,000 g x 10

min, 4°C). Counting of the cells was performed in a hemocytometer after

resuspension of the pellets and staining with 1% gentian violet. The cell differentials

were performed on cytocentrifuge preparations fixed in methanol and stained with Diff

Quick (Dade Behring, Marburg, Germany). The supernatant was stored at - 20 °C

until further analysis.

The presence of pulmonary edema was assessed by the wet-to-dry weight

ratio. The non-lavaged left lung was removed and placed into a preweighed glass

tube for measuring wet lung weight, and dry lung weight (after 24 h at 80 °C) (34).

The wet-to-dry weight ratio was calculated as follows (35):

wet-to-dry weight ratio = (wet weight – dry weight)/wet weight.

Statistics

Data are expressed as means ± SEM. Comparisons between groups were

performed by one way analysis of variance (ANOVA), followed by Newman-Keuls

multiple range test. P values <0.05 are considered significant.

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Results

Particle characterization Transmission electron microscopy of the DEP showed numerous small

aggregates of carbonaceous particles less than 100 nm. Most of these aggregates

were <1 µm in largest diameter (figure 1).

Effect of DEP on blood pressure

The systemic administration of DEP induced a significant decrease of blood

pressure in DEP-exposed rats at doses of 0.02 (- 28 mmHg, p<0.05), 0.1 (- 32

mmHg, p<0.05) and 0.5 mg/kg (- 24 mmHg, p<0.05) compared with mean blood

pressure observed in saline-treated rats (figure 2).

Effect of DEP on heart rate

Figure 3 shows that the administration of DEP at doses of 0.02, 0.1 and 0.5

mg/kg, in rats, resulted in a significant reduction of the heart rate to 348 ± 13

(p<0.05), 348 ± 8 (p<0.05) and 339 ± 12 bpm (p<0.05) compared to 389 ± 11 bpm

recorded in saline-treated rats.

Effect of DEP on tail bleeding time

Figure 4a illustrates a shortening of the tail bleeding time in rats exposed to

0.02, 0.1 and 0.5 mg/kg of DEP. The shortening, which has been shown to be

platelet dependent (13; 33; 45) , was significant at the dose of 0.02 (305 ± 17s,

p<0.01), 0.1 (283 ± 55s, p<0.01) and (255 ± 35s, p<0.01) 0.5 mg/kg compared to

control group (533 ± 63s). Platelet counts in blood did not significantly increase

following DEP administration (figure 4b).

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Effect of DEP on WBC and RBC numbers

No significant effect of DEP at the doses of 0.02, 0.1 and 0.5 mg/kg on the

numbers of granulocytes, monocytes or lymphocytes compared to saline-treated rats

(figure 5).

Similarly, the numbers of RBC were not significantly affected by the DEP

administration compared to the control group (figure 5).

Effect of DEP on pulmonary inflammation

Depending on the systemic treatment performed, the cells found in BAL were

primarily macrophages and PMN (figure 6). Lymphocytes were not found in control

rat BAL. No other cells were observed microscopically.

The systemic administration of DEP resulted in a marked cellular influx in the

lung at doses of 0.02, 0.1 and 0.5 mg/kg. Although it did not reach statistical

significance, the number of macrophages increased at the dose of 0.5 mg/kg (figure

6a). Figure 6b shows that the PMN numbers increased significantly at 0.02 mg/kg

(2.9 ± 0.3 x104/ml, p<0.05), 0.1 mg/kg (3.4 ± 0.4 x104/ml, p<0.05) and 0.5 mg/kg (5.6

± 1.2 x104/ml, p<0.001), compared to saline-treated rats (0.9 ± 0.4 x104/ml).

Wet-to-Dry Weight Ratio

Figure 7 shows the results of the lung wet-to-dry weight ratio. A significant

increase of this relation was observed following the administration of the doses of

0.02, 0.1 and 0.5 mg/kg of DEP (p < 0.05).

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Discussion

In this study, we provide evidence that the systemic administration of DEP in

the circulation affect the blood pressure, heart rate and haemostasis, 24 h later. We

have also demonstrated that the presence of such particles in the circulation trigger

pulmonary oedema and lung inflammation evaluated by BAL fluid analysis.

Exposure of human subjects to DEP results in an acute inflammatory response

characterized by neutrophil and mast cell influx into the airways (38; 39). Moreover, it

has been demonstrated that DEP impairs the regulation of vascular tone and

endogenous fibrinolysis (20). We recently showed in hamsters that pulmonary

exposure to DEP cause lung inflammation and enhance the occurrence of arterial and

venous thrombosis, and that these effects persisted up to 24 hours (22; 27).

Pretreating hamsters with diphenhydramine, a histamine H1 receptor antagonist,

strongly reduced lung inflammation at all time points investigated (ie, 1 h, 6 h, 24 h).

Such pretreatment reduced the thrombotic events at 6 and 24 hours but not at 1 hour

after DEP administration. The findings at 1h are compatible with direct platelet

activation by DEP, having presumably penetrated the systemic circulation. However,

the effects observed at 6 and 24 h are related to lung inflammation. We have also

confirmed that antiinflammatory pretreatment can abrogate the peripheral

thrombogenicity by preventing histamine release from mast cells and PMN influx in

the lung (24). Because we have achieved the inhibition of pulmonary inflammation

and peripheral trhrombosis by i.p. injection of dexamethsone, diphenhydramine or

cromoglycate, we may well have inhibited the effect of DEP that have translocated

into the systemic circulation. Moreover, at 24 h time point, when we pretreated

hamsters with intratracheal instillation of dexamethasone, before exposing them to

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DEP, the observed inhibition of both PMN influx in the lung and thrombosis was only

partial (24). Thus, it is plausible to hypothesize that the translocated DEP (and their

associated constituents), that have presumably occurred within 1 h, could contribute

in the observed pulmonary and extrapulmonary effects at 24 h.

Consequently, in the present study, we wanted to investigate whether and to

which extent the presence of DEP in the systemic circulation can trigger

cardiovascular and pulmonary inflammatory changes at 24 h. To this end, we used

an admittedly less physiologic mode of administration, namely, the intravascular

route, because we wanted to mimics the effect of inhaled particles translocated from

the lungs into the systemic circulation (9; 14; 15; 23; 28; 30). The advantage of this

approach is that it circumvents effects related to the pulmonary accumulation of

particles, e.g., release of inflammatory mediators, and it allows to study, in vivo, the

direct effect of DEP on the heart, haemostasis and whether the presence of particles

in blood can contribute in the development of lung inflammation.

The electron microscopy analysis of the DEP used in the present study

revealed the presence of a substantial amount of ultrafine (nano) sized particle

aggregates (figure 1). These particles are comparable to the DEP (NIST; SRM 1650)

we previously used (22; 24; 27). Therefore, it seems reasonable to postulate passage

of these particles as it has been demonstrated to occur (9; 14; 15; 23; 28; 30). The

lowest dose of DEP used in the present study, i.e. 8 µg/rat can presumably be

achieved in the blood after pulmonary exposure to 32-100 µg/rat (28; 30).

To minimize aggregation, particles were always sonicated for 15 min and

vortexed immediately (< 1 min) before their dilution in saline containing tween 80 %

(0.1%), as well as prior to intravascular administration. Although the EM analysis

clearly demonstrated the presence of UFP and larger particle aggregates (< 1 µm in

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largest diameter), we do not know how much of the total injected dose consists in

UFP or larger aggregates, and whether the observed effects are caused by UFP or

larger aggregates.

Our data show that 24 h following their systemic administration, DEP cause a

decrease of blood pressure and heart rate. This effect could be explained by the

production of reactive oxygen species in the heart, responsible for cardiac

dysfunction (11; 34). Analogous finding have been made in spontaneously

hypertensive rats intratracheally instilled with combustion-derived particles, in which

the decrease of blood pressure and heart rate did not return to pre-exposure values

until 72 and 48 h after dosing, respectively (49). Similarly, it has been shown that the

exposure of healthy rats by instillation to PM2.5 or by inhalation to concentrated

ambient particles was responsible for a decrease of blood pressure and heart rate

within the first and second hour of particle exposure (5; 36). However, others have

reported an increase of heart rate in pulmonary hypertensive rats after exposure to

concentrated ambient particles (5). Interestingly, these discrepancies seem to confirm

the epidemiological observations which found both decrease and increase of blood

pressure in relation to air pollution exposures (well-reviewed by Delfino et al. (6)).

These disparities were related to the differences between subject populations, type of

regional air pollutants or underlying pathology (healthy, asthma or chronic heart

disease). Additional studies are needed to uncover the pathophysiological

mechanisms of particle-induced changes in heart rate and blood pressure.

We have recently shown that DEP enhance arterial and venous thrombosis

after intratracheal instillation both in vivo and ex-vivo. Moreover, we also reported that

DEP induce platelet aggregation in vitro (22; 27). Nevertheless, the effect of systemic

administration of DEP has not been addressed. Here, we show that the presence of

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DEP in the systemic circulation shortens the bleeding time, which has been shown to

be platelet dependent (13; 33; 45). Our findings corroborate with previous studies

which showed that the intravascular administration of positively charged ultrafine

polystyrene particles or carbon black particles are capable to trigger thrombotic

complications (16; 26; 43). In agreement to our previous findings (22), the number of

platelets did not significantly change following DEP administration. It is likely that

DEP which are taken up by phagocytosis or the open canalicular system of platelets,

might predispose them to aggregation and thrombosis (1; 48).

An important finding of our study is that we show that the intravascular

administration of DEP cause pulmonary inflammation and oedema. In line of this

results, diffusional movement of UFP administered intravascularly to the alveolar

space has been reported in vivo in rabbits and in an ex-vivo model of isolated

perfused rabbit lungs (12; 21). We recently reported that pulmonary inflammation and

peripheral thrombosis caused by intratracheal instillation of DEP are correlated at 6

and 24 h, but the prothrombotic tendency observed 1 h resulted from direct platelet

activation by DEP, having presumably translocated into the circulation (27). Based on

the present results, we suggest that the pulmonary inflammation we previously

observed at 24 h after pulmonary deposition of DEP (27), could result, at least partly,

from the translocated DEP-associated components or by DEP particles themselves.

We conclude that the presence of DEP particles in the systemic circulation

leads not only to cardiovascular and haemostatic changes but it also triggers

pulmonary inflammatory reaction. Further studies, are needed to establish which

constituents are responsible for the effect of DEP (i.e. the physical and/or chemical

properties of DEP) and what mechanism is involved.

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Figure legends

Figure 1. Transmission electron micrographs of the DEP suspension showing the

presence of numerous small aggregates of carbonaceous particles.

Figure 2. Blood pressure in WKY rats, 24 hours after the systemic administration of

saline or diesel exhaust particles (DEP). Mean ± SEM (n=6-7). Statistical analysis by

Newman-Keuls test.

Figure 3. Heart rate in WKY rats, 24 hours after the systemic administration of saline

or diesel exhaust particles (DEP). Mean ± SEM (n=6-7). Statistical analysis by

Newman-Keuls test.

Figure 4. Tail bleeding time (a) and Platelet numbers (b) in WKY rats, 24 hours after

the systemic administration of saline or diesel exhaust particles (DEP). Mean ± SEM

(n=6-7). Statistical analysis by Newman-Keuls test.

Figure 5. Red blood cells, monocytes, granulocytes and lymphocytes number in

WKY rats, 24 hours after the systemic administration of saline or diesel exhaust

particles (DEP). Mean ± SEM (n=6-7).

Figure 6. Macrophages (a) and PMN (b) numbers in BAL fluid in WKY rats, 24 hours

after the systemic administration of saline or diesel exhaust particles (DEP). Mean ±

SEM (n=6-7). Statistical analysis by Newman-Keuls test.

Figure 7. Left lung wet/dry weight ratio per 100 g body weight (BW) in WKY rats, 24

hours after the systemic administration of saline or diesel exhaust particles (DEP).

Mean ± SEM (n=6-7). Statistical analysis by Newman-Keuls test.

Page 23 of 30

24

Figure 1

a b

c d

Page 24 of 30

25

Control 0.02 0.1 0.50

70

140

DEP (mg/kg)

Figure 2

P<0.05

P<0.05

P<0.05B

lood

Pre

ssur

e (m

mH

g)

Page 25 of 30

26

Control 0.02 0.1 0.5200

250

300

350

400

Figure 3

DEP (mg/kg)

P<0.05

P<0.05

P<0.05H

eart

Rat

e (b

pm)

Page 26 of 30

27

Control 0.02 0.1 0.50

200

400

600

a

Figure 4

P<0.01

P<0.01

DEP (mg/kg)

P<0.01B

leed

ing

time

(s)

Control 0.02 0.1 0.50

100

200

300

400b

DEP (mg/kg)

Plat

elet

num

bers

(x10

3 /µl)

Page 27 of 30

28

Control 0.02 0.1 0.5

0

3

6

MonocytesGranulocytes

LymphocytesRed Blood CellsFigure 5

DEP (mg/kg)

5000

7500

10000

Cel

ls (x

103 /µ

l Blo

od)

Page 28 of 30

29

Control 0.02 0.1 0.50

10

20

30

DEP (mg/kg)

aFigure 6

Mac

roph

ages

in B

AL

(x10

5 /ml)

Control 0.02 0.1 0.50.0

3.5

7.0

DEP (mg/kg)

b

P<0.05

P<0.001

P<0.05

PMN

in B

AL

(x10

4 /m

l)

Page 29 of 30

30

Control 0.02 0.1 0.50.0

0.1

0.2

DEP (mg/kg)

Figure 7

P<0.05

P<0.05

P<0.05

Wet

/Dry

Wei

ght

(per

100

g B

W)

Page 30 of 30