Compositions and oxidative damage of condensate, particulate and semivolatile organic compounds from gasoline exhausts

Environmental Toxicology and Pharmacology 24 (2007) 11–18

Compositions and oxidative damage of condensate, particulate and semivolatile organic compounds from gasoline exhausts Wangjun Che, Zunzhen Zhang ∗ , Hao Zhang, Mei Wu, Ying Liang, Fang Liu, Ya Shu, Na Li Department of Environmental Health, West China College of Public Health, Sichuan University, Chengdu, People’s Republic of China Received 31 August 2006; received in revised form 20 December 2006; accepted 2 January 2007 Available online 14 January 2007

Abstract The effects of extracts of condensate, particulate and semivolatile organic compounds from absolute gasoline exhausts on DNA single strand break, levels of malondialdehyde (MDA), carbonyl protein and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were investigated in lungs and brains of adult Sprague-Dawley rats of both sexes. In addition, the non-conventional components of the extracts and concentrations of 13 polycyclic aromatic hydrocarbon (PAHs) in gasoline exhaust were measured by GS/MS. Extract of gasoline exhaust at different doses (5.6, 16.7 and 50.0 L/kg) were given to administered animals by intratracheal instillation once a week for 4 weeks, while blank control and solvent groups were given with physiological saline and dimethyl sulfoxide (DMSO). Our results showed that gasoline exhaust increased DNA single strand break, promoted lipid peroxidation and oxidative protein damage and decreased activities of SOD in lungs and brains. While, it decreased the activities of GPx in lungs but not in brains. The present data suggested that gasoline exhaust exposure could cause oxidative damage to lung and brain of rats. That was to say that gasoline is a toxin to brain of mammals, not only to lung. © 2007 Published by Elsevier B.V. Keywords: Vehicle emissions; Gasoline exhaust; Oxidative damage; Rat; Comet assay

1. Introduction With rapid development of transportation, vehicle emissions related air pollution has become a more and more serious problem in many cities. Epidemiologic studies have proved that chronic exposure to automobile emissions increased the risk of pulmonary and extrapulmonary cancer as well as non-cancer health effects (Netterstrom and Suadicani, 1993; De et al., 2003; Hansen, 1993; Hansen et al., 1998; Guo et al., 2004; Lee et al., 2004). In 1989, based on animals and epidemiological results, IARC classified gasoline exhaust as a possible human carcinogen (2B) (IARC, 1989). Vehicle emissions can be classified gasoline, diesel, and natural gas engine exhaust and so on. It was showed that gasoline exhaust had become the major air pollutants in many urban envi-

∗ Corresponding author at: Department of Environmental Health, West China College of Public Health, Sichuan University, No. 16, Section 3, Ren Min Nan Road, Chengdu 610041, People’s Republic of China. Tel.: +86 28 85501298. E-mail address: [email protected] (Z. Zhang).

1382-6689/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.etap.2007.01.001

ronments atmosphere instead of diesel engine exhaust (Alan and Gertler, 2005; Kunzli et al., 2000). Due to smaller particles diameter (Bunger et al., 2000), gasoline exhaust can penetrate deeper into respiratory system and is more likely to be retained there, enhancing harmful toxicological effects (Ferin et al., 1992; Donaldson et al., 1998). Gasoline exhausts can be divided into three major components: gaseous phase, soot particles, and semivolatile organics, which are distributed between the particulate and the gaseous phase. Correspondingly, extracts of it include condensate (CD), particulate matter (PM) and semivolatile organic compounds (SVOC). But previous studies on gasoline exhausts focused primarily on the single component such as PM, CD and SVOC. The studies on combination of these components were few. In addition, efforts to reduce the total emissions rate have led to modifications in fuel, engine, and after-treatment technology. It was well known that biological effects of vehicle emissions would depend on the physical and chemical properties of the emissions. So, knowledge of the chemical components in vehicle emissions, especially in relation to different types of fuel used, is of great importance in the understanding of these adverse effects.

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Compared with diesel engine exhaust, data on toxic effects of gasoline exhaust were little (IARC, 1989). Previous studies had shown that gasoline exhausts induced bacterial mutagenesis, chromosome aberrations, sister chromatic exchanges, micronucleus formation in vitro and vivo, base oxidation, DNA single strand break, cell morphologically transform and DNA adducts formation in vitro (Cheng et al., 2004; Liu et al., 2005; Kuo et al., 1998; Liu et al., 2005; Ye et al., 1999). Meanwhile, it had also been shown that PM was capable of inducing oxidative stress in vivo (Seagrave et al., 2002). For SVOC, current data showed that the SVOC had the same potencies as PM (Liu et al., 2005). CD had mutagenic effect in Ames assay, too (Ye et al., 1999). Moreover, combined PM and SVOC intratracheal instillation in rats indicate multiple endpoints cytotoxicity, inflammatory and oxidative damage besides mutagenicity for Strain TA98, TA100 (Seagrave et al., 2002). Inflammatory responses and oxidative stress have all been reported as potentially important consequences and mechanisms of emissions exposure (Seagrave et al., 2002). Recently, several studies had shown that PM could produce reactive oxygen species (ROS) in vitro (Kuo et al., 1998; Tzeng et al., 2003; Cheng et al., 2004). Meanwhile, antioxidants inhibited PM induced genotoxicity (Cheng et al., 2004). All these indicated that oxidative stress played an important role in vitro toxic effect of gasoline exhausts, which may be an important factor to understand the mechanism of toxic effect of gasoline exhausts. We do know, the biological effects of emissions are contributed to components, which are dependent on the temperature, the test vehicles, cycles used, the fuels and also the exhaust aftertreatment technology (McDonald et al., 2004). Therefore, we determined the concentrations of every chemical from gasoline exhaust so that we would further understand the relationship between toxic effects and chemical components of gasoline exhaust. So far, the health effects of the extracts of CD + PM + SVOC from gasoline exhaust have not been published yet. No report is available on oxidative damage in rat brains induced by gasoline exhaust. The purpose of this study was to evaluate the biological macromolecules oxidative damage in the lung and brain of rat treated with the extracts of gasoline exhaust by intratracheal instillation under the condition of determination of chemicals from gasoline exhaust. Enzymatic (SOD and GPx) antioxidant defenses were evaluated in rat lung and brain homogenates. The rates of tailed cells, levels of MDA and carbonyl protein were, respectively, used to assess DNA, lipid and protein oxidative damage. 2. Materials and methods 2.1. Chemicals Chemicals were obtained from the following sources: thiobarbituric acid (TBA), 1,1,3,3-theramethoxypropane (TMP) and 5,5-dithiobis(2-nitro-benzoic acid) from Sigma–Aldrich Inc. (St. Louis, USA). Normal melting point agarose (NMP) and low melting point agarose (LMP) from Amresco (Solon, USA). SOD and GPx kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals used including solvents were analytical in grade.

2.2. Collection and preparation of organic extracts of gasoline exhaust The vehicle used in this study was a passenger car obtained from Chengdu transit systems. The vehicle was an in-use (110,381 km) 1996 model Guangzhou passenger car with a Dongfeng Gasoline Series 155 kw engine and no exhaust catalytic converter. The vehicle was operated at an ambient temperature of ∼25 ◦ C. Samples were collected about 10 min after the car started with a coldstart. Gasoline exhaust without dilution was collected in a constant volume sampler system from a passenger car fuelled with 90-octane lead-free gasoline supplied by China Petroleum. The sampling apparatus consisted of, in sequence, a 200-(long), 0.6 cm (diameter) condensation tube which attached directly to exhaust-pipe, an empty middle volume air sampler for collecting condensation, then, a filter holder with a glass fiber filter that was followed by a 21-(long), 1.1 cm (diameter) “U” glass tube containing a plug of polyurethane foam (PUF) which was used to collect semivolatile compounds and cross-linked divinyl benzene (XAD-2). A vacuum pump was at the end of the apparatus. The condensation tube and air sampler were kept in water–ice bath. The engine was running at idle status on an empty load, and the pump was set at a flow rate of 6 l/min to collect gasoline exhaust. The effects of temperature on volume of emissions collected were investigated by measuring the temperature and pressure of sampling pipeline. The condensation liquid collected was extracted three times with dichloromethane in a separatory funnel. The particulates were extracted into dichloromethane by sonication and gentle brushing from filters, and resin (PUF and XAD-2) absorbed matters were repeatedly extracted with dichloromethane under sonication. In succession, the three fractions were recombined into a single sample and then concentrated by evaporation with a rotary evaporator in low air pressure and constant temperature bath at 45 ◦ C. The final residues were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 200 l/ml (i.e. per ml solution contains extracts generated by 200 l emissions). The aliquots were stored at −20 ◦ C in the dark until use.

2.3. Animals treatment and preparation of homogenates Fifty healthy adult Sprague-Dawley rats (8 weeks of age, weighing 190–220 g) were obtained from Laboratory Animal Center of Sichuan University (Chengdu, China). Animals were housed in stainless-still cages in temperature and humidity regulated air-conditioned room under standard laboratory conditions (12 h light, 12 h dark and 23 ± 3 ◦ C). Body weights were assessed once a week. The protocol of this study was approved by the local Ethics Committee. All experimental procedures were conducted in accordance with the Guide to the Care and Use of Laboratory Animals. The rats were fed with standard commercial rat diet (pellet form, in the sack, Laboratory Animal Center of Sichuan University, Chengdu, China). Feed and tap water were provided ad libitum. Animals were acclimated for 7 days prior to experimentation. The rats were randomly divided into five groups; each group containing 10 rats (five females and five males). Various doses for toxicological testing were prepared by diluting 200 l/ml stock solution of extracts with physiological saline. The final volume for instillation was 0.3 ml/rat. The extracts of gasoline exhaust which were homogenized in 0.3 ml physiological saline, were intratracheally instilled to rat lungs at the doses of 5.6, 16.7 and 50 l/kg. The blank and solvent control groups were instilled with 0.3 ml/rat physiological saline and 0.25 ml/kg body weight DMSO with the same pathway, respectively. Every group received an intratracheal instillation once a week for 4 weeks. Twentyfour hours after the last instillation rats were anesthetized (sodium pentobarbital, 40 mg/kg, ip), bled from femoral and sacrificed by cervical decapitation. The lungs were removed. The left bronchus was temporarily clamped. The right lung was then lavaged with two 3 ml aliquots of phosphate buffered saline. The recovered bronchoalveolar lavage fluid (BALF) was pooled and the cellular pellet was collected by centrifuged at 400 × g for 10 min. And then, the right lung and brain was excised, immediately cooled in ice, and homogenized in Teflon-glass homogenizer with 120 mM KCl, 30 mM phosphate buffer (pH 7.4). Then, the homogenates were centrifuged at 700 × g for 15 min. The supernatant fraction was stored on ice for use.

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2.4. Intratracheal instillation

2.8. Determination of protein carbonyl

DMSO and extracts of gasoline exhaust were dilute with physiological saline to an equivalent volume: 0.3 ml then administered according to Bell et al. (2000) described with some modification. Briefly, animals were anesthetized with sodium pentobarbital (20 mg/kg, ip) and placed on a slanted board suspended from their maxillary incisors. The tongue of the animal was pressed down with a speculum in order to obtain a clear view of the pharynx. A syringe with a blunt 18-gauge stainless steel ball-tipped needle was inserted transorally into the trachea. Tracheal rings were sensed against the needle confirming its proper insertion. Following fluid delivery, the rat was removed from the board and observed until consciousness was regained.

The levels of protein carbonyls were analyzed by 2,4-dinitrophenylhydrazine (DNPH) method as described by Levine et al. (1990). Briefly, 300 ␮l of tissue supernatant was pipetted into the tubes, to which 300 ␮l of 10 mM DNPH in 2N HCL was added. Adding with 2N HCL only made the blank. Samples were then incubated at room temperature for 1 h, stirred every 10 min, then the proteins were precipitated with 10% trichloroacetic acid (final concentration) and subsequently washed three times with 1 ml ethanol/ethyl acetate (1:1, v/v). The pellet was dissolved in 1 ml of 6 M guanidine HCL in 10 mM sodium phosphate buffer (pH 2.3). Insoluble debris was removed by centrifugation. The difference in absorbance at 366 nm between the DNPH-treated sample and the HCL treated control was determined. Results were expressed as nmol carbonyl groups/mg protein based on an extinction coefficient of 22.0 mM−1 cm−1 for aliphatic hydrazones.

2.5. Chemical characterization of gasoline exhaust Components in extracts from gasoline exhaust were analyzed by HP6890/5973 gas chromatograph–mass spectrometer (Hewlett-Packard Co., USA) using a DB-35MS capillary column. Calibration of PAHs components was achieved using a PAHs standard (supplied from Supelco, USA).

2.6. Single cell gel electrophoresis (comet assay) The lungs were removed and washed with ice-cold phosphate-buffered saline (PBS) to remove superficial blood. The organs were minced and washed in ice-cold PBS to remove the red blood cells. The minced organs were forced through a wire mesh screen to obtain single-cell suspensions. For all samples, cell viability was >95% as determined with a Malassez haemocytometer, using the Trypan-blue dye-exclusion technique. The alkaline version of the comet assay was carried out according to the procedure of Singh et al. (1988) with some modifications. Briefly, the singlecell suspensions were mixed with 0.6% molten LMA at 37 ◦ C and spread on the ground microscope slides precoated with 0.8% NMP. The slides were covered with a coverslip and allowed to solidify for 10 min at 4 ◦ C; then the coverslips were removed and the slides were placed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100, 10% DMSO; the last two components were freshly added) for 1 h at 4 ◦ C. After lysis, the slides were subjected to freshly prepared electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 30 min to allow unwinding of DNA before electrophoresis. To electrophorese the DNA, an electric field of 25 V and 300 mA was applied for 30 min. All of these steps were conducted in the dimmed light to prevent additional DNA damage. After electrophoresis, the microgels were neutralized in distilled water. Just prior to analysis the DNA was stained with 30 ␮l of ethidium bromide (20 ␮g/ml). The comets were analyzed at 200× magnification under fluorescence microscope (Leica, Germany) attached to digital camera (Nikon, Japan) and connected to a personal computer. Two hundreds randomly selected cells were scored from each slide (two slides per dose) and rate of caudate cells was calculated. Quantification of the DNA damage was estimated by measuring DNA migration length (␮m) according to the equation: DNA migration length or trail length (␮m) = (total length of the comet) − (head length). Tail length of 30 randomly selected caudate cells was mensurated by calibrated reticule to evaluate the length of DNA migration.

2.7. Lipid peroxides Lipid peroxide was measured by thiobarbituric acid test for MDA according to a modified method by Ohkawa et al. (1979). Procedure was as follows: 0.1 ml of 10% tissue homogenate of lung was added, 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution, and 1.5 ml of 0.8% aqueous solution of TBA. The mixture was made up to 4.0 ml with distilled water, and then heated in water bath at 95 ◦ C for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of n-butanol and pyridine (15:1, v/v) were added and shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance at 532 nm was measured, TMP was used as an external standard, and the level of lipid peroxides was expressed as nmol MDA/g protein.

2.9. Activity of antioxidant enzymes The activity of SOD and GPx was determined spectrophotometrically according to the method of the Nanjing Jiancheng Bioengineering Institute with a spectrometer. SOD activity was assayed spectrophotometrically at 550 nm by use of a xanthine and xanthine oxidase system. One unit of SOD activity was defined as the amount of SOD required for 50% inhibition of the xanthine and xanthine oxidase system reaction in 1 ml enzyme extraction of per milligram of protein. GPx was assayed spectrophotometrically by use of glutathione (GSH) as substrate by measurement of the decrease of enzymatic reaction of GSH (except the effect of non-enzymatic reaction) at 412 nm. One unit of GPx activity was defined as the decrease amount of 1 nmol/l GSH (except the effect of non-enzymatic reaction) in system of enzymatic reaction of 1 mg protein per minute.

2.10. Protein measurement Protein content was assayed by the method of Lowry et al. (1951), using bovine serum albumin as standard.

2.11. Statistical analysis Data were expressed as the mean ± S.D. for the number of experiments indicated. Statistical analyses of the data were analyzed by one-way analysis variance (ANOVA). P < 0.05 was considered significantly different.

3. Results 3.1. Gasoline exhaust composition Ninety-nine chemicals were found in extracts of gasoline exhaust by GC/MS (see Appendix A). In order to get a better overview, the single compounds were grouped into monocyclic aromatics, alkanes and the alkenes, indene, aldehyde and PAHs five substance classes. The component of the abstracts from gasoline exhaust was dominated by the fraction of the aromatic compounds (78.44%) followed by the alkanes and thealkenes (12.28%). The amount of PAHs was less than 0.5%. The relative proportion of each part was summarized in Fig. 1. In addition, the concentrations of 13 PAHs were measured (Table 1). The data indicated that naphthalene and m-xylene were the most main substance in the two substance classes, respectively.

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Fig. 1. Element proportion in extracts of gasoline exhaust.

Table 1 Concentrations of PAH components per m3 of gasoline exhaust (¯x ± s) Component

Concentration (␮g/m3 )

Naphthalene Acenaphthylene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Chrysene Benzo(a)anthracene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Benzo(g,hi)perylene

405.6 55.8 5.8 27.8 5.3 25.5 94.6 10.5 3.0 3.3 5.9 7.7 10.8

± ± ± ± ± ± ± ± ± ± ± ± ±

11.7 2.4 0.2 1.2 0.3 1.5 5.6 0.7 0.1 0.2 0.2 0.4 0.7

3.2. Comet assay DNA damage in lung and brain was summarized in Fig. 2. A similar trend with regard to DNA damage was observed in the lung and brain of rats exposed to gasoline exhausts. The rates of tailed cells in lung and brain dose-dependently increase. The mean values of rates of tailed cells in lung of rats exposed to 15.6 and 50.0 l/kg of gasoline exhaust were 41.03 ± 7.69% and 82.53 ± 6.87%. These differences were significant compared with the solvent group (20.23 ± 5.96%). But at low dose

Fig. 3. Malondialdehyde levels in lung and brain of rats exposed to gasoline exhaust. Data represent mean ± S.D. (n = 10). * P < 0.05 compared with the solvent group.

(5.6 l/kg) the increase of rates of tailed cells was not statistically significant. For brains, the rates of tailed cells of rats exposed to every test dose of gasoline exhaust, respectively, were 40.98 ± 4.69%, 61.21 ± 7.66% and 75.32 ± 7.40%, which were all significantly increased compared to solvent group (11.21 ± 2.36%) (P < 0.05). Moreover, for tail length of tailed cells in lungs and brains, our results also showed that there were both no significant differences in length of DNA migration in tailed cells between gasoline exhaust groups and solvent control (data not show). 3.3. Lipid peroxidation Fig. 3 indicates the results of lipid peroxidation. MDA contents in lung of rats exposed to various test doses of gasoline exhaust increased by 9, 20 and 18% as compared with solvent control (3.80 ± 0.77). There was a dose–response relationship between lung MDA contents and exposure doses. In addition, the increases of MDA were statistically significant at the dose of 16.7 and 50.0 l/kg (P < 0.05). For brains, the levels of MDA were not changed obviously at the dose of 5.6 and 16.7 l/kg. But for 50.0 l/kg, the levels of MDA significantly increased by 42% compared with solvent control (3.18 ± 0.477) (P < 0.05). 3.4. Carbonyl protein The amounts of carbonyl protein in lung and brain were shown in Fig. 4. The mean carbonyl protein values of solvent control in lungs and brains were 2.45 ± 0.64 and 2.02 ± 0.38. The carbonyl protein contents in lungs and brains of rats exposed to 50.0 l/kg of gasoline exhaust had significantly increased by 263.67 and 274.26% compared with solvent controls. In addition, compared with solvent control, the differences were no statistical significance in lung and brain at 5.6 and 16.7 l/kg treatment groups. 3.5. Antioxidase activities

Fig. 2. Rates of tailed cells in lung and brain of rats exposed to gasoline exhaust. Data represent mean ± S.D. (n = 6). * P < 0.05 compared with solvent group.

Results of SOD activities in lung and brain are presented in Fig. 5. In contrast to MDA and carbonyl protein, lung and brain

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produced a significant decrease by 65.48% (P < 0.05). On the other hand, GPx activities in brain of rats treated with all test concentrations were not significantly changed compared with solvent control. 4. Discussion

Fig. 4. Carbonyl protein contents in lung and brain of rats exposed to gasoline exhaust. Data represent mean ± S.D. (n = 10). * P < 0.05 compared with the solvent group.

Fig. 5. SOD activities in lung and brain of rats exposed to gasoline exhaust. Data represent mean ± S.D. (n = 10). * P < 0.05 compared with the solvent group.

SOD activities progressively decreased with the dose increase of gasoline exhaust. The decreases of SOD activities in the lung were no significance except for the 50.0 l/kg group. SOD activities in brain of rats treated with various doses of gasoline exhaust all decreased. In addition, there were significant differences in brain SOD activities between all three treatment groups and solvent group (P < 0.05). GPx activities in lung and brain were summarized in Fig. 6. GPx activities showed a different profile compared with SOD. Under 50.0 l/kg concentration, the GPx activities of lung were not modified compared with solvent control. But the GPx activities in lung of rats exposed to 50.0 l/kg of gasoline exhaust

Fig. 6. GPx activities in lung and brain of rats exposed to gasoline exhaust. Data represent mean ± S.D. (n = 10). * P < 0.05 compared with the solvent group.

Large numbers of people in the world continue to be exposed to gasoline exhausts containing many known or suspected carcinogens. Although the relationship between long-term exposure to gasoline exhaust and lung cancer in animals or humans is unclear, its adverse effects on health have been understood. In our study, a dose ranges from 5.56 to 50.0 l/kg was used to examine the effects of the rat lung and brain from the mixture. The justification was as follows. According to vehicle emissions inhalation exposure guideline (EPA, 2001), the maximum concentration should lie in the range of a ratio between 1:5 and 1:50 emissions to clean air, the minimum should be in the range between 1:100 and 1:150. The mean values of rat tidal volume and respiration frequency, respectively, are 1.5 ml and 85 times per minute (James et al., 1984). So 50.0 l/kg corresponded to exposure of 8 h/day a ratio about 1:40 raw exhaust to clean air. However, gas inhaled would be largely trapped in respiratory tract and then cleaned through inhalation. Based the conclusion of Leong et al. (1998), the dose of 50 l/kg approximately corresponds to exposure to less than 14% dilution by nose-only inhalation. So the maximum dose was no less than some high occupational exposure level known to induce oxidative DNA damage in healthy individuals (Lai et al., 2005). The reason that lung and brain were chosen to observe is as follows. The lung is a most important target organ by inhalation exposure. Compounds were deposited, accumulated and metabolically activated after exposure. The brain is particularly vulnerable to oxygen free radical attack since this organ consumes 20% of the body’s oxygen. Meanwhile the brain has large amounts of polyunsaturated fatty acids. Furthermore, the data on effects of gasoline exhaust on brain have not been shown, especially in vivo. The current results suggest that gasoline exhaust could induce lung and brain oxidative damage. It is well known that free radical can be formed in a number of ways under the conditions of exposure to vehicle emissions (EPA, 2002). Compared with inhalation administration, the intratracheal instillation is a simple, fast and accurate respiratory administration approach. For pulmonary absorption studies that dosage precision is of primary concern, the intratracheal instillation is more suitable than the nose-only inhalation (Leong et al., 1998). So, in this study intratracheal instillation was chosen for optimal control of exposure dose. Oxidative damage was caused by exposed to ROS intermediates, which could occur when antioxidant potential decreased and/or when ROS increased and then caused damage to proteins, nucleic acids, and cell membranes. This would result in numerous human diseases. There was still no consensus about the mechanism of toxicity of gasoline exhaust. An alternative mechanism was correlated with the generation of reactive oxygen species. Gasoline exhaust was a mixture that consisted of a variety of chemicals. Some

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chemicals or metabolic activation from gasoline exhaust, such as PAHs, nitro-PAHs, quinine and so on, could generate ROS via cytochrome p450 1A1 dependent transformation or electron transfer pathway in respiratory chain (Chesis et al., 1984). And particles might induce production of ROS by macrophage in vivo. Transition metals such as iron are also capable of catalyzing the generation of ROS by Harber–Weiss reaction of Fenton type. Moreover, inflammation is also an important source of ROS. ROS possess high chemical reactivity with biological macromolecules, which may result in oxidative damage. Most of all, ROS was also known to play a role in tumor promotional stage of carcinogenesis (Cerutti, 1985). ROS could attack DNA bases or deoxyribose residues to produce base oxidation, DNA adducts and DNA strand break. It was possible that an accumulation of oxidative macromolecules damage would increase probability of mutagenesis, even carcinogenesis. Moreover, some PAHs, after being metabolically activated, were known to react with DNA and form DNA adducts (Conney, 1982), which were thought to be relevant with respect to chemical carcinogenesis (Miller, 1970; Lawley, 1989). Recently, lipid peroxidation had become subject of interest and had been investigated as a marker of oxidative damage in vitro and in vivo, and in human studies (Salonen, 2000; Diplock, 2000; Orhan et al., 2004; Kadiiska et al., 2005). MDA was a major aldehyde product of lipid peroxidation and also a mutagen in bacterial and mammalian cells and carcinogen in rat (Basu and Marnett, 1983; Yau, 1979). In present study, lipid peroxidation results showed exposure to gasoline exhaust dosedependently increased the contents of MDA in rat lungs and brains. And brains of rats exposed to gasoline exhaust were found to increase with the exposure dose. This may be because the brain contains the large amount of easily oxidizable polyunsaturated fatty acids, the abundance of redox-active transition metal ions, and the relative dearth of antioxidant defense system (Samuel et al., 2005). And for lung, administration manner leads to that lung exposure doses were higher than that of the others. The increase in protein carbonyl levels was further evidenced that enhanced intracellular oxidative stress occurs in the lungs and brains of rats treated with 50 l/kg gasoline exhaust. Protein carbonyls resulted from oxidative damage to proteins and were widely used biomarkers of oxidative stress (Butterfield et al., 1998). The possible sources of the carbonyl protein generation were as follows: ROS react with the side chains of lysine, arginine, proline, threonine and glutamic acid residues of proteins leads to the formation of carbonyl derivatives (Stadtman and Berlett, 1997). Furthermore, aldehydes, such as 4-hydroxy-2nonenal or malondialdehyde produced during lipid peroxidation could be incorporated into proteins by reaction with either the amino moiety of lysine or the sulfhydryl group of cysteine residues to form carbonyl derivatives (Uchida and Stadtman, 1993). Carbonyl groups could also be introduced into proteins by glycation and glycoxidation reactions (Baynes, 1991). Our results showed that carbonyl protein significantly increased only at dose of 50.0 l/kg. Since carbonyl levels were relatively stable under non-pathological conditions, the increase of 263.67 and 274.26% of this chemical indicated a significant intensification

of intracellular oxidative stress in lung and brain. Therefore, these results suggested that the accumulation of peroxidants was over the function of antioxidants and led to the break of the balance between oxidative stress and protein oxidation and antioxidation, with the increase of concentration of gasoline exhaust. Seagrave et al. (2002) showed that gasoline exhaust could induce oxidative stress and increased levels of carbonyl protein in rat bronchoalveolar lavage fluid. SOD and GPx were main protective enzyme against oxygen free radical-induced damage (Powers and Lennon, 1999). SOD catalyses the dismutation of superoxide anion to hydrogen peroxide, and GPx catalyses the reduction of hydrogen peroxide to water, which should result in decrease of the formation of hydrogen radical. In this study that the treatment of gasoline exhaust lowered SOD and GPx activities in lungs of rats. The causes might be that chemicals from gasoline exhaust promoted ROS production and accumulation to deplete more antioxidants. Moreover, oxidative stress could modify enzyme proteins to make enzyme structural alteration and functional inactivation. These would result in decrease of activities of the enzymes in tissues. Consistent with our findings, Ueng et al. (2004) showed that inhalation of gasoline exhaust diluted would increase levels of MDA and inhibit SOD activities in rat lungs, but have no effect on GPx. Unfortunately, the effects of gasoline exhaust on activities of GPx and levels of protein carbonyl in brain and lung did not follow linear dose–response curves. And the changes with statistics significance were only in the highest dose group. It also implied that there was a limit to protein damage and activities of GPx inhibition induced by gasoline exposure in the both organs. Comet assay was a direct, sensitive, simple, rapid and powerful technique used in experimental and epidemiological studies (Rojas et al., 1999). Gasoline exhaust could induce genotoxic activities in vitro cells by causing DNA single strand breaks (Liu et al., 2005). But the effects on DNA strand breaks have not been investigated by comet assay in vivo. Our study showed that levels of DNA single strand breaks were dose-dependently increased in cells from lung and brain of rats exposed to gasoline exhaust with intratracheal instillation, indicating gasoline exhaust may be a genotoxic mixture. Zhao et al. (1998) also observed the increase of the frequencies of micronuclei and sister-chromatid exchanges in peripheral blood lymphocytes from traffic policemen. In addition, antioxidants were found to inhibit genotoxic effects of gasoline exhaust; therefore, it was likely that an accumulation of oxidative DNA and chromosomal damage in cells is the critical factor inducing genotoxicity (Cheng et al., 2004). In summary, the present study had demonstrated that collected CD + PM + SVOC gasoline exhaust samples from passenger car contained many known mutagenic and genotoxic chemicals, and could induce lipid peroxidation in rat lung and brain tissue and increased levels of carbonyl protein, MDA and DNA strand breaks, and further caused the decrease of SOD and GPx activities. Meanwhile, our results suggested that gasoline exhaust was a toxin to brain of mammals, not only to lung. In addition, further study is needed so that we can better understand the health effects and toxic mechanism of gasoline exhaust.

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Appendix A (Continued )

Acknowledgement This research was supported by the National Science Funds of China (30571535). Appendix A. Entire unconventional composition data set presented as weight proportion from extracts of gasoline exhaust

Component

Proportion (%)

Methyl cyclopentane Toluene Ethylbenzene o-Xylene, m-xylene 3-Methyl octane p-Xylene Nonane 3,5-Dimethyl octane 2,6-Dimethyl octane n-Propylbenzene 1-Ethyl-4-methylbenzene 1,3,5-Trimethylbenzene 1-Ethyl-2-methylbenzene 3-Methyl nonane 1,2,3-Trimethylbenzene Decane 1,2,4-Trimethylbenzene 2,3-Dihydroindene Indene 2-Methyl decane 1,2-Diethyl benzene 1-Methyl,3-propylbenzene Diethyl benzene 2-Ethyl,1,4-dimethylbenzene 1-Methyl-2-propylbenzene 5-Methyl decane 1-Methyl-2(1-dimethylethyl)benzene 2,3-Dihydro-1-methylindene 1-Ethyl,2,3-dimethylbenzene 3-Methyl decane 2-Isopropyl-1-methylbenzene 1,2,3,4-Tetramethylbenzene 1,2,4,5-Tetramethylbenzene 2-Ethylene-1,4-dimethylbenzene Naphthalene 2-(4 -Methylphenyl)propionaldehyde 2-Methyl nonane Acetylene benzene Styrene 1-Ethynyl-3-methylbenzene n-Undecane Benzaldehyde 1-Methyl-3-propylbenzene 1,3,-Diethylbenzene 1-Methyl-4-isopropylbenzene 2-Ethyl-1,3-dimethylbenzene 1-Methyl,2-phenyl cyclopropane 1-Ethynyl-3-ethylbenzene 1-Methyl,4-propenylbenzene 3-Methylbenzaldehyde 4-Methylbenzaldehyde 7-Methylbenzofuran 2-Methylbenzaldehyde 1,2,3,5-Tetramethylbenzene

5.679 0.411 1.892 9.58 0.47 6.21 1.347 0.323 0.45 1.605 11.005 5.848 5.381 1.099 15.666 0.938 3.652 2.054 2.552 0.46 0.764 1.684 1.028 2.668 0.668 0.215 2.937 1.646 2.003 0.526 0.656 1.268 1.31 0.436 0.104 0.059 0.049 0.14 0.048 0.158 0.026 0.657 0.161 0.029 0.266 0.128 0.689 0.065 0.036 0.173 0.559 0.094 0.258 0.029

Component 2,3-Dihydro-4-methyl indene 1-Methylindene 4-Ethylbenzaldehyde 3,4-Mimethylbenzaldehyde 2,4-Mimethylbenzaldehyde 2,6-Di(1,1-dimethylethyl)hydroxybenzene 1-Cyclopentyl-1-yl-benzene 1-Methoxy-2-propenylbenzene 2-Methylnaphthalene 1-Methylnaphthalene 1,1 -Biphenyl 2,7-Dimethylnaphthalene 1,8-Dimethylnaphthalene Acenaphthylene Fluorene 2-Phenylbenzaldhyde 2-Amino-5-phenypridine Phenanthrene Cyclopentyl [def]phenanthrene Chalcone 2,3,6-Trimethyl carbazole Fluoranthene Pyrene Phenylnaphthylamine 2-Methylpyrene 1-Methylpyrene Dibenzo(a,h)anthracene Chrysene Benzo(ghi)fluoranthene Benzo(a)anthracene Phenylanthrancene-7-one 4,5-Methanochrysene 1-Formyloxy-2-methoxy-9-methylanthrancene Benzo(k)fluoranthene Benzo(a)pyrene 1-Fluorophenyl-2,3-dimethylnaphthalene 1-(3,4-Dicyanobenzyl)naphthalene Ethyldiphenylboroxin Indeno(1,2,3-cd)pyrene Benzo(g,hi)perylene Dibenzo(ghi,pqr)perylene Diethyl phthalate Diisobutyl phthalate Dibutyl phthalate Di-2-ethylhexyl phthalate Total

Proportion (%) 0.042 0.255 0.15 0.06 0.052 0.091 0.02 0.002 0.013 0.014 0.002 0.004 0.03 0.014 0.002 0.001 0.001 0.003 0.002 0.002 0.001 0.012 0.038 0.002 0.001 0.006 0.124 0.002 0.001 0.003 0.001 0.003 0.002 0.006 0.024 0.002 0.021 0.006 0.024 0.07 0.01 0.002 0.033 0.001 0.01 100.00

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