Comparison of cytotoxicity and genotoxicity induced by the extracts of methanol and gasoline engine exhausts

Toxicology in Vitro 21 (2007) 1058–1065 www.elsevier.com/locate/toxinvit

Comparison of cytotoxicity and genotoxicity induced by the extracts of methanol and gasoline engine exhausts q Zunzhen Zhang *, Wangjun Che, Ying Liang, Mei Wu, Na Li, Ya Shu, Fang Liu, Desheng Wu 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 Received 6 July 2006; accepted 2 April 2007 Available online 14 April 2007

Abstract Gasoline engine exhaust has been considered a major source of air pollution in China, and methanol is considered as a potential substitute for gasoline fuel. In this study, the genotoxicity and cytotoxicity of organic extracts of condensate, particulate matters (PM) and semivolatile organic compounds (SVOC) of gasoline and absolute methanol engine exhaust were examined by using MTT assay, micronucleus assay, comet assay and Ames test. The results have showed that gasoline engine exhaust exhibited stronger cytotoxicity to human lung carcinoma cell lines (A549 cell) than methanol engine exhaust. Furthermore, gasoline engine exhaust increased micronucleus formation, induced DNA damage in A549 cells and increased TA98 revertants in the presence of metabolic activating enzymes in a concentration-dependent manner. In contrast, methanol engine exhaust failed to exhibit these adverse effects. The results suggest methanol may be used as a cleaner fuel for automobile.  2007 Elsevier Ltd. All rights reserved. Keywords: Genotoxicity; Cytotoxicity; Gasoline engine exhaust; Methanol; Vehicle emissions; Micronucleus test; Comet assay; Ames test

1. Introduction Automobiles play an important role in modern life. However, they can also generate hazards to the environment and human health by producing engine combusting exhaust (WHO, 2000). Epidemiological studies showed that chronic exposure to automobile emissions increased the risk of pulmonary and other pulmonary-associated cancers as well as non-cancerous health effects (Netterstrom and Suadicani, 1993; De Rosa et al., 2003; Hansen, 1993; Hansen et al., 1998; Guo et al., 2004; Lee et al., 2004). Based on animal and epidemiological studies, IARC classified gasoline engine exhaust as a possible human carq This research was supported by the National Science Funds of China (30571535). * Corresponding author. Tel.: +86 2 85501298. E-mail address: [email protected] (Z. Zhang).

0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.04.001

cinogen (2B) and diesel as a probable human carcinogen (2A) (IARC, 1989). The genotoxicity of gasoline engine exhaust was initially studied in the 1930s (Mauderly, 1994). However, the study has not been as extensive as that for diesel engine exhaust. Recently several studies have shown the mutagenicity of PM from gasoline engine exhaust by using the Salmonella mutagenicity assay (Pohjola et al., 2003a). It also can induce DNA damage, create DNA adducts, enhance micronucleus formation, as well as promote chromosome aberration that has been thought to be relevant with carcinogenesis. (Miller, 1970; Lawley, 1989; Pohjola et al., 2003b). Recent studies have demonstrated that the semivolatile organic compounds (SVOC) from automobile engine exhaust had the same genotoxic effects as PM (Liu et al., 2005; Seagrave et al., 2002; Cheng et al., 2004). In addition, the condensate from gasoline combustion exhaust also was found to be mutagenic by Ames assay (Ye et al., 1999).

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Methanol is rich in sources and has similar physical and chemical properties with gasoline. Methanol exhaust contains lower concentrations of particulate matters and nitrogen oxides than gasoline exhaust. Thus it may be considered as a potentially cleaner substitute for gasoline in regarding to control of air pollution. Little is known about the toxicity of absolute methanol engine exhausts. Maejima et al. found that sub-chronic inhalation of low concentration of methanol engine exhausts increased the amount of carboxyhemoglobin in the erythrocytes and decreased P-450 level in rat lung (Maejima et al., 1994). However, it is unknown that what kind of cyto- and genotoxicity absolute methanol engine exhausts could generate. Vehicle exhausts are constituted by three major components: gaseous components, soot particles, and semivolatile organic substance that are distributed in the phase in between the particulate and the gaseous component. Previous studies on engine emission samples primarily focused on toxicity of the PM and PM-associated organic material (Bernson, 1983; Crebelli et al., 1991). However, given the overlap in chemical composition and concurrent exposure, the toxicity of gasoline combustion exhaust that include all three components, SVOC, PM and condensate should be explored. This study has two aims: (1) to evaluate the relative geno- and cytotoxic potencies of automobile emissions with combination of PM, condensate and SVOC collected from gasoline and methanol engine exhausts for further understanding the relative contributions of gasoline and methanol engine exhausts to the adverse effects on human health, (2) to provide scientific evidence for decision-making on the substituting methanol for gasoline based on the toxicities of the two types of vehicle emissions. 2. Material and methods

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40 passengers. The vehicle is a 1996 model of Guangzhou passenger bus with a Dongfeng Gasoline Series 155 kW engine that has no exhaust catalytic converter. The vehicle had a mileage of 68,602 (110,381 km). The samples were collected at 25 C after the bus was ignited with a coldstart for 10 min. Gasoline engine exhaust was collected using a constant volume sampler system from the vehicle fueled with No. 90-octane unleaded gasoline supplied by China Petroleum. The sampling apparatus consisted of a 200- (long) 0.6-cm (diameter) condensation tube that is directly attached to exhaust-pipe, an empty middle volume air sampler for collecting condensation, and a holder with a glass fiber filter, followed by a 21-(long) 1.1-cm (diameter) ‘‘U’’ shape tube that is filled with polyurethane foam (PUF) and crosslinked divinyl benzene (XAD-2) resin for trapping semivolatile compounds. A vacuum pump was then used 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 for collecting gasoline engine 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. The particulate matters and resin-absorbed matter were extracted for three times with dichloromethane under sonicating condition. Subsequently, all the extracted matters were combined and concentrated by evaporation in a rotary evaporator under low air pressure and constant temperature of 45 C maintained by water bath. The residual matters after evaporation were dissolved in dimethyl sulphoxide (DMSO) at a stock concentration of 200 L/ml (i.e. 1 ml solution contains the extract from 200 L emissions). The samples were aliquoted and stored at 20 C in the dark until use.

2.1. Materials Dulbecco’s modified eagle medium (DMEM), acridine orange (AO), ethidium bromide (EB), 3-[4,5-dimetho-thiazol-2-yo]-2,5-diphenyl tetrazolium bromide (MTT) and nicotinamide adenine dinucleotide phosphate (NADP) were all purchased from Sigma–Aldrich (St. Louis, MO, USA). Fetal calf serum was from HyClone Chemical Company (Logan, UT, USA). Normal melting point agarose (NMP) and low melting point agarose (LMP) were from AMRESCO Inc. (Solon, Ohio, USA). All other chemicals were analytical grade. Microplate Reader was from BioRad (Hercules, CA USA), CO2 incubator was from Sanyo (Japan), and fluorescence microscope was a product of Leica (Germany).

2.3. Collection and preparation of organic extract of methanol engine exhaust Methanol engine exhaust was collected using the same bus as used for collection of gasoline engine exhaust except that absolute methanol was used as fuel. The sampling apparatus is the same as that for collection of gasoline engine exhaust except that a middle-size air sampler filled with 5 ml water was utilized, and the ‘‘U’’ shape tube was filled with active carbon and silica gel. These alterations in sampling procedure were made to improve sampling efficiency. All other procedures for sampling and matter extraction were the same as that used for gasoline engine exhaust. The final sample was collected in water and stored at 20 C for use.

2.2. Collection and preparation of organic extracts from gasoline engine exhaust

2.4. Cell culture

The vehicle used in this study is a transit bus provided by Chengdu City Transit system that can accommodate

The human lung adenocarcinoma A549 cell line was purchased from Chengdu Hoist Inc. Ltd. Cell was cultured

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in DMEM (containing 10% fetal calf serum, 100 units/ml of penicillin and 100 lg/ml of streptomycin, pH 7.4). Cell was maintained in a humid incubator at 37 C with 5% CO2. When the cells grew to 5 · 106/ml (about 85% confluence), they were harvested by trypsinization with 0.25%trypsin–0.02%EDTA and pelleted by 5-min centrifugation at 1000 rpm. The cell pellet was subsequently resuspended with phosphate buffer saline (PBS). The cell suspension was adjusted to an appropriate concentration for various experiments and assays. 2.5. MTT assay The cytotoxicity of gasoline and methanol engine exhaust was examined by MTT assay according to manufacturer’s instruction (ATCC, Manassas, America) with some modifications. Briefly, the cell monolayer was trypsinized. Cells were then harvested and resuspended in fresh growth medium. 200-ll cell suspension (5 · 104/ml) was aliquoted into each well of a 96 well culture plate. After 24 h incubation, the medium was replaced with fresh medium having various concentrations of gasoline or methanol engine exhausts. The cells were then incubated for another 2 or 24 h. At the end of the experiments, cells were washed with phenol red-free minimum essential medium for two times. 180 ll medium and 20 ll MTT (0.5 mg/ml final concentration) were then added into each well. The samples were incubated for another 4 h. The medium was removed, and 150 ll DMSO was added. The plate was shaken slightly for 2 min to facilitate the dissolution of the blue formazan particles. The absorbance was determined at 570 nm using a Microplate Reader. 2.6. Micronucleus assay The micronucleus test was conducted according to the method of Michael Fenech (Fenech, 2000). Briefly, 105 cells were seeded into 25 ml cell culture flask with 4 ml DMEM culture medium and incubated at 37 C with 5% CO2. When the cells grew to about 85% confluence, they were washed thoroughly with PBS. Then 4 ml fresh medium was added into flask. Subsequently, 40 ll various concentrations of gasoline or methanol engine exhaust extract were added to each culture flask. Untreated cells were used as negative control. Cells treated with DMSO and mitomycin C (0.5 lg/ml) were used as solvent and positive controls, respectively. The final concentration of DMSO in culture medium did not exceeded 1%. The cells were harvested after 24 h incubation. Cells were resuspended in 0.075 M KCl solution and incubated at 4 C for 5 min (mild hypotonic treatment). Subsequently, the cells were pelleted by centrifugation and resuspended and fixed with 3:1 of methanol:acetic acid (v/v) for 20 min. This fixation step was repeated for three times. Finally, cells were resuspended in a small volume of methanol/acetic acid solution at a ratio of 95/5. 100 ll cell suspension was dropped

onto a clean slide. The slide was air dried at room temperature and stained for several minutes with 150 ll 40 lg/ ml acridine orange. Thousand randomly selected cells from each concentration were scored under fluorescent microscopy (400· magnification) for identifying micronuclei and micronucleated cells. The micronuclei were scored according to the method of Tolbert et al. (1992) with some modifications. Briefly, the diameter of the micronucleus must be no larger than one-third or no less than one-twentieth the main nuclei. The micronucleus must be nonrefractile, thus excluding small stain particles. The color of micronucleus must be the same as or brighter than the main nuclei, and micronucleus must be located within the cytoplasm but not in contact with the main nuclei. The fluorescent images were obtained by fluorescent microscope with a digital camera connected to microscope (Leica, Germany). The experiment was repeated for three times. 2.7. Single cell gel electrophoresis (comet assay) The alkaline version of the comet assay was conducted according to the procedure of Singh et al. (1988) with some modifications. Briefly, after exposed to different concentrations of gasoline or methanol engine exhaust for 2 h, cells were washed twice with PBS and harvested by trypsination and centrifugation at 2000 rpm for 5 min. Subsequently, 10 ll cell suspension containing 5 · 104 cells were mixed with 150 ll 0.6% LMP pre-warmed at 37 C and 70 ll mixture was spread on each of the duplicated spots on frosted microscope slides that was pre-coated with 0.8% NMP. The cell-gel layer was covered with a coverslip allowing gel solidification for 10 min at 4 C. Then the coverslips were removed and the slides were immersed in cold fresh lysing 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 at least 1 h at 4 C in a dark chamber. 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. Electrophoresis was performed in the same buffer for 30 min at 25 V and 300 mA. All these steps were conducted in the dimmed light to prevent additional DNA damage. After electrophoresis, the slides were washed with distilled water and allowed to dry at room temperature. Just prior to analysis the DNA was stained with 30 ll of ethidium bromide (20 lg/ml). The comets were analyzed at 200· magnification under a fluorescence microscope (Leica, Germany) attached to digital camera (Nikon, Japan) and connected to a personal computer. Two hundred randomly selected cells were scored from each slide (two slides per dose), and the percentage of comet cell (comet rate) was calculated. Tail length (comet length) of 30 randomly selected comet cells was also measured by calibrated scale in the ocular of the microscope to evaluate the length of DNA migration.

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2.8. Ames test

3. Results

2.8.1. S9 fraction preparation To obtain the liver microsomal fraction, male Wistar rats were injected through ip with polychlorinated biphenyl (Aroclor1254). Rat livers were homogenated, and centrifuged at 9000g for 10 min at 4 C. The supernatant was then aliquoted. The aliquots were stored at 80 C until use. The final preparation of the metabolizing system (S9 mixture) was made in accordance with the protocol of Ames et al. (1975). The composition and final concentrations of the S9 mixture used for the Ames test were as follows: glucose-6-phosphate, 4.4 mM; NADP, 0.84 mM; KCl, 30 mM; NaHCO3, 0.032%; and S9 fraction, 10%.

3.1. MTT assay

2.8.2. Ames Salmonella/microsome test The mutagenicity test was performed using TA98 and TA100 with or without S9 mixture. The method we used followed the recommendations of Maron and Ames (1983) with some modifications. Briefly, the Salmonella typhimurium bacteria and histidine auxotrophic strains TA98 and TA100 were grown at 37 C with continuous shaking until bacteria grew to a density of 1–2 · 109 cells/ ml indicated by OD600 absorbance of 0.2–0.3. The top agar was made by mixing 2.5 ml of heated agar, 0.1 ml of test chemical and 0.1 ml of bacterial suspension with or without 0.5 ml of S9 solution, and then added to three different minimal glucose agar plates. All plates were incubated at 37 C for 72 h. The number of bacterial colonies was determined. The entire experiment was repeated on a different day with a total of six plates used for each concentration of test substances with or without S9. Each tested strain was routinely checked to confirm its features for optimal response to known mutagenic chemicals. 2,4,7-Trinitro-9-fluorenone (0.2 lg/plate) and sodium azide (1.5 lg/plate) were used as positive controls for TA98 and TA100 with S9, respectively. In the presence of S9, 2-aminofluorene (20 lg/plate) was used as positive control for the both strains. A test compound was judged to be mutagenic in the plate test if it produced, in at least one concentration and one strain, a response equal to twice (or more) of the control incidence with a dose–response relationship considered to be positive (De Serres and Shelby, 1979; Suter et al., 2002). 2.9. Statistical analysis Data are expressed as the mean ± standard deviation. Statistical analysis of the data of micronucleus assays, comet assays and Ames test was performed by using u-test of Poisson distribution, Student’s t-test and v2 test, and P < 0.05 was considered significantly different. Statistical comparisons of the effects of methanol or gasoline exhaust and the control on MTT assay were analyzed by ANOVA.

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The cytotoxicity of gasoline and methanol engine exhaust at concentrations of 0.05–0.8 L/ml was examined by measuring cell viability via MTT assay at 2 and 24 h. The results were illustrated in Table 1. No significant difference was found in the absorbance between the groups with 2 h methanol engine exhaust treatment and negative control at any tested concentration (P > 0.05). However, the absorbance from the group with 2 h gasoline engine exhaust treatment was significantly reduced compared with that from the control at the highest concentration (0.8 L/ml) (P < 0.05). The difference was more obvious in the groups with 24 h treatment of gasoline exhaust. At the concentrations that are higher than 0.2 L/ml, all the groups exhibited significantly reduced absorbance compared with the control. Furthermore, a clear dose–response relationship was also identified under these doses. The absorbance from the groups with 24 h methanol engine exhaust treatment was significantly reduced only when the concentration of the exhaust reached to 0.8 L/ml. At the same concentration,

Table 1 Absorbances of A549 cells treated with different concentrations of gasoline and methanol engine exhaust Dose (L/ml) 0 0.05 0.1 0.2 0.4 0.8

A570 of MEE

A570 of GEE

2h

24 h

2h

24 h

0.998 ± 0.094 1.000 ± 0.058 0.989 ± 0.068 1.042 ± 0.104 0.898 ± 0.093 1.036 ± 0.102

1.060 ± 0.081 1.069 ± 0.098 1.051 ± 0.101 0.994 ± 0.107 1.071 ± 0.029 0.858 ± 0.106*

1.004 ± 0.089 1.011 ± 0.078 0.994 ± 0.072 0.992 ± 0.087 1.010 ± 0.055 0.796 ± 0.062*

0.996 ± 0.089 1.002 ± 0.067 0.954 ± 0.072 0.832 ± 0.074* 0.698 ± 0.082* 0.524 ± 0.076*

GEE: gasoline engine exhaust; MEE: methanol engine exhaust. * Significant with respect to negative control (P < 0.01).

Table 2 Rates of micronucleated cells in A549 cells treated with different concentrations of gasoline and methanol engine exhaust Group

Doses (L/ml)

Rates of micronucleated cells (%)

GEE

0.025 0.05 0.1 0.2

2.65 ± 0.21* 4.35 ± 0.40* 4.95 ± 0.32* 5.85 ± 0.35*

MEE

0.025 0.05 0.1 0.2

1.35 ± 0.31 1.40 ± 0.30 1.30 ± 0.26 1.40 ± 0.36

Negative control DMSO MMC

0 0 0.5 lg/ml

1.30 ± 0.26 1.35 ± 0.21 4.90 ± 0.36*

GEE: gasoline engine exhaust; MEE: methanol engine exhaust; DMSO: dimethylsulphoxide; MMC: mitomycin C. * Significant with respect to negative control (P < 0.01).

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the absorbance from gasoline engine exhaust was lower than that from methanol engine exhaust. Thus, it is obvious that the cytotoxicity resulting from gasoline engine exhaust was stronger than that from methanol engine exhaust.

quency of micronucleated cells (4.90%). For the negative control and DMSO control only a low rate of micronucleus was identified as 1.30% and 1.35%, respectively. 3.3. Comet assay

3.2. Micronucleus assay Results of the micronucleus assay are summarized in Table 2. The results indicated that gasoline engine exhaust significantly increased the formation of micronucleated cells at all the doses used in the experiments. Furthermore, a dose–response relationship was observed between gasoline engine exhaust exposure and formation of micronucleus. Methanol engine exhaust, however, did not exhibit a significant impact on the formation of micronucleus at all tested concentrations (P > 0.05). Under the same exposure dosage, the rate of micronucleated cells resulting from gasoline engine exhaust was much higher than that from methanol exhaust (P < 0.05). Mitomycin C used as a positive control (0.5 lg/ml) significantly increased the fre-

Table 3 illustrates the results of comet assay. There is significantly difference in comet rate and comet length at any tested concentration between the group exposed to gasoline engine exhaust and the control (P < 0.05). However significant difference was not found between the group exposed to methanol engine exhaust and the control (P > 0.05). A549 cells exposed to gasoline engine exhaust had higher rate of tailed cells and length of DNA migration than those exposed to methanol engine exhaust. Under our experimental conditions, methanol engine exhaust did not exhibit any effect of DNA damage on A549 cells at any concentration we examined that was equivalent to the one used for gasoline exhaust exposure. 3.4. Ames test

Table 3 Comet rate and comet length in A549 cells treated with different concentrations of gasoline and methanol engine exhaust Group

Doses (L/ml)

Comet rate (%)

Comet length (lm)

GEE

0.025 0.05 0.1 0.2 0.4

78.3 ± 5.2* 89.1 ± 6.8* 100* 100* 100*

40.2 ± 4.6 51.5 ± 5.4* 75.2 ± 8.9* 102.6 ± 15.7* 162.9 ± 19.8*

MEE

0.025 0.05 0.1 0.2 0.4

16.2 ± 3.2 15.3 ± 3.6 12.7 ± 2.8 14.4 ± 2.5 16.0 ± 3.0

7.0 ± 3.1 6.9 ± 2.9 5.8 ± 1.2 6.5 ± 2.7 7.8 ± 3.1

Negative control DMSO K2Cr2O7

0 0 0.4 mmol/L

17.3 ± 2.2 12.4 ± 2.0 100*

6.6 ± 2.2 6.3 ± 2.4 136.4 ± 13.5*

GEE: gasoline engine exhaust; MEE: methanol engine exhaust; DMSO: dimethylsulphoxide; K2Cr2O7: potassium dichromate (it is used as a positive control). * Significant with respect to negative control (P < 0.01).

The results on mutagenicity of gasoline and methanol engine exhaust extract examined by using Salmonella typhimurium test strain TA98 and TA100, with S9 and without S9 activation, are summarized in Tables 4 and 5. In the absence of S9, gasoline engine exhaust only significantly increased colony formation in strain TA98 at the concentration of 10 L/plate and 20 L/plate. However, in the presence of S9, it significantly increased colony formation at all tested concentrations except 0.625 L/plate. Furthermore, a dose–response relationship between the concentration and number of revertants was identified at the concentrations ranging from 0.625 to 10 L/plate (r = 0.95, P < 0.05). The mutagenicity of gasoline engine exhaust was higher in TA98 with S9 activation than without S9 activation (P < 0.05). However, gasoline engine exhaust did not exhibit mutagenicity in strain TA100 at all exposure levels ranging from 0.625 to 20 L/plate. Methanol engine exhaust did not increase colony formation in both TA98 and TA100 strains in the absence or

Table 4 Revertants in two strains of Salmonella typhimurium treated with different concentrations of gasoline engine exhaust Dose (L/plate)

TA98 S9

0.625 1.25 2.5 5.0 10 20 DMSO Positive control

33.0 ± 4.6 47.3 ± 3.8 49.7 ± 7.4 53.7 ± 3.8 69.0 ± 1.0a 96.0 ± 9.2a 31.0 ± 3.6 1673.3 ± 110.2a

TA100 +S9 36.7 ± 4.7 73.0 ± 6.2a 85.3 ± 8.4a 96.0 ± 5.3a 133.7 ± 11.5a 86.0 ± 12.0a 35.0 ± 4.4 1763.3 ± 90.2a

DMSO: dimethylsulphoxide. a Spontaneous reverse number was at least 2 times higher than DMSO.

S9 118.7 ± 13.0 132.7 ± 12.7 144.5 ± 18.5 160.3 ± 14.2 147.0 ± 14.1 90.7 ± 6.4 123.0 ± 17.3 1290.0 ± 108.2a

+S9 138.7 ± 13.2 159.7 ± 4.0 160.3 ± 9.5 166.3 ± 10.2 175.0 ± 7.5 125.0 ± 13.1 137.0 ± 18.1 1413.3 ± 80.2a

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Table 5 Revertants in two strains of Salmonella typhimurium treated with different concentrations of methanol engine exhaust Dose (L/plate)

TA98 S9

0.3125 0.625 1.25 2.5 5.0 10.0 20.0 Negative control Positive control a

29.3 ± 3.1 36.3 ± 3.5 39.0 ± 2.6 2.7 ± 2.1 0 0 0 32.3 ± 2.6 1673.3 ± 110.2a

TA100 +S9

S9

36.7 ± 6.1 37.7 ± 2.5 40.0 ± 5.6 39.7 ± 6.7 39.0 ± 6.1 38.5 ± 5.4 37.1 ± 6.2 36.1 ± 3.8 1763.3 ± 90.2a

126.3 ± 9.1 124.0 ± 15.1 135.0 ± 14.2 128.1 ± 9.0 0 0 0 121.8 ± 15.4 1290.0 ± 108.2a

+S9 143.7 ± 14.0 152.3 ± 7.5 138.0 ± 15.1 152.0 ± 12.5 160.3 ± 14.1 152.4 ± 13.8 149.1 ± 15.6 132.6 ± 12.6 1413.3 ± 80.2a

Spontaneous reverse number was at least 2 times higher than negative control.

presence of S9 (Table 5) at all exposure levels. It exhibited cytotoxicity in the absence of S9 and inhibited colony formation at concentrations above 2.5 L/plate. Thus, these results suggest that methanol exhaust did not exhibit obvious mutagenic effects in TA98 and TA100 under our experimental conditions.

4. Discussion Large human population in the world continues to be exposed to pollutant mixtures containing known or suspected carcinogens. Epidemiological studies over the last 50 years suggest consistently that general ambient air pollution, mainly due to the incomplete combustion of fossil fuels, may be responsible for increased rate of lung cancer (Cohen and Pope, 1995). The substances in the automobile combustion exhaust may exist as a form of complex mixture that include carcinogenic compounds such as nitrofluorene, polycyclic aromatic hydrocarbons (PAH) benzene, heterocyclic amine, various aromatic nitroso compounds and many others. They have been suggested to increase lung cancer risk in humans (Pope et al., 2002; Gu et al., 1992). In many big cities in China, the major pollutants in urban air are derived from automobile exhausts. Thus, this study is focused to determine the cytotoxic effects of gasoline and methanol engine exhaust and their mutagenicity. It will further help to evaluate the potential application for using methanol as a substitute of gasoline. Genotoxicity should be evaluated under non-toxic conditions. To evaluate cell viability, colorimetric MTT assay was utilized in our study to measure mitochondrial activity in viable cells. This method is based on the conversion of the MTT to MTT–formazan crystal by mitochondrial enzyme. Because MTT is metabolized to formazan only by viable cells, a reduction of measured optical density at 570 nm in toxicant treated cells in comparison to untreated cells would indicate a loss of cell growth and viability. Therefore, the magnitude of the absorbance at 570 nm is proportional to the number of living cells and can be used as an index of the cell viability.

At the 2 h time point, both gasoline and methanol engine exhaust had no effect on the reduction of MTT except the highest exposure level of gasoline, suggesting that at this time point, little effect on cell viability resulting from both types of exhaust could be detected. However, at the 24 h time point, significant concentration-dependent decreases in MTT were observed in gasoline engine exhaust-treated groups at concentrations ranging from 0.2 to 0.8 L/ml. Thus, the gasoline engine exhaust was apparently more toxic than methanol engine exhaust to A549 cells. In contrast, methanol engine exhaust exhibited little or no cytotoxicity to A549 cells under the same conditions. DNA breaks in A549 cells treated with gasoline engine exhaust could be detected by using comet and micronucleus assay at the concentrations by which no cytotoxicity was observed. The results from comet assay have demonstrated that gasoline engine exhaust increased the rate of tailed cells and length of DNA migration in A549 cells in a dose-dependent manner. Significant difference was found in the rate of tailed cells and length of DNA migration at any tested concentrations between the groups treated with gasoline engine exhaust and control, but not for the groups treated with methanol engine exhaust groups. These results indicated that gasoline engine exhaust induced DNA damage, whereas methanol engine exhaust did not. The cause for the genotoxicity resulting from gasoline engine exhaust is not known. But it is well known that reactive oxygen species (ROS) plays a crucial role in DNA damage progression. Metabolic activation of gasoline engine exhaust may lead to generation of ROS that are responsible for its genotoxicity. It is known that gasoline engine exhaust contains PAH and metal ions. Metabolic activation of PAH may result in the production of ROS via cytochrome P4501A1-dependent transformation and redox cycling between quinines and semi-quinone radicals (Segura-Aguilar et al., 1998; Chesis et al., 1984; Monks et al., 1992). Metal ion can also induce the production of ROS by Fenton reactions (Lloyd and Phillips, 1999). ROS and some metabolic activated form of PAH may react with DNA to produce adducts (Kim and Lee, 1997). If the DNA

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adducts were not correctly repaired by the DNA nucleotide excision repair or DNA base excision repair, it would lead to DNA single strand break (Eastmen and Barry, 1992). Cheng et al. (2004) showed that PM which is one of the major components of gasoline engine exhaust also could induce genotoxicity through a ROS-dependent pathway, which can be further augmented by metabolic activation. In supporting of this, antioxidant has been found to inhibit PM-induced genotoxicity. The clastogenic effects of gasoline engine exhaust on chromosomes were also examined by using in vitro micronucleus test in A549 cells. Micronucleus is displaced chromatin, resulting from chromosome loss or breakage that fail to be incorporated into daughter nuclei as a cell divides. Our results showed that the groups treated with various doses of gasoline engine exhaust significantly increased the micronucleus formation whereas methanol engine exhaust did not exhibit any effect on formation of micronucleus at all tested concentrations. For many years, the Ames test has been widely used as a screening tool to determine the mutagenic potential of new chemicals and drugs, since it has a predictive value for rodent carcinogenicity (Zeiger et al., 1990). Previous studies indicated that PM or SVOC exhibited mutagenicity in strains TA98 and TA100 (Seagrave et al., 2002). In our study, gasoline engine exhaust in the absence and presence of S9, was found to exhibit mutagenicity only in strain TA98, but not in strain TA100. This suggested that the mutagens in our samples led to frameshift rather than base substitution mutation. In the presence of S9, the number of reverse mutation resulting from gasoline exhaust-treated plates was increased in TA98 in a concentration-dependent manner. The concentration needed to induce mutation in the presence of S9 is much lower than that in the absence of S9. This suggests that gasoline engine exhaust may contain less direct- and more indirect-mutagenic substances. In contrast, methanol engine exhaust did not cause mutagenic effect in strains TA98 and TA100 in the absence and presence of S9 at all tested concentration. At the concentration above 5.0 L/plate, in the absence of S9, normal colony formation was not detected in strains TA98 and TA100, indicating that normal colony formation was completely inhibited. In order to further understand the toxicity of methanol engine exhaust, another round of Ames test was performed to examine gene mutation of pure methanol. Our data indicated that pure methanol in the absence and presence of S9 was not mutagenic and cytotoxic in both strains at concentration of 100 ll/plate (data not shown). Thus, the bacterial cytotoxicity of methanol engine exhaust appears to result from methanol combustion products rather than methanol itself. In conclusion, the gasoline engine exhaust exhibited a stronger cytotoxicity to A549 cells than methanol engine exhaust at 2 h and 24 h exposure time points. Gasoline engine exhaust exhibited genotoxicity, whereas, methanol engine exhaust did not. Our results suggest that methanol may be used as a cleaner fuel to substitute gasoline in future.

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