Mutagenicity of diesel engine exhaust in the Ames ...

1 downloads 0 Views 535KB Size Report
Sep 6, 2011 - This article was downloaded by: [HINARI]. On: 02 November 2011, At: 10:59. Publisher: Taylor & Francis. Informa Ltd Registered in England ...
This article was downloaded by: [HINARI] On: 02 November 2011, At: 10:59 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Toxicological & Environmental Chemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gtec20

Mutagenicity of diesel engine exhaust in the Ames / Salmonella assay using a direct exposure method Mamadou Fall b

a b

c

a

, Hasnaà Haddouk , Stéphane Loriot , Amadou d

c

Diouf , Frédéric Dionnet , Roy Forster & Jean-Paul Morin

a

a

INSERM U644, Faculty of Medicine and Pharmacy, Université de Rouen, Rouen, France b

Laboratoire de Toxicologie, Faculty of Medicine and Pharmacy, Université Cheikh Anta DIOP Dakar, Dakar, Senegal c

Centre International de Toxicologie, Evreux, France

d

CERTAM, Sainte Etienne du Rouvray, France

Available online: 06 Sep 2011

To cite this article: Mamadou Fall, Hasnaà Haddouk, Stéphane Loriot, Amadou Diouf, Frédéric Dionnet, Roy Forster & Jean-Paul Morin (2011): Mutagenicity of diesel engine exhaust in the Ames / Salmonella assay using a direct exposure method, Toxicological & Environmental Chemistry, 93:10, 1971-1981 To link to this article: http://dx.doi.org/10.1080/02772248.2011.619538

PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,

Downloaded by [HINARI] at 10:59 02 November 2011

demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Toxicological & Environmental Chemistry Vol. 93, No. 10, December 2011, 1971–1981

Mutagenicity of diesel engine exhaust in the Ames / Salmonella assay using a direct exposure method Mamadou Fallab*, Hasnaa` Haddoukc, Ste´phane Loriota, Amadou Dioufb, Fre´de´ric Dionnetd, Roy Forsterc and Jean-Paul Morina a

INSERM U644, Faculty of Medicine and Pharmacy, Universite´ de Rouen, Rouen, France; Laboratoire de Toxicologie, Faculty of Medicine and Pharmacy, Universite´ Cheikh Anta DIOP Dakar, Dakar, Senegal; cCentre International de Toxicologie, Evreux, France; d CERTAM, Sainte Etienne du Rouvray, France

Downloaded by [HINARI] at 10:59 02 November 2011

b

(Received 27 January 2011; final version received 16 August 2011) The aim of this study was to investigate the potential mutagenic activity of diesel engine exhaust in the Ames/Salmonella assay using a direct aerosol exposure system. So, TA 98 and TA 100 strains, with or without added S9 mix, were exposed to diesel emissions after varying degrees of filtration. Variants of these two strains, deficient in nitroreductase (TA 98NR and TA 100NR) or overexpressing O-Acetyl Transferase (YG 1024 and YG 1029), were also exposed to total (unfiltered) diesel exhaust to highlight the putative mutagenicity of any nitro-PAHs present in these emissions. Mutagenic activity of the diesel exhaust was demonstrated on Salmonella typhimurium, strains TA 100 and variants TA 100 NR and YG1029. The use of a particle filter did not modify the genotoxicity of the diesel emissions, indicating a major contribution of the gas phase to the mutagenicity of these diesel emissions. The prominent role of the particulate-associated nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) claimed by some authors working on diesel exhaust organic extracts was not confirmed by our results with native diesel exhaust exposure. Our results show that the gas phase is potentially more mutagenic than the particles alone. Keywords: diesel exhaust; Ames test; aerosol exposure; mutagenicity

Introduction The complexity and diversity of air pollution, which contains thousands of gaseous or particle-associated compounds, makes the evaluation of their impact on health difficult. Toxicological studies of diesel exhaust present numerous problems, such as the sampling and exposure procedures, evaluating the contribution of the particulate and/or gaseous phases, and identifying the compounds or families of compounds contributing to total engine emission toxicity. As a consequence of the difficulties in testing diesel exhaust directly in vitro, studies are generally carried out on particulate fractions. In order to minimize artifacts due to sampling, some authors have worked on continuous flows of diesel exhaust (Morin et al. 1999; Abe et al. 2000). This approach avoids modification of the physico-chemical characteristics and chemical bioavailibility of the engine emissions prior to bacterial exposure.

*Corresponding author. Email: [email protected] ISSN 0277–2248 print/ISSN 1029–0486 online ß 2011 Taylor & Francis http://dx.doi.org/10.1080/02772248.2011.619538 http://www.tandfonline.com

Downloaded by [HINARI] at 10:59 02 November 2011

1972

M. Fall et al.

The genotoxic potency of environmental mixtures is often monitored by use of the Salmonella typhimurium mammalian microsome assay Ames test. Organic solvent extracts of airborne particulates generated by combustion processes contain a large number of compounds that are mutagenic in short-term bioassays and carcinogenic in laboratory animals (Chou and Lee 1990; Lee et al. 1994). Diesel exhaust particle (DEP) extracts have been found to be both direct-acting and indirect-acting in the Ames bacterial mutation assay (Ball et al. 1990, Lapin et al. 2002). To highlight the genotoxicity of nitro-PAHs, some authors have used Salmonella strains which are nitroreductase-deficient (NR) (TA 98NR, TA 100NR) or which over-express O-acetyl transferase (OAT) (YG 1024, YG 1029), or both of these at the same time (YG 1041, YG 1042). These enzymes mediate the bio-activation of the nitropyrenes (Masahiko, Ishidate, and Nohmi 1990; Josephy, Gruz, and Nohmi 1997). Thus, strains YG 1024 and YG 1029 are found to be 20–30 times more responsive to the action of these mutagens than their respective homologues without enzyme over-expression, TA 98 and TA 100 (Scheepers, Theuws, and Bos 1991, Houk et al. 1992). Few studies have been carried out on the mutagenicity of diesel exhaust using a direct exposure method to evaluate the total or gaseous phase of engine emissions. These include the studies of Jones et al. (1985) and Courtois et al. (1993). This study was conducted to investigate the potential mutagenic activity of diesel engine exhaust in the Ames/Salmonella assay using a direct exposure method. So, the impact of particle filter on mutagenic activity of diesel exhaust is evaluated.

Materials and methods Metabolic activation system S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg kg1 by the intraperitoneal route). The S9 mix was prepared at þ4 C, immediately before use, and maintained at this temperature until added to the overlay agar. The final composition of the S9 mix was as follows: glucose-6-phosphate (5 mmol L1), NADP (4 mmol L1), KCl (33 mmol L1), MgCl2 (8 mmol L1), sodium phosphate buffer pH 7.4 (100 mmol L1), and S9 fraction, 10% (v/v).

Bacterials strains Salmonella typhimurium tester strains used were TA 98, TA 100, TA 98NR, TA100NR, YG1024 and YG1029. Strains TA 98 and TA100 were kindly supplied by the B.N. Ames Laboratory (University of California, Berkeley, USA). Nitroreductase-deficient strains TA 98NR and TA 100NR were supplied by the Institut Pasteur (Lille, France). Strains YG1024 and YG1029, which over-express OAT, come from The National Institute of Health Science (Tokyo, Japan). Bacterial suspension (0.1 mL) mixed with S9 mix or phosphate buffer at pH 7.4 (0.5 mL), with 2 mL of overlay agar (containing traces of histidine and biotin), was poured onto a Petri plate containing 20 mL of minimum medium. The plates were covered, inverted, and placed in the rack in the closed jar. Positive controls used to validate the sensitivity of the strains in the various tests are: 2-nitrofluorene (Ega-Chemie, Steinheim, Germany) for the TA 98 strain without S9 mix

Toxicological & Environmental Chemistry

1973

(0.5 mg per plate), sodium azide (Sigma, St. Louis, MO, USA) for the TA 100 strain without S9 mix (1 mg per plate), and 2-Aminoanthracene (Sigma, St. Louis, MO, USA) for both TA 98 and TA 100 strains with S9 mix (2 mg per plate).

Downloaded by [HINARI] at 10:59 02 November 2011

Diesel engine A power-generating unit Honda EX4D with a 4-stroke, 230 cm3 diesel engine was used, and in order to apply a reproducible load to the engine, it was connected to seven independent lamps, the lighting of which made it possible to modulate the load imposed on the engine. Under the experimental conditions of this study, the electric output developed in full load was 3500 Watts. The engine was supplied with standard diesel fuel containing less than 500 ppm of sulfur. To evaluate potential differences in the mutagenic effects of gaseous and DEP fractions from diesel exhaust on bacterial strains, DEPs were continuously removed from the sampled exhausts with a silicone carbide diesel particle filter placed on the taking line, downstream from the dilution device. This procedure did not alter the gas phase pollution content, but it removed more than 99% of the particles.

Exposure and dilution system The apparatus used for the gaseous phase exposure diesel exhaust Ames test is shown in Figure 1 and consists of a 2.5 L capacity polycarbonate jar (Merck, Darmstadt, Germany), a lid with a clamp and O-ring gasket, and a Petri-dish rack for up to 12 standard Petri dishes. This device is usually used for the anaerobic culture of bacteria. These jars had previously been used in our laboratory to assess gaseous phase mutagenicity (Fall et al. 2007). In this study they were modified for direct exposure to the engine emissions, passing so from a static system to a dynamic exposure system. Thus holes were drilled in the lid of the jar, on the side (gas entry), and in the center (gas exit). The entry was extended to the

Figure 1. Diagram of the exposure system used to perform the diesel exhaust gas Ames test.

Downloaded by [HINARI] at 10:59 02 November 2011

1974

M. Fall et al.

Figure 2. description of the diluting system used to perform the Diesel exhaust gas Ames test.

bottom of the exposure system by a polyamide pipe to ensure the good diffusion of gases, by going back into the whole volume of the jar. Diesel exhaust was diluted with purified air using mass flow regulators (MFRs) (Figure 2). This provides, at the same time, the functions of a flowmeter and an electronically controlled valve. MFR1 was controlled to inject the volume of air necessary to ensure the desired dilution. MFR2 ensured the total output of gas (air þ exhaust) which passed into the circuit.

Pollutants measurements Soot emission in the exhaust line was measured by a mass monitoring system (AVL 415 refractometer). Regulated pollutant emissions, including hydrocarbon (HC: flame photometric detector), carbon monoxide (CO: infrared analyzer), nitrogen oxides (NO and NOx: chemiluminescent detection method) were evaluated with a HORIBA MEXA7000 analyzer. The aerodynamic size distribution of DEPs was assessed in the exhaust line using a scanning mobility particle sizer (SMPS).

Experimental procedure Exhaust was evacuated from the exposure chambers under a constant flow rate of 2.5 L min1 and the duration of exposure to the diluted exhaust was usually 5 h. The exposed plates were taken out of the jar and were then maintained at 37 C until a total incubation time of 48 h had elapsed. All the plates were scored after incubation using an automatic counter (Cardinal counter, Perspective Instruments, Suffolk CB9 7BN, UK), and a reproducible twofold increase in the number of revertants, when compared with the vehicle control

Toxicological & Environmental Chemistry

1975

Downloaded by [HINARI] at 10:59 02 November 2011

Figure 3. Particle size distribution in whole exhaust and the gaseous phase.

(plates exposed to purified air only), together with evidence of a dose-relationship was considered as a positive result. Statistical analysis The statistical test used for the comparison of the results derived from the difference techniques was Student’s t-test with a p-value 0.05.

Results The mean levels of HC, NO, NOx, and CO were, respectively, 590  43, 306  27, 326  18 and 220  25 ppm. Average levels of DEPs in the raw exhaust line were 19.6  3 mg m3. The size distribution of DEPs in the exhaust line is shown in Figure 3. Typical log-normal distribution with a maximum value at 100 nm was recorded. Moreover, downstream from the filter, more than 99.99% of DEPs were removed from the diesel exhaust. The mutagenic activity of unfiltered and filtered diesel exhaust on strain TA 98, with or without S9 mix, are reported in Table 1. These results show that no significant increase in the number of revertants was observed in the TA98 strain without S9 mix. With S9 mix, a weak, dose-related increase up to 1.97-fold in the number of revertants was noted with the unfiltered exhaust. Strain TA 100 exposure to diesel exhaust gave the results reported in Table 2. These results show that the TA 100 strain was more sensitive than TA98 under the exposure conditions of this study. Exhaust genotoxicity was observed from 10% of exhaust, especially for filtered exhaust. A statistically significant difference was observed between unfiltered and filtered exhaust without S9 mix. The presence of metabolic activation seems to decrease the mutagenicity of filtered exhaust. The genotoxic activity of unfiltered diesel exhaust on TA 98 NR and YG 1024 strains when compared to TA 98 strain is shown in Figure 4. These results show the mutagenic effect of diesel exhaust on the YG 1024 strain. Therefore this strain, which over-expresses OAT, is more sensitive than the TA 98 strain. Figure 5 presents results obtained with TA 100 NR and YG 1029 strains, and compared with TA 100. These results show the mutagenic effect of diesel exhaust on these

1976

M. Fall et al.

Table 1. Mutagen activity of unfiltered and filtered diesel exhaust on TA 98 without and with S9 mix. Without S9 mix Unfiltered % Exhaust

Downloaded by [HINARI] at 10:59 02 November 2011

0 10 20 30 Positive controls

With S9 mix

Filtered

Unfiltered

Filtered

Revertants/ Revertants/ Revertants/ Revertants/ plate ratio plate ratio plate ratio plate ratio 25  4 29  3 33  5 32  10 þ

– 1.16 1.32 1.28

24  6 33  3 35  5 39  4

– 1.32 1.40 1.56

26  4 47  4* 49  5* 47  8 þ

– 1.89 1.97 1.89

25  3 37  2 34  5 45  3#

– 1.48 1.36 1.80

Notes: The mean of the results from three independent experiments (three plates per experience) are expressed here as revertants/plate  SEM. The ratio ¼ revertants with diesel exhaust/revertants with purified air. The positive control was tested in the standard Ames assay to validate the sensitivity of the strains (in the absence of S9 mix) or the metabolic capability of the S9 mix. Statistically significant different with S9 mix using the t-test, between: (*) unfiltered and filtered at 10 and 20% exhaust; (#) 30 and others % at filtered exhaust.

Table 2. Mutagen activity of unfiltered and filtered diesel exhaust on TA 100 without and with S9 mix. Without S9 mix Unfiltered % Exhaust 0 10 20 30 Positive controls

With S9 mix

Filtered

Unfiltered

Revertants/ Revertants/ Revertants/ plate ratio plate ratio plate ratio 112  9 263  8* 271  12* 314  18* þ

– 2.35 2.42 2.80

408  20 419  32 435  27

– 3.64 3.74 3.88

215  10 258  18 290  18 þ

– 1.92# 2.30 2.59

Filtered Revertants/ plate ratio 295  17 379  22 359  14

– 2.63 3.38 3.21

Notes: The mean of the results from three independent experiments (three plates per experience) are expressed here as revertants/plate  SEM. The ratio ¼ revertants with diesel exhaust/revertants with purified air. The positive control was tested in the standard Ames assay to validate the sensitivity of the strains (in the absence of S9 mix) or the metabolic capability of the S9 mix. Statistically significant different using the t-test, between: (*) unfiltered and filtered exhaust without S9 mix; (#) 10 and others % at filtered exhaust with S9 mix.

three strains and demonstrate the greater sensitivity of strains TA 100 and YG 1029. A weak decrease is observed with TA 100 NR. Macroscopic examination of plates showed that revertants were localized uniformly across the medium. Microscope examination of the bacterial lawn did not show any signs of exhaust toxicity, even with the total emissions (data not shown). No precipitate resembling diesel particles was visible.

Downloaded by [HINARI] at 10:59 02 November 2011

Toxicological & Environmental Chemistry

1977

Figure 4. Mutagen activity of unfiltered diesel exhaust on TA 98, TA 98NR and YG 1024 without S9 mix. The mean of the results from two independent experiments are expressed here as revertants/ plate þ/ SEM. Note: *Ratio  2 ( ¼ revertants with exhausts/revertants with control).

Figure 5. Mutagen activity of unfiltered diesel exhaust on TA 100, TA 100NR and YG 1029 without S9 mix. The mean of the results from two independent experiments are expressed here as revertants/ plate þ/ SEM. Note: *Ratio  2 (¼ revertants with exhausts/revertants with control).

Discussion The aim of this study was to develop a more appropriate model to evaluate the mutagenicity of diesel exhaust, based on continuous aerosol emission flow from the engine exhaust line. The advantage of this model is to mitigate the disadvantages of sampling and/or extraction of diesel exhaust particles. Gas phase exposure takes place in this system by simple diffusion in the agar layer. The contact between particles and agar may be considered as limited, especially as the dishes containing the bacterial strain were covered. Use of a low flow rates (2.5 L min1) with renewal of the atmosphere in the jar each minute permitted a good diffusion of gases in the agar. The small size of diesel soot particles probably allows good diffusion of the soot also. With regards to the engine used in this study, HC and CO levels found were relatively higher than those obtained with the most recent light-duty engines. This difference can be attributed to lower combustion efficiency due to the lower injection pressure of this type of

Downloaded by [HINARI] at 10:59 02 November 2011

1978

M. Fall et al.

engine opposite to ‘‘High Direct Injection’’ (HDI) used in new motor technology. The removal (by filtration) of more than 99.99% (both in number and mass) of DEPs from the diesel exhaust suggests that these sampling and exposure conditions allow differentiating the respective contributions of the gaseous phase and particle fractions. The most commonly used bacterial strains are TA 98 and TA 100. Strain TA 98 appeared to be less sensitive to Diesel exhaust mutagenicity than strain TA 100, since a twofold increase in the number of revertants was obtained only from 50% of unfiltered emissions (data not shown). At this exhaust concentration the formation of condensation in the dilution system could not be avoided, and this may have resulted in alteration of the physico-chemical properties of the exhaust sample. Rannung, Sundvall, and Westerholm (1983) showed that the gas phase of gasoline emissions, collected by condensation after distillation and filtration, was mutagenic on strains TA 98 and TA 100. Seagrave et al. (2002), by reconstituting diesel emissions with particles and a volatile semi organic fraction of the gas phase collected by condensation, showed that this mixture was mutagenic on strains TA 98 and TA 100. With the direct exposure method of diesel exhaust used in the present study, a mutagenic effect of the emissions on TA 100 was shown from 20% of unfiltered exhaust and 10% of filtered exhaust on the same strain. This mutagenicity of the filtered aerosol emissions strongly indicated that the gas phase, under these exposure conditions, represents a major contribution to the mutagenic potential of engine emissions on S. typhimurium strains. Use of a monolith ceramic-type particle filter in this study increased the mutagenic activity of diesel exhaust on TA 100. The cooling of exhaust and their residence time in the particle filter could be at the origin of the formation of secondary chemical species, like nitro-HAPs, by reaction between primary chemical species. Matsushita et al. (1986) found that the filtered emissions of diesel exhaust were mutagenic on TA 98 and TA 100 strains, but only without S9 mix. Scheepers, Theuws, and Bos (1991) showed that the gas phase and its condensate were more mutagenic without metabolic activation. One may wonder if the particle filter does not act as a chemical reactor, as positive results obtained from 10% of exhaust show a more significant mutagenic activity with filtered emissions. This mutagenicity of the gaseous phase on Salmonella was estimated at less than 50% of the mutagenicity of the total emissions by Rannung, Sundvall, and Westerholm (1983) and at more than 90% by Matsushita (1986). This variation could be explained by the nature of the samples tested, as there may have been a difference in the physico-chemical contents of the condensate: semi-volatile compound extract or total emissions. In addition, the chemical species present in the gas phase of engine emissions are numerous, and many show mutagenic activity in various biological models. Compounds often found in the gas phase are ethylene 1,3 butadiene, acrolein, and many monocyclic aromatic hydrocarbons (MAH) and PAHs. The mutagenic activity of formaldehyde was highlighted by direct exposure of bacteria to the gas phase of a main mixture of this aldehyde (Glass et al. 1986). Acetaldehyde was found to be mutagen on Escherichia coli, but not on S. typhimurium. On the other hand the exposure of bacteria to a gas mixture of acetaldehyde-NOx showed the mutagenic effect of photooxydation products of acetaldehyde (Shepson et al. 1986). Thus, part of the mutagenic activity of the gas phase of diesel exhaust could probably be due to aldehydes. Sasaki, Endo, and Koido (1980), after an exposure of Bacillus subtilis spores to gasoline vehicle gas phase emissions, showed that the mutagenic activity of these gases was mainly due to the presence of nitrogen dioxide. MAHs such as benzene or anthracene are not themselves mutagenic on bacteria. Some PAHs with three (phenanthrene) or four (pyrene,

Downloaded by [HINARI] at 10:59 02 November 2011

Toxicological & Environmental Chemistry

1979

fluoranthene) cycles, found in the gas phase, do not show direct mutagenic activity in the Ames Salmonella assay. Matsushita et al. (1986), in their study on the individual mutagenic activity of several compounds, found in diesel emissions that 61 out of 94 nitrated derivatives of benzene were mutagenic, particularly on TA 100 without metabolic activation, and that 28 of the 50 PAHs tested were mutagenic and required the presence of metabolic activation. Surprisingly, in the present study, the presence of the metabolic activator with the filtered emissions tended to decrease the mutagenic activity of the exhaust. Pornet et al. (1995) reported toxicity to bacterial strains during exposure with the relatively short duration of 20 minutes. These authors could not demonstrate the mutagenicity of emissions of gasoline and diesel vehicles under the working conditions of their engines. The toxicity observed could have been related to agar dehydration, because of the gas flow in their system (1000 m3 h1) directed over the uncovered agar. With the system used in this study, no dehydration or toxicity were demonstrated, even after 5 hours of exposure with total exhaust (results not shown). As another effect observed with the particle filter is exhaust condensation even at weak concentrations, the presence of the filter may be responsible for a faster cooling of the exhaust and bringing the aerosol closer to the dew-point, thus promoting water condensation and gas phase compound dissolution into water (like NO). The presence of a filter may also alter the kinetics of pollutant interaction with the water vapor, leading to solubilization of the pollutants in the condensation. Variants of TA 98 and TA 100 strains, such as nitroreductase-deficiencies strains, were used by some authors to demonstrate the main contributing factor of the nitro-PAHs in the mutagenic activity of engine emissions (Jones et al. 1985). In this study, no mutagenic effect was observed on strain TA 98 or TA 98NR. On the other hand, strain YG 1024 showed positive results; this strain overexpresses OAT, an enzyme which transforms the nitro-PAHs into aryl hydroxylamines. The use of variants of TA 100, such as TA 100NR and YG 1029, seems to implicate nitro-PAHs in the mutagenicity of diesel exhaust. The reduction in the rate of TA 100 NR revertants and the reactivity of YG 1029 contributed toward the confirmation of this assumption. However, this prominent role of nitro-PAHs suggested by Crebelli et al. (1991) was not demonstrated in this study according to the results obtained because variants considered as more sensitive to nitro-PAHs (YG 1029) did not show any mutagenic potential. It is claimed that these nitro-PAHs are greatly responsible for the mutagenic activity of DEP extract (Crebelli et al. 1991). For Jones et al. (1985), these nitro-PAHs triggered up to 80% of the mutagenicity of diesel exhaust on strain TA 98. This was demonstrated by the use of nitroreductase-deficient strains. As a matter of controversy, other authors state that these nitropyrenes alone only play a minor contribution in the mutagenicity of diesel particles (Siak et al. 1985). The formation of some chemical compounds, such as nitro-PAHs, at the time of particle collection with a particle-filter is supposed by Risby and Lestz (1983) and Schuetzle and Prez (1983). Some authors are particularly interested in the impact of the fuel (Claxton 1983; Crebelli and al. 1995) or in the working of the engine (Courtois et al. 1993). Thus, according to Claxton (1983), depending on the fuel, the mutagenicity of the exhaust could be multiplied by 100. For Crebelli (1995), the mutagenicity of diesel emissions in the Ames test is largely dependant on the composition of the aromatic compounds; polyaromatic compounds are thought to increase this mutagenicity. As for Courtois et al. (1993), the direct exposure of diluted effluents demonstrated that the mutagenicity of exhaust could be influenced by the engine load, which could be responsible for both the amount and the quality of the soluble organic fraction adsorbed by the diesel soot and the amount of

Downloaded by [HINARI] at 10:59 02 November 2011

1980

M. Fall et al.

non-methane hydrocarbons emitted. Thus, mutagenic activity of the diesel emissions would be stronger with weak loads, where more unburnt fuel condensates may occur. The present study however did not conclude on the impact of engine speed on the mutagenic activity. Direct exposures of S. typhimurium to the emissions from unleaded gasoline vehicles showed clear mutagenic activity toward strains TA 98 NR and TA 98/1.8 DNP6 (Jones et al. 1985). Moreover, extracts of particles resulting from emissions of gasoline without and with lead showed the same genotoxic profiles in the Ames test (Yuan, Zhou and Ye 2000). Lin et al. (2002) found that the addition of methanol in diesel oil increased the toxicity of the gas phase of the fuel, but had no influence on the mutagenicity of the gas phase. In the context of the present study, it is difficult to evaluate mutagenic contribution of DEPs, but the system used is appropriate for mutagenicity study of complex volatile compounds. However, to improve this exposure system, it is necessary to regulate and follow the temperature to maintain high Reynolds numbers and keep conditions above the dew points of pollutants and water, as described by Bion et al. (2002), so as to avoid any condensation of pollutant and aggregation of particulate matter.

Acknowledgments This work was supported by Multidisciplinary Approach to Airborne Pollutant Health Related Issues (MAAPHRI) European project QLK4-CT-2002-02357.

References Abe, S., H. Takizawa, I. Sugawara, and S. Kudoh. 2000. Diesel exhaust (DE)-induced cytokine expression in human bronchial epithelial cells: A study with a new cell exposure system to freshly generated Diesel exhaust in vitro. American Journal of Respiratory Cell and Molecular Biology 22: 296–303. Ball, J.C., B. Greene, W.C. Young, J.F.O. Richert, and I.T. Salmeen. 1990. S9-activated Ames assays of diesel-particle extracts. Detecting indirect-acting mutagens in samples that are directacting. Environmental Science & Technology 24: 890–4. Bion, A., M. Fall, F. Gouriou, E. Le Prieur, F. Dionnet, and J.-P. Morin. 2002. Biphasic culture of rat lung slices for pharmacotoxicological evaluation of complex atmospheres. Cell Biology and Toxicology 18: 301–14. Chou, M.C., and H. Lee. 1990. Mutagenicity of airborne particles from four cities in Taiwan, Proceedings of the National Science Council B (ROC) 14: 142–50. Claxton, L.D. 1983. Characterization of automotive emissions by bacterial mutagenesis bioassay: A review. Environmental Mutagenesis 5: 609–31. Courtois, Y., B. Molinier, M. Pasquereau, P. Degobert, and B. Festy. 1993. Influence des conditions de fonctionnement d’un moteur Diesel sur les effets mutage`nes de ses effluents. The Science of the Total Environment 134: 61–70. Crebelli, R., L. Conti, B. Crochi, A. Carere, C. Bertoli, and N. Del Giacomo. 1995. The effect of fuel composition on the mutagenicity of diesel engine exhaust. Mutation Research 346: 167–72. Crebelli, R., S. Fuselli, G. Conti, L. Conti, and A. Carere. 1991. Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts. Mutation Research 261: 237–48. Fall, M., H. Haddouk, J.P. Morin, and R. Forster. 2007. Mutagenicity of benzyl chloride in the Salmonella/microsome mutagenesis assay depends on exposure conditions. Mutation Research 633: 13–20. Glass, L.R., T.H. Connor, J.C. Theiss, C.E. Dallas, and T.S. Mathey. 1986. Genotoxicity of the offgasing product of particle board. Toxicology Letters 31: 75–83.

Downloaded by [HINARI] at 10:59 02 November 2011

Toxicological & Environmental Chemistry

1981

Houk, V.S., S. Goto, O. Endo, L.D. Claxton, J. Lewtas, and H. Matsushita. 1992. Detection of direct-acting mutagens in ambient air: A comparison of two highly sensitive mutagenicity assays. Environmental and Molecular Mutagenesis 20: 19–28. Jones, E., M. Richold, J.H. May, and A. Saje. 1985. The assessment of mutagenic potential of vehicle engine exhaust in the Ames Salmonella using a direct exposure method. Mutation Research 155: 35–40. Josephy, P.D., P. Gruz, and T. Nohmi. 1997. Recent advances in the construction of bacterial genotoxicity assays. Mutation Research 386: 1–23. Lapin, C.A., M. Gautam, B. Zielinska, V.O. Wagner, and R.O. McClellan. 2002. Mutagenicity of emissions from a natural gas fuelled truck. Mutation Research 519: 205–9. Lee, H., S.Y. Su, K.S. Liu, and M.C. Chou. 1994. Correlation between meteorological conditions and mutagenicity of airborne particulates samples in a tropical monsoon climate area from Kaoshiung City, Taiwan. Environmental and Molecular Mutagenesis 23: 200–7. Lin, T.C., and M.R. Chao. 2002. Assessing the influence of methanol-containing additive on biological characteristics of diesel exhaust emissions using microtox and mutatox assays. The Science of the Total Environment 284: 61–74. Masahiko, W., M. Ishidate Jr, and T. Nohmi. 1990. Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyl transferase levels. Muationt Research 235: 337–48. Matsushita, H., S. Goto, O. Endo, J.H. Lee, and A. Kawai. 1986. Mutagenicity of Diesel exhaust and related chemicals. Developments in Toxicology & Environmental Science 13: 103–18. Morin, J.P., F. Fouquet, C. Monteil, E. Leprieur, E. Vaz, and F. Dionnet. 1999. Development of new in vitro system for continuous exposure of lung tissue to complex atmospheres: Application to diesel exhaust toxicology. Cell Biology and Toxicology 15: 143–52. Pornet, P., C. Beaubestre, Y. Courtois, B. Festy, H. Ing, B. Lopez, J.L. Marduel, et al. 1995. Impact des conditions de conduite sur l’efficacite´ des pots catalytiques de ve´hicules a` essence et Diesel. The Sciences of the Total Environment 169: 321–9. Rannung, U., A. Sundvall, and R. Westerholm. 1983. Some aspects of mutagenicity testing of the particulate phase and gas phase of diluted and undiluted automobile exhaust. Environmental Science Research 27: 3–16. Risby, T.H., and S.S. Lestz. 1983. Is the direct mutagenic activity of Diesel particulate matter a sampling artefact? Environmental Science & Technology 17: 621–4. Sasaki, Y., O. Endo, and Y. Koido. 1980. Direct mutagens in the gaseous component of automobile exhaust detected with Bacillus subtilis spores. Mutation Research 79: 181–4. Scheepers, P.T.J., J.L.G. Theuws, and R.P. Bos. 1991. Mutagenicity of urine from rat after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024. Mutation Research 260: 393–9. Schuetzle, D., and J.M. Perez. 1983. Factors inflenciung the emissions of nitrated-polynuclear aromatic hydrocarbons (nitro-PAH) from diesel engines. Journal of the Air Pollution Control Association 33: 751–5. Seagrave, J.C., J.D. McDonald, A.P. Gigliotti, K.J. Nikula, S.K. Seilkop, M. Gurevich, and J.L. Mauderly. 2002. Mutagenicity and in vivo toxicity of combined particulate and semivolatile organic fractions of gazoline and diesel engine emissions. Toxicological Sciences 70: 212–26. Shepson, P.B., T.E. Kleindienst, E.O. Edney, and C.M. Nero. 1986. Acetaldehyde: The mutagenic activity of its photooxidation product. Environmental Science & Technology 20: 1008–13. Siak, J.S., J.L. Chan, T.L. Gibson, and G.T. Wolff. 1985. Contribution to bacterial mutagenicity from nitro-PAH compounds in ambient aerosols. Atmospheric Environment 19: 369–76. Yuan, D., W. Zhou, and S. Ye. 2000. Effect of leaded and unleaded gasoline on the mutagenicity of vehicle exhaust particulate matter. Journal of Environmental Pathology Toxicology and Oncology 19: 41–8.