Plate Incorporation Assay Research Articles

Influence of CYP1A2 and NAT2 Metabolic Phenotypes on Smokers' Urinary Mutagenicity

The influence of the metabolic phenotypes NAT2 and CYP1A2 on urinary mutagenicity of 118 smokers was studied. Mutagenicity of urine samples was determined by Ames test (preincubation plate incorporation assay on YG1024 Salmonella typhimurium strain with S9 mix). Urinary nicotine plus its metabolites were determined to check cigarette smoke intake. The N -acetyltransferase (NAT2) and cytochrome P450 1A2 (CYP1A2) phenotypes were measured by the molar ratio of urinary caffeine metabolites, determined by HPLC analysis. Urinary mutagenicity was significantly higher in smokers CYP1A2 extensive (EM) than in CYP1A2 poor metabolizers (PM) (Mann-Whitney U test, p = 0.020). Linear multiple regression analysis shows that an increase in urinary mutagenicity levels was significantly related to cigarette smoke intake and to CYP1A2 N -hydroxylation activity ( t = 5.06, p < 0.001, and t = 2.33, p = 0.021), but not to NAT2 acetylation phenotype. In conclusion, phenotypic differences in metabolic activation of tobacco smoke mutagens are able to modulate the presence of mutagens in urine of cigarette smokers and, consequently, the potential genotoxic risk.

Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China

The aim of this research is to investigate the impact of air pollution on the population in Shanghai. The genotoxicity of extractable organic matter (EOM) from the air particles was investigated by the means of the Salmonella plate incorporation assay, rat hepatocyte unscheduled DNA repair assay, and mice micronuclei test. The airborne particles were collected in 13 locations during the summer of 1992 and winter of 1993. The crude extracts were fractionated by acid–base partitioning into acid, base and neutral fractions. The neutral fractions were further fractionated by resin–silica gel column chromatography into three subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The mutagenicity was detected with TA98, but was not detected with TA100. The mutagenic activity was the greatest in the acid, aromatic and polar fractions from summer samples. The fractions from the winter samples did not show clear differences. There was no substantial location-related variance in the mutagenic potencies of EOM, but substantial location- or time-related variances in the mutagenic potencies of the airborne particles per cubic meter air were found. While rat hepatocyte unscheduled DNA synthesis (UDS) assay revealed genotoxicity for all the samples, there was no big variance in the genotoxicity of the fractions. The mouse micronuclei test showed results similar to the UDS assay. The difference of locality did not have statistical significance.

Evaluation of the potential effects of ingredients added to cigarettes. Part 3: In vitro genotoxicity and cytotoxicity

Cigarette mainstream smoke from blended cigarettes with and without the addition of ingredients was assayed for its cytotoxicity and genotoxicity. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was added at a low and a high level to the test cigarettes. The mutagenicity of the particulate phase of the resulting cigarette smoke was assayed in the Salmonella plate incorporation (Ames) assay with tester strains TA98, TA100, TA102, TA1535 and TA1537. The cytotoxicity of the gas/vapor phase and the particulate phase was determined in the neutral red uptake assay with mouse embryo BALB/c 3T3 cells. Within the sensitivity and specificity of the test systems, the in vitro mutagenicity and cytotoxicity of the cigarette smoke were not increased by the addition of the ingredients.

A comparative assessment of Boise, Idaho, ambient air fine particle samples using the plate and microsuspension Salmonella mutagenicity assays

The primary objective of this study is to characterize the genotoxic potential of the ambient air aerosols collected within an air shed impacted primarily by wood smoke and automotive emissions. The study also examines the relative merits of a microsuspension assay and the standard plate assay for monitoring the presence of airborne particle-bound mutagens. Wintertime ambient air particulate samples collected from Boise, Idaho, USA, were shown to contain extractable organic matter that is mutagenic in the Salmonella typhimurium microsuspension and plate-incorporation assays. Differences in the results from the primary sites, auxiliary sites and the background site demonstrate that the particle-bound mutagens are not evenly distributed within the air shed and are more associated with the location of sampling than with the time of sampling or the type of bioassay used to evaluate the samples. This study also demonstrates that the bioassay protocol used in such studies should depend upon the characteristics of the air shed's mutagens and the purpose of the study. For example, the microsuspension assay gave somewhat more variable results between samples but was approximately threefold more sensitive than the plate assay. When strain TA98 was used in the microsuspension assay, the mutagenic response was greater without an exogenous activation system. The reverse was true for the plate assay in which the use of an exogenous activation system increased the mutagenicity response. TA100 in the microsuspension assay provided results comparable to those with TA98. This is important because TA100 can also be used to bioassay semivolatile and volatile organics associated with ambient air mutagenicity. This, in turn, allows a comparison of the mutagenicity of organics collected by differing methods due to their volatility. Future studies should be directed toward correlation of mutagenicity results with other analytical results in order to further develop methods for better characterization of the genotoxicity of ambient air.

Genotoxicity of urban air pollutants in the Czech Republic: Part I. Bacterial mutagenic potencies of organic compounds adsorbed on PM10 particulates

As part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region in Northern Bohemia, mutagenicity of extractable organic matter (EOM) from air particles PM10 was investigated by the means of Salmonella typhimurium indicator strains TA98 and YG1041 using the Ames plate incorporation assay. The air samples were collected in both the polluted and the control districts during the summers and winters of 1993–1994. In the polluted district, the collection was repeated during the winter of 1996–1997. The crude extracts from filters pooled according to the locality and the season were fractionated by acid–base partitioning into acid, base, and neutral fractions. The neutral fractions were further fractionated by silica gel column chromatography into five subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The indirect mutagenic potency of aromatic subfractions containing polycyclic aromatic hydrocarbons (PAHs) was generally low. The mutagenic potency detected with TA98 was more distinct only in the winter sample 1993–1994 from the polluted area, where the aromatic subfraction accounted for 23% of total mutagenicity. In both strains, the highest direct-acting mutagenicity was found in slightly polar fractions containing nitro-PAHs. The mutagenic potency detected with YG1041 was about two orders of magnitude higher than that detected with TA98. No substantial locational- or time-related variances in the mutagenic potencies of EOM, or in the spectrum of chemical components identified in individual fractions were found. The polluted district, in comparison to the control district, was found to have higher amounts of EOM, carcinogenic PAHs and mutagenicity of air particles (rev/m 3). The fractionating process, combined with the bacterial mutagenicity test, confirmed that nitro-derivatives are the most important contributors to the bacterial mutagenicity of air particles. However, this study did not fulfill the expectancy to bring substantially new, clear-cut information on the composition and the biological activity of air pollution in both districts.

Comparison of the mutagenic specificity induced by four nitro-group-containing aromatic amines in Salmonella typhimurium his genes

Four nitrated aromatic amines (2-nitro- p-phenylenediamine [2NPD], 3-nitro- o-phenylenediamine [3NPD], 4-nitro- o-phenylenediamine [4NPD] and 4,4′-dinitro-2-biphenylamine [DNBA]) are direct-acting mutagens in Salmonella typhimurium strain TA100. These compounds were tested further using the Xenometrix strains of S. typhimurium: TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006, with and without S9 mix in the plate incorporation assay. The direct-acting mutagenicity of 2NPD, 4NPD, and DNBA was detected with TA7002, TA7004 and TA7005. 2NPD and DNBA showed some activity in TA7006; DNBA also showed some activity in TA7003. Mutagenicity was generally decreased in these strains when S9 was added. 3NPD was mutagenic in TA7004 without S9 and in TA7005 with and without S9. These data suggest that 2NPD, 4NPD and DNBA induced TA→AT and CG→AT transversions as well as GC→AT transitions in the his gene. 3NPD induced CG→AT transversions and GC→AT transitions. 2NPD and DNBA also induced a small portion of CG→GC transversions.

Novel nitrated derivatives of 5,8-diazabenzo[c]phenanthrene and 9,14-diazadibenz[a,e]acephenanthrylene: new classes of potent mutagenic compounds.

We report the synthesis of 4-nitro-5,8-diazabenzo[c]phenanthrene (4-NDBP) and 11-nitro-9,14-diazadibenz[a,e]acephenanthrylene (11-NDDA) and the remarkable mutagenic activity of the latter. These two compounds and their non-nitrated parents, 5, 8-diazabenzo[c]phenanthrene (DBP) and 9,14-diazadibenz[a, e]acephenanthrylene (DDA), were screened in Ames plate incorporation assays against Escherichia coli WP2uvrA and Salmonella typhimurium TA98 both in the presence and absence of S9 liver fraction from Aroclor 1254-induced rats. None of the four compounds were cytotoxic up to the limits of their solubility and none showed mutagenic activity in E.coli WP2uvrA, which suggested that any such activity they may have had was not mediated via a base substitution mechanism. DBP and DDA also displayed a lack of activity in TA98 up to their precipitating doses (560 and 33.5 microg/plate, respectively). The two nitrated compounds, however, were genotoxic. 4-NDBP was active at a dose of 500 ng/plate, in the absence of S9, producing 80.0 +/- 28.0 prototrophic organisms (equivalent to 44 revertants/nmol) and at 0.5 ng/plate, in the presence of S9, giving 147 +/- 6.6 revertants (equivalent to 81 000/nmol) and allowed the description of this tetracycle as a potent mutagen. Much more striking was the activity of 11-NDDA: in the absence of S9 a dose of 8.0 ng produced 2000 revertants/nmol and, remarkably, in the presence of S9 80 pg produced the equivalent of 643 000 revertants/nmol. This makes the hexacyclic 11-NDDA the most potent mutagen to date, in the Ames procedures described here.

Genotoxicity tests with 6-acetyl-1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran

6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran (HHCB), synthetic fragrance ingredients, were evaluated for potential genotoxicity in a battery of short-term tests. Salmonella typhimurium/ Escherichia coli plate incorporation and liquid preincubation assays were conducted on AHTN using tester strains TA97, TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA±S9 activation at doses from 8 to 5000 μg/plate. The plate incorporation mutagenicity assay was conducted on HHCB using tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2 uvrA±S9 activation at doses from 10 to 5000 μg/plate. An in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells was conducted with AHTN and HHCB at three concentrations each with ±S9 activation. In the non-activated study, the exposure/harvest periods were 4/20-, 20/20- and 44/44-h. In the S9 activated study, the exposure/harvest periods were 4/20- and 4/44-h. In vitro unscheduled DNA synthesis (UDS) assays were conducted in primary rat hepatocytes at concentrations between 0.15 and 50 μg/ml for AHTN and HHCB. In vivo mouse micronucleus assays were conducted with high doses of 1600 mg AHTN/kg and of 1500 mg HHCB/kg in corn oil. No positive responses were observed in any of the tests with HHCB. With AHTN, no positive responses were observed except for cells with structural aberrations in the in vitro cytogenetics assay in CHO cells with S9 activation at the treatment/harvest time of 4/20 h. In initial studies with AHTN, the high dose of 7.8 μg/ml showed 0.5% aberrant cells, with the mitotic index at 41% relative to vehicle control and cell growth inhibition in the range of 25–50%. Thus the genotoxicity findings with AHTN were limited to this one positive response; all other genotoxicity tests with AHTN were considered as negative. In particular, the negative finding in the in vivo assay supports AHTN as not likely to be mutagenic in mammalian systems. These considerations, along with other negative published data, lead to the conclusion that both AHTN and HHCB do not have significant potential to act as genotoxic carcinogens.

Oxymetholone: I. Evaluation in a comprehensive battery of genetic toxicology and in vitro transformation assays.

Oxymetholone is generally assumed to be a nongenotoxic carcinogen. This assumption is based primarily on the results of an Ames test, existing data in repeat-dose toxicology studies, and the predicted results of a 2-yr National Toxicology Program (NTP) rat carcinogenicity bioassay. To provide a comprehensive assessment of its genotoxicity in a standard battery of mutagenicity assays, oxymetholone was tested in microbial and mammalian cell gene mutation assays, in an in vitro cytogenetics assay (human lymphocytes), and in an in vivo micronucleus assay. Oxymetholone was also tested in an in vitro morphologic transformation model using Syrian hamster embryo (SHE) cells. These studies were initiated and completed prior to the disclosure of the results of the NTP bioassay. Oxymetholone was tested at doses up to 5,000 microg/plate in the bacterial plate incorporation assay using 4 Salmonella strains and the WP2 uvrA (pKM101) strain of Escherichia coil. There was no induction of revertants up to the highest dose levels, which were insoluble as well as toxic. In the L5178Y tk+/- mouse lymphoma assay, doses up to 30 microg/ml reduced relative survival to approximately 30% with no increase in mutants. Male or female human lymphocytes were exposed in vitro to oxymetholone for 24 hr without S9 or 3 hr with S9 and evaluated for the induction of chromosomal aberrations. There was no increase in aberration frequency over control levels and no difference between male and female cells. Peripheral blood from Tg.AC transgenic mice treated dermally for 20 wk with 0, 1.2, 6.0, or 12.0 mg/day of oxymetholone and from p53 transgenic mice treated orally by gavage for 26 wk with 125, 625, or 1,250 mg/kg/day of oxymetholone was evaluated for micronuclei in polychromatic and normochromatic erythrocytes. There was no difference in micronuclei frequency between control and treated animals. These results confirm that oxymetholone is not genotoxic in a comprehensive battery of mutagenicity assays. In the SHE assay, oxymetholone produced a significant increase in morphologically transformed colonies at dose levels of 13-18 microg/ml. The lack of genotoxicity of oxymetholone, the positive response in the in vitro transformation assay, and the results of transgenic mouse carcinogenicity assays will provide an interesting perspective on the results of an on-going NTP rat carcinogenicity bioassay.

Alternatives in the induction and preparation of phenobarbital/naphthoflavone-induced S9 and their activation profiles.

With the aim of optimizing the efficiency of S9 fractions used in in vitro mutagenicity assays, different schemes for the induction of liver enzymes in rats were tried and the amount of S9 fraction required was assessed. The activity of 2-anthramine (2AA), 2-acetylaminofluorene (2AAF), 3-methylcholanthrene (3MTCL) and benzo[a]pyrene in bacterial mutagenicity tests was compared with the enzymatic activity in S9 fractions obtained from rats treated with either phenobarbital (NaPB), beta-naphthoflavone (betaNF) or combinations of both. Three pool systems prepared with different amounts of NaPB-induced S9 and betaNF-induced S9 were also analyzed for their activation capacities. Profiles of standard plate incorporation assays with Salmonella typhimurium TA98 increased with the amount of S9 fraction added for all drugs tested, except for 2AA, which showed a maximun of activity at low protein concentrations. According to these profiles, an optimal S9 protein content of 700-1000 microg/plate was estimated. For 2AAF and 3MTCL an S9 fraction obtained following a simultaneous treatment with NaPB (i.p.) and betaNF (oral gavage) (NaPB + betaNF) yielded the greatest response. This preparation was the only one which produced positive activation with 3MTCL as test drug. With the other test drugs all the S9 fractions were very active, including the NAPB + betaNF-induced S9. Both Phase I and Phase II cytochrome P450 enzymatic activities were enhanced in this S9 fraction. These results suggest that the simultaneous treatment (NaPB + betaNF) would be an adequate inducer for in vitro activation when used at 700-1000 microg protein/plate.

Exposure to mutagenic airborne particulate in a rubber manufacturing plant

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined.

Multi-bioassay approach for assessing the potency of complex mixtures of polycyclic aromatic hydrocarbons

The chick embryotoxicity screening test (CHEST) and the Salmonella/microsome bioassay were used to evaluate embryotoxic and mutagenic endpoints from crude coal tar (CT) and its fractionated polycyclic aromatic hydrocarbon (PAH) mixtures (designated as A, B, C, D and E). In the CHEST assay, CT and PAH mixtures were injected into the egg yolk. A dose-dependent increase in embryo mortality was observed for all fractions. The E fraction resulted in 47% embryo mortality at a dose of 0.125 mg/kg and was more toxic than CT. At a dose of 1 mg/kg, 85–100% embryonic deaths occurred in fractions C and D and these two fractions were more potent than fractions A and B. The main visual toxic manifestations were liver lesions, discoloration of the liver, and edema. Both CT and fractionated PAH mixtures were also tested in the Salmonella/microsome plate incorporation assay with Salmonella typhimurium strain TA98 and were evaluated with and without metabolic activation at five dose levels. In the presence of S9, the CT and fractions C, D and E induced a dose-dependent positive response. Results from the Salmonella/microsome assay were in good agreement with findings from the CHEST assay suggesting that these two bioassays in combination may facilitate the rapid detection and ranking of complex PAH mixtures.

Base‐substitution profiles of externally activated polycyclic aromatic hydrocarbons and aromatic amines determined in a lacZ reversion assay

Using an improved set of lactose-auxotrophic Escherichia coli tester strains, the proportion of the six possible transitions and transversions after mutagen exposure was assessed. Mutagenic specificity was determined in plate-incorporation assays using lactose-containing minimal medium for the selection of revertants, either after application of directly acting mutagens or by including a metabolic activation system with rat liver S9-extract. The differential and dose-dependent response of the six tester strains was shown by treating the bacteria with described diagnostic mutagens and other directly DNA damaging substances, e.g., N-methyl-N-nitrosoguanidine (MNNG) and benzo[a]pyrene-diolepoxide (BPDE). Polycyclic aromatic hydrocarbons and aromatic amines were investigated in the presence of an external metabolic activation system. Benzo[a]pyrene (BaP) yielded similar mutation profiles as its ultimate mutagen BPDE, if 100-fold increased doses were applied. In contrast to the mutation profile of BaP, which was dominated by G:C–T:A transversions, mutagenesis with benzo[c]phenanthrene (BcPh) produced predominantly A:T–T:A transversions. The same base change was observed with 5-methylchrysene and found to be missing with 5,6-dimethylchrysene, while both compounds caused G:C–A:T transitions. The aromatic amines 4-aminobiphenyl (4-ABP), 2-aminoanthracene (2-AA) and 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) yielded similar yet distinguishable mutation profiles. Base-substitution reversion profiles of the chemical mutagens were in agreement with those obtained in other systems and with molecular analysis of mutants induced by these agents. Environ. Mol. Mutagen. 33:75–85, 1999 © 1999 Wiley-Liss, Inc.

Base-substitution profiles of externally activated polycyclic aromatic hydrocarbons and aromatic amines determined in alacZ reversion assay

Using an improved set of lactose-auxotrophic Escherichia coli tester strains, the proportion of the six possible transitions and transversions after mutagen exposure was assessed. Mutagenic specificity was determined in plate-incorporation assays using lactose-containing minimal medium for the selection of revertants, either after application of directly acting mutagens or by including a metabolic activation system with rat liver S9-extract. The differential and dose-dependent response of the six tester strains was shown by treating the bacteria with described diagnostic mutagens and other directly DNA damaging substances, e.g., N-methyl-N-nitrosoguanidine (MNNG) and benzo[a]pyrene-diolepoxide (BPDE). Polycyclic aromatic hydrocarbons and aromatic amines were investigated in the presence of an external metabolic activation system. Benzo[a]pyrene (BaP) yielded similar mutation profiles as its ultimate mutagen BPDE, if 100-fold increased doses were applied. In contrast to the mutation profile of BaP, which was dominated by G:C-T:A transversions, mutagenesis with benzo[c]phenanthrene (BcPh) produced predominantly A:T-T:A transversions. The same base change was observed with 5-methylchrysene and found to be missing with 5,6-dimethylchrysene, while both compounds caused G:C-A:T transitions. The aromatic amines 4-aminobiphenyl (4-ABP), 2-aminoanthracene (2-AA) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) yielded similar yet distinguishable mutation profiles. Base-substitution reversion profiles of the chemical mutagens were in agreement with those obtained in other systems and with molecular analysis of mutants induced by these agents.

Mutagenicity assay in Salmonella and in vivo sister chromatid exchange in bone marrow cells of mice for four pyrazolone derivatives

Phenylbutazone (PB), oxyphenbutazone (OPB), antipyrine (AP) and dipyrone (DP) are four important pyrazolone derivatives mainly used as anti-inflammatory, antipyretic and analgesic drugs. At present these are the most widely used pyrazolone derivatives throughout the world. The widespread use of these drugs are of great concern for human health problems. In the present study these four drugs were tested in mutagenicity assays in Salmonella strains TA97a, TA98, TA100 and TA102 using a plate incorporation assay both with and without S-9 mix and for in vivo sister chromatid exchanges (SCE) in bone marrow cells of mice. The first three drugs were negative in all the tester strains but dipyrone showed a weak mutagenic activity at higher concentrations in all four strains both with and without metabolic activation. In the in vivo SCE assay in male mice, all four drugs showed a statistically significant increase in SCE in bone marrow cells when compared with control.

The use of YG bacterial tester strains for the monitoring of drinking water mutagenicity

The organic extract from the 50 drinking water specimens taken in four cities were tested for mutagenicity in the Ames test (plate incorporation assay) using the parent TA98 and TA100 strains, and derived YG1041 and YG1042 strains. Four dose levels of extractable organic matter (EOM) with duplicate plate per dose were used. Slopes (revertants/mg EOM) were calculated by the Bernstein linear regression rejection model using GeneTox Manager software. The mutagenicity observed in the conventional strains TA98 and TA100 did not reach the significant increase in all tested samples with the higher mutagenic response found in TA100-S9. With the YG1041 and YG1042 tester strains, the results obtained demonstrated the clear-cut direct dose-related mutagenicity response in all tested drinking water extracts. Compared with TA98 and TA100 strains, the numbers of YG induced revertants were ≈20 times higher. The high sensitivity of the YG tester strains could facilitate the mutagenicity monitoring in drinking water extracts, and help reduce the volume of sample required. However, to identify the chemical contaminants in drinking water responsible for the mutagenicity further studies are required.

Mutagenic and cytotoxic effects of exhaust particulate matter of biodiesel compared to fossil diesel fuel

The mutagenic and cytotoxic effects of diesel engine exhaust (DEE) from a modern passenger car using rapeseed oil methyl esters (RME, biodiesel) as fuel were directly compared to DEE of diesel fuel (DF) derived from petroleum. Combustion particulate matter was collected on glass fiber filters coated with polytetrafluoroethylene (PTFE) from an exhaust dilution tunnel using three different engine test cycles on a chassis dynamometer. Filters were extracted with dichloromethane in a soxhlet apparatus for 12 h. The mutagenicity of the extracts was tested in the Salmonella typhimurium/mammalian microsome plate-incorporation assay using strains TA97a, TA98, TA100, and TA102. The toxicity to the established cell line L929 (mouse lung fibroblasts) was investigated in the neutral red assay. In the tester strains TA98 and TA100 a significant increase of mutations resulted for the particle extracts of both fuels, but for DF the revertants were significantly higher compared to RME. The highest levels of revertants were observed in tests including a cold start phase. This was probably due to incomplete combustion in the cold engine and a lower conversion rate of the cold catalytic converter. Testing with activated liver S9 fraction induced a slightly lower increase of revertants in most experiments. TA97a and TA102 showed no significant enhancement of spontaneous mutations. In the FTP-75 test cycle RME extracts showed slightly higher toxic effects to the L929 cells than DF, whereas in the other tests no significant differences were observable. These results indicate a higher mutagenic potency of DEE of DF compared to RME. This is probably due to the lower content of polycyclic aromatic compounds (PAC) in RME exhaust, although the emitted masses of RME were higher in most test procedures applied in this study.

Catabolite repressors are potent antimutagens in Escherichia coli plate incorporation assays: experiments with glucose, glucose-6-phosphate and methyl- α- d-glucopyranoside

Having previously found that the yields of spontaneous valine-resistant (Val r) Escherichia coli mutants which appeared on plates containing 40 μg/ml of valine were always much lower when glucose was present in the glycerol-containing defined medium normally used to select them, we now sought to determine whether or not the global regulatory mechanism known as catabolite repression (formerly also called glucose repression) might be involved. We therefore tested glucose (the archetypal catabolite repressor), glycerol (a non catabolite-repressing substrate), glucose-6-phosphate (G6P, an exceptionally powerful catabolite repressor) and methyl- α- d-glucopyranoside ( αMG, a strongly catabolite-repressing but non-utilisable glucose analogue), as potential inhibitors of spontaneous mutagenesis in plate incorporation assays, using three distinct mutation detection systems. We found that the numbers of spontaneous Val r and Lac + mutations appearing on the selective plates tended to be highest when the medium contained only a non-repressing primary carbon source (glycerol in the Val s→Val r system, lactose in the Lac −→Lac + system) and lowest when it had been supplemented with a strongly catabolite-repressing compound such as αMG, G6P or glucose. These results would seem to establish that catabolite repression is an important factor in determining the outcome of the spontaneous mutation generation process in E. coli and hence that the numbers of spontaneous mutations which can be expected to arise in any given set of mutation assay conditions may often be dependent upon the levels of catabolite repression which prevail during the course of the assay. The implications of these results for conventional plate-incorporation mutation assays are discussed.

Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15 000 μg/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 μg/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 μg/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 μg/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.

Development of a standard reference material for diesel mutagenicity in the Salmonella plate incorporation assay

The present study documents the mutagenicity of a new National Institute of Standards and Technology (NIST) standard reference material (SRM) in the Salmonella plate incorporation assay. This study is in response to a previous recommendation by the World Health Organization to develop large batches of new SRMs for biological and chemical research. SRM 1975 is a dichloromethane (DCM) extract of 5.6 kg of filter-collected combustion particulate matter (SRM 2975) from operating forklifts with diesel engines. The mutagenicity and a summary of the related chemical analysis of mutagens in SRM 1975 is presented in this paper, and are available from the NIST. Mutagenicity test conditions were: Salmonella typhimurium TA98, TA100 (standard strains); TA98NR, TA100NR (nitroreductase (NR) gene deficient); and YG1021 and YG1026 (NR gene addition); 10 dose levels in the linear portion of the dose–response curve; duplicate plates per dose; and S9 at 6.4% or 1.1 mg of protein/plate. Four rounds of testing were conducted. Rounds were conducted at least 1 week apart. Slopes (revertants/μg) were calculated by the linear regression rejection model of Bernstein and by the Stead and Krewski models which analyze non-linear data. The GeneTox Manager software package developed at the EPA was used to record the data and calculate the slopes. Results demonstrated: (1) the ranking of slopes without S9 was: YG1021 > TA98 > TA98NR > YG1026 > TA100 > TA100NR in all three statistical models; (2) the mutagenic activity of SRM 1975 was significantly increased by the presence of the NR gene; (3) the slope values for the TA100 series were significantly less than for the TA98 series; (4) in general, the addition of the S9 significantly reduced mutagenic activity; (5) the mutagenic activity of the SRM 1975 was stable over time and variability was low (generally less than 20% in slope values over the 4 rounds); and (6) agreement of the slope values among the three models was excellent due to the linear nature of the data. These data will be useful in ranking other diesel and air samples for mutagenic activity, for quality assurance of data generated in different laboratories, for quality control within a laboratory, and as positive control values for future air and automotive emission studies.