mutation spectra of Glu-P-1 in Salmonella: Induction of hotspot frameshifts and site-specific base substitutions

We used colony probe hybridization and PCR/ DNA sequence analysis to determine the mutations in —1,640 revertants of the -1 frameshift allele hisD3052 and -260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2-amino-6-methyldipy-rido[1,2-a:3′,2′-d]imidazole (Glu-P-1). All of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu-P-1-induced mutations at the frameshift allele in strains TA1978 (uvr⁺) and TA1538 (uvrB) and 99% in TA98 (uvrB, pKM101). To explain the induction of these hotspot mutations by Glu-P-1, we describe here a more detailed version of our recently proposed correct incorporation/ slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu-P-1-adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu-P-1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu-P-1 at the frameshift allele, it is required by Glu-P-1 to revert the base substitution allele, where Glu-P-1 induces G-C-→T-A transversions (75%) and G-C→T.A transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20–30 times less) base substitution mutagenic potency of Glu-P-1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu-P-1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu-P-1-induced rat colon tumors. © 1994 Wiley-Liss, Inc.     

Mutagenicity in vitro of 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid), a chlorine disinfection by-product in drinking water

The mutagenicity of chlorinated humic drinking waters is accounted for mainly by a single contaminant, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), as assessed in Salmonella typhimurium strain TA100. In the present study, 3,4-dichl-oro-5-hydroxy-2(5H)-furanone (mucochloric acid, MA), another drinking water contaminant much less potent as a mutagen in TA100 than MX, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (hprl) locus to 6-thioguanine resistance (TG'). Unexpectedly, MA induced TG mutants in CHO cells with a potency comparable to that reported previously for MX. In subsequent experiments with S. typhimurium, the presence of pKM1O1 plasmid in strain TA100 increased susceptibility to the mutagenicity of MA, but much less than to that of MX, relative to the parental strain TA1535 lacking pKMlOl. The difference between the two compounds in TA100 thus appears to be due to a higher enhancement of the mutagenicity of MX than that of MA by pKM101 mediated error-prone DNA repair. © 1995 Wiley-Liss, Inc.     

A correlation of Salmonella mutagenicity with DNA adducts induced by the cooked-food mutagen 2-amino-1-methyl-6-6-phenylimidazo[4,5,-b]pyridine

The correlation of bacterial mutagenicity with DNA adducts fromthe heterocyclic amine cooked-food mutagen 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) was investigated in Salmonella typhimurium strains TA98(uvrB deficient) and TA1978 (uvrB proficient). Bacterial cellswere exposed to PhIP using a modification of the Ames/ Salmonellamicrosuspension assay. Half of the cells, generated from a 90min pre-incubation and washing, were plated for revertant formationwhile the remaining half was subjected to DNA adduct analysisvia ³²P-postlabeling. In TA98, DNA adducts were detected atan RAL (relative adduct labeling) of 10x10^–7 and 21x10^–7at PhIP concentrations of 5.5 and 17 µM, respectively.This corresponded to 28.8 and 20.9 adducts/revertant, respectively.These values were based on the assumption that only four repeatingGC bases within a 75 DNA base region is the gene target sitefor PhIP induced mutations. In TA1978, no revertants above backgroundwere detected at any concentration of PhIP tested. DNA adducts,however, were detected at 11x10^–7 and 21x10^–7 adductsper nucleotide at 223 and 1116 µM PhIP, respectively.The lack of detectable revertants, but the presence of DNA adducts,suggests pre-mutational lesions did occur during the 90 minpre-incubation. Presumably, when the S9 activating system andPhIP were removed (via washing with phosphate buffered saline)prior to plating, the cells containing an intact uvrB repairsystem repaired the lesions during the incubation time on theplates. In conclusion, the induction of revertants by adductsappears quite efficient, as     

Glutathione S-transferase-mediated induction of GC → AT transitions by halomethanes in salmonella

Halomethanes are among the most common mutagenic and carcinogenic disinfection by-products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH₂Cl₂) and bromodichloromethane (BrCHCl₂) can be mediated by a theta-class glutathione S-transferase (GSTT1-1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base-substitution strain TA1535 (hisG46, rfa, δuvrB), into which has been cloned the GSTT1-1gene from rat. In the present report, we have ex tended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr₃) and chlorodibromomethane (ClCHBr₂), are also mediated by GSTT1-1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr₃ ≈ ClCHBr₂ > BrCHCl₂ ≈ CH₂Cl₂. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by per forming colony probe hybridizations of ∼100 revertants induced by each compound. The majority (96–100%) of the mutations were GC → AT transitions, and 87–100% of these were at the second position of the CCC/GGG target. In contrast, only15% of mutants induced by CH₂Cl₂ were GC → AT transitions in the absence of the GSTT1-1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1-1 to mediate the mutagenicity of these di- and trihalomethanes and the induction of almost exclusively GC → AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST-mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH-DNAadducts. Environ. Mol. Mutagen. 30:440–447, 1997 Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and as such, is in the public domain in the United States of America.

     

Studies of error-prone DNA repair in Escherichia coli K-12 and Ames Salmonella typhimurium strains using a model alkylating agent

4-Acetoxy-3-acetoxymethyl acetophenone (AAMAP) is muta-genicin Ames Salmonella typhimurium tester strains TA100 and TA98,which carry plasmid pKM101, but not in the isogenic plasmid-lessstrains TA1535 and TA1538. Similarly, no AAMAP-induced reversionof the his-4 allele is detectable in Escherichia coli K-12 umuCstrains in the absence of the plasmid, even when the strainsare treated with ethylene-diaminetetraacetate to increase permeability,or when the uvrB allele is introduced to increase error-proneDNA repair. AAMAP is, however, mutagenic in umuC⁺ strains orin umuC strains in which plasmid pKM101 has been introduced,suggesting that the plasmid-encoded MucAB or the chromosomallydetermined UmuDC proteins are required for mutagenesis. Mutationfrequencies are higher in E. coli umuC (pKM101) strains, whichresemble Ames tester strains of S.typhimurium, than in E.coliumuC⁺ or even umuC⁺ (pKM101) strains. Therefore, providing thatthe recommended pKM101-containing tester strains are used, theapparent absence of Umu-like protein activity in S. typhimuriummay actually increase the sensitivity of the Ames test for thedetection of mutagens that require error-prone DNA repair foractivity. ^* Parts of this paper were communicated to the Fourth InternationalConference on Environmental Mutagens, Stockholm, 1985. ²To whom correspondence should be addressed      

Genotoxic activity of the N-acetylated metabolites of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ)

The genotoxic potential of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline(IQ) and 2-amino-3,4-di-methylimidazo[4,5-f]quinoline (MelQ)and their N-acetylated metabolites (AcIQ and AcMelQ, respectively)has been studied, in order to evaluate whether an initial N-acetylationof IQ or MelQ is important for the overall in vivo genotoxicityof the compounds. When incubated with uninduced (control) rathepatocytes, both the acetylated and the unacetylated compoundsappeared to be relatively stable, whereas water-soluble metabolites(i.e. not extractable by ethyl acetate at alkaline pH) wererapidly formed with hepatocytes from PCB-induced animals. NoDNA damage was induced by IQ or MelQ in hepatocytes isolatedfrom control rats, as measured by alkaline elution. In hepatocytesfrom PCB-pretreated rats, IQ, MelQ, AcIQ and AcMelQ inducedDNA damage at low (10^–6 M) concentrations, with AcIQ beingmore potent than IQ whereas AcMelQ was less potent than MelQ.Similar patterns were observed when unscheduled DNA synthesiswas measured in hepatocytes. The compounds induced sister chromatidexchanges in Chinese hamster V79 cells with PCB-induced hepatocytesas activation system; IQ and AcIQ were equal while AcMelQ hadless activity than MelQ. The compounds were also compared inbacterial mutagenesis test systems (Salmonella typhimurium TA98).With hepatocyte activation, AcIQ was slightly more potent thanIQ, whereas AcMelQ was markedly less mutagenic than MelQ. Withsub-cellular fractions as activation system (rat liver S9 ormicro-somes), the N-acetylated compounds were similar to orless mutagenic than their parent compounds. The mutagenic effectsof AcIQ and AcMelQ in bacteria with microsomal activation weremarkedly reduced by the deacetylase inhibitor paraoxon. Paraoxonalso reduced the DNA strand breaks induced by AcIQ or AcMelQin PCB-induced hepatocytes, but did not affect IQ- or MelQ-inducedDNA damage. The results show that an initial N-acetylation ofIQ or MelQ does not dramatically change the overall genotoxicityof these hetrocyclic aromatic amines.     

Mutational response at the splenic T-Lymphocyte hprt locus in mice treated as neonates: Contrasting effects of the carcinogens N-ethyl-N-nitrosourea,dimethylnitrosamine, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

The newborn mouse tumorigenicity assay, which involves the treatment of animals during the first two weeks after birth and monitoring tumor induction after a year, has been suggested as a cost- and time-effective alternative to the conventional two-year rodent bioassay. In order to evaluate whether or not lymphocyte hprt mutant induction is an accurate predictor of carcinogenicity in the assay, we determined the frequencies of 6-thioguanine-resistant (TG^r) lymphocytes in the spleens of mice neonatally treated with the carcinogenic mutagens N-ethyl-N-nitrosourea (ENU), dimethylnitrosamine (DMN),and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Male C57BL/6 pups were injected on post-natal days 8 and 15, and the frequency of TG^r T-lymphocytes was measured in groups of three animals, sacrificed periodically up to 31 weeks post-treatment. Compared to background frequencies of 1.1–2.9 × 10⁻⁶, mutant frequencies (MFS) reached 155.1 × 10⁻⁶ following a cumulative dose of 49 mg ENU/kg body weight and 172.3 × 10⁻⁶ following a cumulative dose of 142 mg ENU/kg. These results show that TG^r lymphocyte mutations can be induced and measured in mice treated as neonates and that the induced MFs found for mice treated neonatally with ENU are comparable with frequencies reported for the treatment of adult animals with the same chemical. In contrast, treatment with the promutagenic and procarcinogenic compounds DMN (at a maximum concentration of 10.5 mg/kg) and PhIP (26.2 mg/kg) did not result in an increase in lymphocyte MF, suggesting that reactive metabolites of these compounds may not be reaching cells that are sensitive for mutation fixation. The results indicate that the lymphocyte hprt assay may fail to predict the carcinogenicity of some test chemicals in the neonatal mouse bioassay. Environ. Mol. Mutagen. 31:243–247, 1998 © 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The structure--activity relationships of flavonoids as inhibitors of cytochrome P-450 enzymes in rat liver microsomes and the mutagenicity of 2-amino-3-methyl-imidazo[4,5-f]quinoline

The antimutagenicity of 19 naturally occurring flavonoids andtheri derivatives including flavones, flavonols, flavanoes,inoflavones and flavanols were determined using Salmonella typhimuriumTA98 against 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) inthe presence of Aroclor 1254-induced rat hepatic S9. In general,a relationship between the chemical structure of flavonoidsand their antimutagenicity was found for compounds containingone or more of the following featutes: (i) C4 keto group, (ii)aglycone, (iii double bond at positions C2 and C3, (iv) phenylgroup at position C2, and (v) three hydroxyl substituents atpositons C4', C5 and C7. The inhibitory effects of flavonoidson activities of 7-ethoxycoumarin deethylase (ECD) and 7-ethoxyresorufindeethylase (ESD) of Aroclor 12540-induced hepatic microsomeswere also examined. In addition, we studied the effects of flavonoidson the metabolism of IQ by Aroclor 1254-induced microsomes usinghigh-performance liquid chromatography. The antimutagenicitycorrelated with the inhibition of cytochrome P-450IA1-linkedESD and P-450IA2-linked ECD activity in hepatic microsomes,and with an inhibition of N-hydroxy-IQ fromation from IQ metabolismby hepatic microsomes. These reslute indicated that flavonesor flavonols that contasin C5, C7 and C4' hydroxyl groups arepotent inhibitors of P-450 enzyme activities induced by Aroclor1254 (P-450IA1 and P-450IA2), and masy potentially be usefulas chemopreventive agents against heterocyclic amine-inducedmutagenesis or carcinogenesis. ¹To whom correspondence sould be addressed     

Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains

Formaldehyde was examined for bacterial mutagenicity using Escherichiacoli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimuriumTA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absenceof any exogenous source of metabolic activation. Using pre-incubationexposure, clear mutagenicity was seen for TA98, TA100 and TA102,and both E.coli strains. In standard plate-incorporation assays,consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101).No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538using either method of exposure. These data confirm the enhancedability of the pre-incubation method to detect the mutagenicityof formaldehyde both quantitatively, as expressed by numbersof revertant colonies, and qualitatively, in terms of the rangeof indicator strains reverted. The relatively greater sensitivityof the pre-incubation assay is probably due to better containmentof a volatile agent and/or lack of interaction with agar duringthe initial period of exposure. The findings are consistentwith the suggestion that formaldehyde induces lesions in bacteriawhich are, at least to some extent, excision-repairable, andindicate that the presence of the R-factor plasmid may be requiredfor the expression of its mutagenicity in excision repair-deficientSalmonella.     

Studies on mammary carcinogenesis induced by a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, in mice and rats

Ten heterocyclic amines (HCAs) that are produced by heating amino acids, proteins, or proteinaceous food such as fish and meat were examined for carcinogenicity in rats and mice. Three of them, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), have been shown to induce mammary cancer in female F344 and/or SD rats, but none of the HCAs induced mammary cancer in CDF(1) mice. This report reviews our recent studies on mammary carcinogenesis of PhIP in various strains of mice and on the roles of genomic instability in the rat mammary carcinogenesis of PhIP. We demonstrated that the survival time from mammary adenocarcinomas was shorter in PhIP-treated BALB/c mice than that in the untreated control, and with a significantly higher incidence in the C.B-17 strain of mice compared with that of the control. To clarify mechanisms of mammary carcinogenesis, we examined genomic instability in rat mammary cancer induced by PhIP. Mammary cancers were induced in F344 x SD F(1) rats harboring the lacI transgene, and two cell lines were established from two adenocarcinomas. They showed a greater than 10-fold higher frequency of spontaneous mutations than that of the primary culture of normal mammary epithelial cells, in the lacI transgene and the hprt endogenous gene during cell replication. Nucleotide sequencing revealed that almost all types of mutations were increased, with a remarkable increase of A:T --> C:G mutation. This genomic instability was not attributed either to alterations of mismatch-repair enzymes or to p53. These mutational characteristics were also observed in the original tumors. Single-nucleotide instability (SNI) might be implicated in the mammary cancer induced by PhIP.     

Relevance of plasmid pKM101-mediated mutagenicity in bacteria to genotoxicity in mammalian cells

Three structurally related compounds, 4-acetoxy-3-acetoxy-methyl-acetophenone(AAMAP), 1-[4'- hydroxy-3'-hydroxy-methylphenyl]-2-[benzyl-t-butylamino]ethanone hydrochloride (HHBEH) and 1-[4'-hydroxy-3'-hydroxymethyl-phenyl]-2-[benzyl-t-butylamino]ethanol (HHBE), gave positive dose-related mutagenic responsesin the Ames test when Salmonella typhimurium strain TA100 wasused as the test organism. Strain TA100 carries the hisG46 allele,which is revertable by base changes, together with plasmid pKM101,which encodes mucAB genes that are analogous to umuDC, the chromosomalSOS-repair genes of Escherichia coli K-12. None of the compoundswas mutagenic in Ames strain TA1535, which is the plasmid-freederivative of strain TA100. Only AAMAP, and that at only thehighest concentration tested, was mutagenic in strain TA98,which detects frameshift mutations and carries plasmid pKM101.No compound was significantly mutagenic in strain TA1538, whichis the plasmid-free derivative of strain TA98. When the threecompounds were tested for the induction of sister-chromatidexchanges (SCEs) in Chinese hamster cells, the two more potentmutagens, AAMAP and HHBEH were found to increase SCEs, whereasHHBE did not give a significant response at any concentrationtested. Ames test data showing plasmid pKM101-dependent mutagenesisare therefore, at least for these compounds, relevant indicatorsof eukaryotic genotoxicity. Parts of this paper were communicated to the Science Group atthe 123rd British Pharmaceutical Conference, Jersey, 1986.      

Activation of 2-amino-3-methylimidazo (4,5-f) quinoline in rat and human hepatocyte/Salmonella mutagenicity assays: the contribution of hepatic conjugation

The capacity of human liver S9 and hepatocytes to metabol-icallyactivate 2-amino-3-methylimidazo (4,5-f) quinoline (IQ) in Salmonellamutagenicity assays more closely resembles that of preparationsfrom Aroclor-induced rat than control rat. The extent to whichhepatocyte conjugating enzymes contribute to activation in theseassays has been studied. Omission of sulphate or addition ofa ‘specific’ sulphotransferase inhibitor (2,6-DCNP)did not significantly reduce mutagenicity, nor did PAPS enhanceS9-mediated bacterial mutagenicity. Conversely, mutagenicitywas significantly inhibited by PCP (an inhibitor of both sulphotransferaseand acetyltranferase) and an acetylation-deficient Salmonella(TA98/1,8DNP₆) was unresponsive to the mutagenicity of IQ. Thesedata suggest that acetylation but not sulphation is importantin IQ bacterial mutagenesis. The addition of acetyl CoA, PAPS-generatingsystem or ATP paradoxically reduces the mutagenicity of IQ inS9/Salmonella TA98 assays. Therefore, activation by esterificationin hepatocytes does not contribute to the mutagenicity of IQin Salmonella typhimurium possibly due to restricted accessof conjugates into the bacterial cell.     

Mutations induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats. In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats. This is the first report of mutational data from discrete regions of the colon. After 61 days of treatment with 200 p.p.m. PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls. PhIP-induced mutant frequencies increased significantly (overall trend, P < 10(-4)) along the length of the colon of both males and females, with cecum < proximal colon < distal colon. A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females. These mutational spectra consisted predominantly of G:C-->T:A and G:C-->C:G transversions and deletions of G:C base pairs. There were no significant differences between the mutational spectra with respect to sex or position in the colon. Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats.     

SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds

To examine the concordance of two microbial genotoxicity short-termassays, 330 experimental results for the SOS chromotest usingtester strain Escherichia coli PQ37 were compared with the resultsof the Salmonella/mammalian microsome mutagenicity assay withSalmonella typhimurium TA97, TA98, TA100, TA102, TA104, TA1535,TA1537 and/or TA1538. With respect to qualitative features,the concordance between SOS chromotest and Salmonella mutagenicitytest results was 86.4% (sensitivity, 78.6%; specificity, 100%;     

Base-pair mutations caused by six aliphatic epoxides in salmonella typhimurium TA100, TA104, TA4001, and TA4006

Salmonella typhimurium strains TA100, TA104, TA4001, and TA4006 were used to detect the base-pair mutations caused by six aliphatic epoxides: chloropropylene oxide, glycidyl 1-naphthyl ether, glycidyl 4-nitrophenyl ether, 1-naphthyl-propylene oxide, styrene oxide, and trichloropropylene oxide. Dose-mutagenicity relationships could be established for all six epoxides in strains TA100 and TA104 but not in strains TA4001 and TA4006. These results, together with the lack of sensitivity of the TA100 revertants to DL-1,2,4-triazole-3-alanine, indicate CG→TA transitions and/or CG→AT transversions are of major importance for mutations induced by these epoxides in Salmonella TA100 and possibly TA104. In addition, since the reproducibility of the effect of the triazole on TA104 reversions was poor, TA→AT transversions were not eliminated as also contributing to the mutagenicity of these epoxides in this Salmonella strain. © 1993 Wiley-Liss, Inc.     

Characterization of the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline prepared from a 2-methylpyridine/creatinine/acetylformaldehyde model system

A mixture of 2-methylpyridine, creatinine and aldehydes washeated in diethylene glycol containing 5% water for 1 h at 140°C.The mutagenic compounds were purified by XAD-2 column chromatography,acid/base partition, blue cotton treatment, thin layer chromatographyand HPLC. The active substances purified from each step weremonitored by their mutagenicity with Salmonella typhimuriumTA98 in the presence of S9 mix. Among the mutagens collected,2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was isolated fromHPLC, and was identified by its UV and mass spectrum using aphotodiode array detector and mass spectrometry. Our findingsappear to be the first experimental evidence to substantiatethe hypothetical pathway for the formation of IQ mutagens froma heated model system consisting of a pyridine or pyrazine derivative,an aldehyde and creatinine or pyrazine derviative, an aldehydeand creatinine or creatine. ²To whom correspondence should be addressed     

mutation spectra in salmonella of complex mixtures: Comparison of urban air to benzo[a]pyrene

We used an ion-exchange procedure coupled to the Salmonella assay to fractionate the dichlo-romethane-extractable particulate organics from an urban air sample collected in Boise, ldaho. A resulting base/neutral fraction contained 81% of the mutagenic activity but only 36% of the mass of the unfractionated sample. Chemical analysis showed that polycyclic aromatic hydrocarbons (PAHs) accounted for much of the mutagenic activity of the air sample. Colony probe hybridization, PCR, and DNA sequence analysis were then used to determine the mutations induced by the complex mixtures and a model PAH, benzo[a]pyrene (BAP) in ～900 revertants of the frameshift hisD3052 allele and ～400 revertants of the base-substitution hisG46 allele. The majority (93–94%) of the mutations induced at the frameshift allele in strain TA98 by the whole or base/neutral fraction of the urban air sample was a hotspot 2-base deletion of a CG or GC within the sequence CGCGCGCG. The remaining mutations were complex frame-shifts that consisted of ?2 or +1 frameshifts associated with a flanking base substitution. BAP induced a somewhat similar pattern of mutations, with 70% being the hotspot mutation, 23% being complex frameshifts, and the remaining being deletions. The inferred base-substitution specificity associated with the complex frame-shifts at the hisD3052 allele (primarily G · C→T · A transversions) was consistent with the observation that this same transversion was the primary mutation induced by the whole urban air sample and BAP at the base-substitution allele in strain TA100. At the frameshift allele, adducts that promote correct incorporation/slippage could account for hotspot mutations, whereas those that promote misincorporation/slippage could account for complex frameshifts. At the base-substitution allele, a mixture of adducts or of adducts with multiple conformations could account for the observed proportion of transitions and transversions. Combined with the bioassay-directed chemical analysis, these results from the first mutation spectra of a complex mixture suggest that such spectra reflect the dominance of particular classes of chemical mutagens within the mixture. © 1994 Wiley-Liss, Inc.     

Role of nitroreduction in the synergistic mutational response induced by mixtures of 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium

Previous studies showed that binary mixtures of the environmental pollutants 1- and 3-nitrobenzo[a]pyrene produced a synergistic mutational response in the Salmonella reversion assay. Since nitroreduction is believed to mediate the direct-acting mutagenicity of the individual compounds, we have examined the role of nitroreduction in the mutagenicity of mixtures of 1- and 3-nitrobenzo[a]pyrene in the Salmonella plate incorporation assay. While mixtures of 1- and 3-nitrobenzo[a]pyrene induced up to 183% more revertants in strain TA98 than produced by equivalent amounts of the individual compounds, in the nitroreductase-deficient strain TA98NR the same mixtures only induced up to 57% more revertants than the individual compounds. Analysis of mixtures of 1- and 3-nitrosobenzo[a]pyrene (the two-electron reduction products of 1- and 3-nitrobenzo[a]pyrene) for mutation induction in TA98 yielded no evidence of a synergistic effect between the compounds. The mutagenicity of the mixtures was dependent upon the amount of the more mutagenic component. Salmonella cultures were also incubated with mixtures of 1- and 3-nitrobenzo[a]pyrene, as well as with equivalent amounts of the individual compounds. In two experiments, nitroreductive ability, as measured by the amount of 1-nitropyrene metabolized to 1-aminopyrene in 1 hr, was increased 9 to 105% in cultures pretreated with the mixtures as compared with cultures pretreated with the individual compounds. The results of this study support the hypothesis that nitroreduction is a major factor in the synergistic mutational response induced by 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium.     

Lack of uniformity in the mutational spectra of chlorohydroxyfuranones in Salmonella typhimurium strain TA100

The mutational specificity of three chlorohydroxyfuranones foundin chlorinated drinking water, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone(MX), 3-chloro-4(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF)and 3, 4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid,MCA), was examined in Salmonella typhimurium strain TA100. DNAcolony-hybridization of TA100 revertants showed that MX andCMCF both induced predominantly G:C T: A transversions (87and 75% of total, respectively) with a 3: 1 preference for thesecond position of the hisG46 (CCC) target codon. By contrast,MCA produced primarily G:C A: T transitions (66% of the total)with a 4:1 preference for the second position of the CCC codon.The mutational specificity of MCA is consistent with the ideathat chloroacetaldehyde, a degradation product of MCA, is responsiblefor the observed mutations. The chemical mechanism by whicheither MX or CMCF induces G: C T: A transversions remains unknown. ¹To whom correspondence should be addressed     

Selection of agar for use in Salmonella typhimurium and Escherichia coli mutation assays

The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli [WP2 uvrA (pKM101)] was evaluated using Vogel Bonner minimal plates prepared with ten different agars. In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No. 1 agars; and GIBCO select agar were tested. Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E. coli have not been done. The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254-induced rat liver activation system. With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars. However, with E. coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency. Difco Bacto agar appears to be the most consistent agar for use with these strains. As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions. These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains. Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens. Environ. Mol. Mutagen. 32:192–196, 1998 © 1998 Wiley-Liss, Inc.