Comparative mutagenicity of structurally related aliphatic epoxides in a modified Salmonella/microsome assay

Four structurally related aliphatic epoxides (1,2-epoxypropane,1,2-epoxyisobutane, cis- and trans-2,3-epoxybutane) have beentested in the Salmonella/microsome assay, modified for volatilesubtances, using the strains TA1535 and TA100. The aim of thestudy was to evaluate the effect of methylation on the mutagenicityof 1,2-epoxypropane in this vaporization assay, with and withoutexogenous metabolization. All substances induced a significantincrease of revertants in the strains TA1535 and TA100. In termsof mutagenic potency, the following hierarchy was observed inthe standard tester strain TA1535 and in the absence of ratS9: 1,2-epoxypropane > cis-2,3-epoxybutane > 1,2-epoxyisobutane> trans-2,3-epoxybutane. After exogenous metabolization,the mutagenic response of 1,2-epoxyisobutane was substantiallyreduced, while a moderate decrease of cis-2,3-epoxybutane wasobserved in the presence of S9, as compared with the responsewithout S9. No influence of the S9 on the mutagenic responseof trans-2,3-epoxybutane was noticed in both strains TA1535and TA100, while an increased response with 1,2-epoxypropanewas observed in TA100 but not in TA1535. The results suggestthat the vaporization assay may provide more relevant informationconcerning mutagenic potencies of gaseous or volatile compoundsthan the common treat-and-plate or preincubation assays. Moreover,it appears that mutagenicity theories, based only upon inductiveeffects of side groups, may not suffice to explain differencesin mutagenicity. Sterical factors or differential interactionswith metabolizing enzymes could also be important in the evaluationof mutagenic effects. ⁴Present address: Ministrium für Umwelt und Gesundheit,Kaiser Friedrichstrasse 7, D-6500 Mainz, Germany      

Effects of the nitro-group on the mutagenicity and toxicity of some benzamines

The Ames Salmonella/microsomal assay was employed to test the mutagenicity of some benzamines (aniline, and o- and p-phenylenediamine) and their nitro-derivatives (p-nitroaniline, 2-nitro-p-phenylenediamine 3- and 4-nitro-o-phenylenediamine), using strains TA98) and TA100 and their nitroreductase-deficient mutants, TA98NR and TA100NR, in the presence and absence of rat S9 mix. The addition of the nitro-group to benzamine molecules converted them into direct mutagens. Furthermore, the position of the nitro-group affected their mutagenic activities. Cytotoxicity testing with Chinese hamster ovary cells (CHO-K1) showed that the presence of the nitro-group in these compounds had no specific effect on toxicity. The test compounds all showed a dose-related increase in inducing chromosomal aberrations in CHO cells. However, the presence of the nitro-group did not affect potency in inducing chromosomal aberrations. Compounds containing the nitro-group had higher initial oxidation potentials and dipole moments (μ) than their nonnitro-containing counterparts. The mutagenicity and toxicity of these compounds were not related to physico-chemical properties, including oxidation potential, energy difference (ΔE) between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), ionization potential (I.P.), and μ. © 1996 Wiley-Liss, Inc.     

Mutagenic activity in synthetic pyrethroids in Salmonella typhimurium

Four pyrethroids, allethrin, resmethrin, permethrin and fen-valerate,were tested for mutagenicity in bacterial reversion assay systemswith seven strains (TA1535, TA100, TA1538, TA98, TA1537, TA97and TA104) of Salmonella typhimurium. Our results show thatthree pyrethroids, namely resmethrin, permethrin and fenvalerate,were not found to be mutagenic in S. typhimurium in the presenceor absence of a rat liver activation system. Allethrin was foundto be mutagenic with TA100, TA104 and TA97 strains and requiredmetabolic activation (S9 mix) in order to show its activity,mainly with TA100 and TA104 strains.     

Mutagenic potential of Indian tobacco products

The mutagenic potential of aqueous extracts of masheri (ME),chewing tobacco alone (CTE) and a mixture of chewing tobaccoplus lime (CTLE) was tested using the Ames assay. ME exhibitedmutagenicity in Salmonella typhimurium TA98 upon metabolic activationwith aroclor-1254-induced rat liver S9, while nitrosation renderedit mutagenic in TA100 and TA102. CTE exhibited borderline mutagenicityin the absence or presence of S9 in TA98 and TA100 and afternitrosation in TA102, while nitrosation led to doubling of TA98and TA100 revertants. In contrast, CTLE exhibited direct mutagenicityin TA98, TA100 and TA102, was mutagenic to TA98 upon S9 additionand induced mutagenic responses in all three tester strainsafter nitrosation. Experiments using scavengers of reactiveoxygen species (ROS) suggested that CTLE-induced oxidat-ivedamage in TA102 was mediated by a variety of ROS. The high mutagenicpotency of CTLE vis à vis that of CTE may be attributedto changes in the pH leading to differences in the amount andnature of compounds extracted from tobacco. Thus, exposure toa wide spectrum of tobacco-derived mutagcns and promutagensmay play a critical role in the development of oral cancer amongusers of tobacco plus lime. ¹To whom correspondence should be addressed     

Evaluation of the genotoxicity of 4-diethylamino-4'-nitroazobenzene and seven analogues

A series of eight nitroaromatic azo compounds based on 4-diethylamino-4'-nitroazobenzene has been examined for genotoxic activity ina collaborative study conducted under the auspices of the Ecologicaland Toxicological Association of Dyes and Organic Pigments Manufacturers(ETAD). The evaluation has been conducted in two parts, firstlyan examination in vitro to assess any intrinsic genotoxic activityof the compound. The chemicals were examined in the Salmonellaassay in a standard plate incorporation protocol in both thepresence and absence of S9 and in a minimum of the four testerstrains recommended in the OECD guideline for this assay, i.e.TA1535, TA1537, TA98 and TA100. All of the compounds were mutagenicin one or more of the Salmonella tester strains, and all werepositive in TA98 with S9. A considerable range of potency wasseen in this assay. The chemicals were further examined in vitrofor mammalian cell gene mutation at either the HGPRT or TK locusin a standard (CHO, V79 or L5178Y) cell system. Only one ofthe chemicals was mutagenic and only with S9. This chemicalalso showed the most potent response in the Salmonella assay.The second part of the study was an examination in vivo to seewhether any genotoxic activity was expressed in the whole animal.The in vivo rat liver DNA repair (unscheduled DNA synthesis;UDS) assay was chosen as being the most likely to be sensitiveto aromatic nitroazo compounds. All of the materials were negativewhen tested alongside a structurally related positive control.The chemicals were also examined in the mouse bone marrow micronucleusassay in order to provide a second in vivo assessment. Sevenof the chemicals were negative in this assay, however, one produceda positive response. This compound was also the only one detectedas positive in the in vitro mammalian cell gene mutation assay.The data obtained in this study show how genotoxic nitroazocompounds can be evaluated using a structured combination ofin vitro and in vivo assays.     

Investigations into the genotoxic potential of loxtidine, a long-acting H2-receptor antagonist

Loxtidine, a potent, non-competitive histamine H₂-receptor antagonistwas evaluated for genotoxic potential using a range of short-termmutagenicity assays. Unequivocally negative results were obtainedin a Salmonella/plate incorporation assay and a liquid pre-incubationassay (using S.typhimurium strains TA1535, TA100, TA1537, TA1538and TA98), a fluctuation assay [using Escherichia coli strainsWP2, WP2 uvrA (R46) and 343/113 lys60 (R46)], a gene conversionassay (using Saccharomyces cerevisiae JD1) and a human peripherallymphocyte cytogenetic assay. All of these in vitro tests werecarried out in the presence and absence of rat liver S9 mix.In addition, the major metabolites of loxtidine in the rat werealso negative in the same range of microbial mutagenicity assays.Loxtidine was inactive in the mouse micronucleus test afteroral administration. The potential nitrosatability of loxtidinewas investigated using an expanded version of the WHO NitrosationAssay Procedure, and detectable quantities of mutagenic nitroso-specieswere not formed. The subsequent appearance of carcinoid tumourswithin the gastric fundus of rodents treated orally with loxtidinefor most of their natural lifespan, led to additional assaysbeing carried out on this compound to determine whether thetumorigenic effects were due to alternative mutagenic mechanisms.Negative results were obtained in an in vitro unscheduled DNAsynthesis assay using primary rat hepatocytes, and an assayfor spindle damaging agents using Muntjac skin fibroblasts.It can be concluded from these results that loxtidine is unlikelyto be a genotoxic carcinogen. The increase in carcinoid tumourincidence observed in rats and mice after loxtidine treatmentwas probably related to the prolonged achlorhydria producedby this potent unsurmountable histamine H₂-receptor antagonist.     

Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays

Stevioside, a constituent of Stevia rebaudiana, is commonlyused as a non-caloric sugar substitute in Japan. The genetictoxicities of stevioside and its aglycone, steviol, were examinedwith seven mutagenicity tests using bacteria (reverse mutationassay, forward mutation assay, umu test and rec assay), culturedmammalian cells (chromosomal aberration test and gene mutationassay) and mice (micronucleus test). Stevioside was not mutagenicin any of the assays examined. The aglycone, steviol, however,produced dose-related positive responses in some mutagenicitytests, i.e. the forward mutation assay using Salmonella typhimuriumTM677, the chromosomal aberration test using Chinese hamsterlung fibroblast cell line (CHL) and the gene mutation assayusing CHL. Metabolic activation systems containing 9000 g supernatantfraction (S9) of liver homogenates prepared from polychlorinatedbiphenyl or phenobarbital plus 5, 6-benzoflavone-pretreatedrats were required for mutagenesis and clastogenesis. Steviolwas weakly positive in the umu test using S.typhimurium TA1535/pSK1002either with or without the metabolic activation system. Steviol,even in the presence of the S9 activation system, was negativein other assays, i.e. the reverse mutation assays using S.typhimuriumTA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichiacoli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis.Steviol was negative in the mouse micronucleus test The genotoxicrisk of steviol to humans is discussed. ⁹To whom correspondence should be addressed     

Mutagenicity of extracts of brown and black masheri, pyrolysed products of tobacco using short-term tests

Black and brown varieties of masheri, which are pyrolysed tobaccoproducts were analysed for their mutagenic potentials usinga battery of test systems. Both materials were found to be mutagenicin Salmonella typhimurium strain TA98 with metabolic activationand also in V79 Chinese Hamster cells producing 8-azaguanineresistant mutations. Both varieties were found to induce statisticallysignificant increases in micronuclei formation as compared tothose produced in the solvent controls. Both varieties inducedstructural chromosomal aberrations in bone marrow cells of mice.Our data suggest that masheri is a potent mutagen in a varietyof test systems and is likely to have a mutagenic potentialvis à vis humans. ¹To whom correspondence should be addressed     

UDS activity in the rat liver of the human carcinogens benzidine and 4-aminobiphenyl, and the rodent carcinogens 3, 3'-dichlorobenzidine and Direct Black 38

The activity of benzidine and three structurally related carcinogensin an in vivo/in vitro rat liver assay for unscheduled DNA synthesisis described. The first three of these chemicals, benzidine,4-aminobiphenyl (4AB) and Direct Black 38 have been reportedas positive in this assay by other investigators, albeit thedata reported for 4AB were limited. The fourth compound, 3,3’-dichlorobenzidinehas not been studied in this assay before. Each compound gavea clear positive response under conditions of routine testing. ¹To whom correspondence should be addressed     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

Concentrated drinking water extracts, which cause bacterial mutation and chromosome damage in CHO cells, do not induce sex-linked recessive lethal mutations in Drosophila

Concentrated extracts prepared from chlorinated drinking watersamples were tested for their ability to induce sex-linked recessivelethal mutations in Drosophila melanogaster. Adult flies wereallowed to feed on sucrose solutions prepared using neat orhalf-strength water extract. The drinking water extracts usedfor this study were also tested in bacterial fluctuation assaysusing Salmonella typhimurium TA100 and TA98 and in an in vitrocytogenetic assay using CHO cells. Although the water extractsgave positive results in both of these in vitro tests, therewas no evidence of mutagenic activity in the Drosophila studies. ¹Present address: Glaxo Group Research Ltd, Park Road, Ware,Hertfordshire SG12 ODJ, UK      

The effectiveness of Salmonella strains TA100, TA102 and TA104 for detecting mutagenicity of some aldehydes and peroxides

Several aldehydes and peroxides were tested for mutagenicityusing Salmonella typhimurium tester strains TA97a, TA100, TA102and TA104, in the presence and absence of Aroclor-induced liverS9 mix from F344 rats and B6C3F₁ mice, in either preincubationor vapour phase rotocols. Some chemicals were tested in additionalSalmonella strains. Benzaldehyde, butyraldehyde, benzoyl peroxide,4-chlorobenzaldehyde, isobutyraldehyde, propionaldehyde andveratraldehyde were non-mutagenic Acetaldehyde and dicumyl peroxidegave inconsistent results and furfural gave equivocal responsesin TA100 and TA104. Cumene hydroperoxide, formaldehyde and glutaraldehydewere mutagenic in TA100, TA102 and TA104. trans-Cinnamaldehydeexhibited a weak mutagenic response in TA100 with mouse liverS9 only. 2,4,5-Trimethoxybenzaldehyde was mutagenic only instrain TA1538 with rat liver S9. With the exception of butanoneperoxide, which was mutagenic only in TA104, all chemicals mutagenicin strains TA102 and/or TA104 were also mutagenic in TA100.The data do not, therefore, support the preferential use ofstrains TA102 and TA104 for screening aldehydes and peroxidesfor mutagenicity. For a number of these chemicals the advantagesof using TA102 or TA104 was in the increased responses comparedwith those obtained with TA100. Two of the four peroxides weremutagenic and one of these was mutagenic only with TA104. Thissuggests that strains TA102 and TA104 be used if peroxides arenot mutagemc in TA100 or TA97. ⁴Present addresses: ⁴British American Tobacco Ltd, SouthamptonSO15 8TL, UK ⁵FRAME, Nottingham NG1 4EE, UK ³To whom correspondence should be addressed. Tel: +1 919 541 4482; Fax: +1 919 541 2242; Email: zeiger{at}niehs.nih.gov      

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 by caecal reduction of the azo dye D & C Red No. 9 to a bacterial mutagen

D & C Red No. 9 is a monoazo dye used for manufacturingprinting inks, rubber and plastics, and as an additive in cosmeticsand drugs. In an NTP carcinogenicity study in rats and miceit induced splenic sarcomas and liver nodules in male rats;no chemical-related tumours were induced in mice. On the basisof its contradictory responses in a range of in vitro testsand its inactivity in several in vivo genotoxicity assays, ithas been suggested that the dye may act as a non-genotoxic carcinogen.We tested the dye in the Salmonella mutagenicity assay usingseveral different protocols. The dye was not mutagenic whentested using the standard (aerobic) preincubation protocol.Variable responses were seen when the flavin mononucleotide(FMN) reduction protocol was used. A third protocol was providedby incubating the test compound overnight with a rat caecalpreparation under anoxic conditions to reduce the azo bond.Ethyl acetate extracts of this incubation mixture, when testedin the standard preincubation protocol using induced rat liverS9, yielded dose-related mutagenic responses in TA 100, anda weak response in TA98. The presumed major reduction product,1-amino-2-naphthol (1-A-2-N) was mutagenic to TA100, but notTA98, in standard protocols with S9. The results show that itis necessary to use a protocol in which D & C Red No. 9is reduced in order to demonstrate the mutagenicity of thisdye. The non-genotoxicity previously reported for D & CRed No. 9, may have been due to insufficient reductive cleavage.The carcinogenicity of this compound may, therefore, be a consequenceof its genotoxicity, rather than a result of some non-genotoxicprocess. ¹Present address: FRAME, Eastgate House, 34 Stoney Street, NottinghamNG1 1NB, UK ³To whom correspondence should be addressed.      

Genetic differences between the standard Ames tester strains TA100 and TA98

The standard Ames tester strains of Salmonella typhimurium areseparated by many steps in their pedigree, some involving mutagentreatments, and contain independently isolated uvrB-bio-galdeletions and rfa mutations. In this work the araD531 mutationwas introduced into the Ames tester strains TA100 and TA98.The responsiveness of the resulting strains (BA15 and BA14)to a number of chemical mutagens was then assessed by monitoringthe induction of forward mutations to L-arabinose resistance(Ara test). Here we have shown that these two strains of theAmes test differ greatly in their responses to mutagens, inways that are not associated with the mutagenic specificitiesof the original his mutations. In general, the genetic backgroundof strain TA100 appears to be more sensitive to the killingeffects of chemicals than that of TA98. The greatest differenceswere found with nifurtimox (NFX) and its analogue, compound1K. The Ara test responded to the mutagenic effects of thesetwo nitrofurans when carried out in the genetic background ofstrain TA98 but not in that of TA100. A higher sensitivity tothe lethal effects of NFX and 1K together with the greater nitroreductioncapability of strain TA100 as compared with TA98 might explainthe differences. In conclusion, our results indicate that thestandard Ames S. typhimurium tester strains are not isogenicand that genetic differences at loci other than his might besignificant for mutagenicity testing. To this respect the routineuse of the isogenic set of S. typhimurium strains constructedby Popkin et al. (Mut. Res., 224, 453–464, 1989) and derivedfrom strain hisD3052 (as the standard TA98) seems advisable.     

Mutagenicity of red, wine in the L-arabinose resistance test with Salmonella typhimurium

The forward mutation assay to L-arabinose resistance (Ara test)in Salmonella typhimurium detected a Spanish red table wine(Rioja) as a direct-acting mutagen. The best mutagenic responsewas obtained by preincubating strain BA13 with the wine samplein the presence of sodium phosphate buffer and in the absenceof any external metabolic activation. In fact, the S9 mixtureabolished most of the mutagenic activity of red wine in theAra test. Such an inactivating capacity seems to be independentof microsomal enzymes and mediated through some kind of heat-stablecomponent(s) in the S9 fraction. Both regular wine (directlyfrom the bottle) and lyophilized wine were strong mutagens inthe Ara test, inducing 4914 and 2739 Ara^R mutants/ml. Both pKMlOland uvrB were critical factors in the detection of the mutagenicityof wine, exhibiting a synergic effect in strain BA13. The mutagenicityof red wine was somehow pH-dependent, increasing with the pHvalue of the preincuba-tion mixture. In comparison with theAra test, the His reverse mutation assay (Ames test) was muchless sensitive to the mutagenicity of lyophilized red wine,TA102 being the most (448 His⁺/ml) and TA98 the least (38 His⁺/ml)sensitive strain. TA100, TA104 and TA97 manifested intermediatemutagenicities to red wine. Previous reports have identifiedstrain TA98 as the His strain most sensitive to the apolar fraction(e.g. XAD-2-bound) of red wine. Based on these results we proposethat TA98 mainly detects glycosides of mutagenic flavonols presentin red wine (quercetin, rutin, etc.), which do not constitutethe major direct-acting mutagens detected with the Ara test.In contrast, the Ara test (and possibly TA102) are more sensitiveto other chemical(s) present in complete regular wine or lyophilizedwine. In this respect, it is possible that the direct-actingmutagenicity of red wine in the Ara test could be due to oxidativechemicals.     

Testing human faecal extracts for genotoxic activity with the SOS Chromotest: the importance of controlling for faecal enzyme activity

Two aqueous extracts of human faeces were prepared from a healthymale donor and assayed in the SOS Chromotest. Both extractswere positive in microtitre fluctuation tests in Salmonellatyphimurium TA100 and Escherichia coli WP2uvrA(pKM101). Differenceswere observed in the induction factors of these samples whenp-nitrophenyl-/5–D-galacto-pyranoskle (p-NPG) and o-nhTophenyl–3-D-galactopyranoside(o-NPG) were used as substrates for the /3-galactosidase assayin the SOS Chromotest. With one sample, a positive inductionfactor was reproducibly obtained using p-NPG but not o-NPG.When the bacterial cells were washed with fresh LB broth beforeenzyme assay, the positive induction factor obtained with p-NPGwas reduced to an insignificant level. During the 2-h treatmentperiod, both faecal samples enhanced bacterial growth abovethat of the zero-dose control. When SOS Chromotest assays wereperformed with no bacteria or with 5. typhimurium TA100 or hisG46(non-lactose fermenting organisms) in place of E. coli PQ37,it was found that the extracts contained significant levelsof endogenous 3–galactosidase and alkaline phosphatase,which, due to their carry-over in the bacterial pellet (aftercentrifugation to remove the coloured extract) gave rise tothe positive induction factor obtained with p-NPG. The resultsobtained in these experiments indicate that where the SOS Chromotestis applied to biological samples, care should be taken in theinterpretation of the data and that a washing step should beincluded to prevent possible errors occurring due to exogenousenzymes in the sample.     

Errata

The first paragraph of the Discussion should read: "BZD and DAT were predicted to be mutagenic for both strainsTA98 and TA100 while CDA and DAB were predicted to be non-mutagenicin these strains. This is similar to the overall evaluationof the Second UKEMS Collaborative Study (Parry et al., 1985).The latter found that in Salmonella (strain TA1538 which forthe purpose of this discussion is taken to be similar to TA98)(Ames et al., 1975), both BZD and DAT were mutagenic. CDA wasnon-mutagenic, while DAB was difficult..."     

Effects of epoxide hydratase inhibitors in forward and reversion bacterial mutagenesis assay systems

Cyclohexene oxide (CCHO) and 1,1,1-trichloropropene-2,3-oxide (TCPO) are inhibitors of epoxide hydratase and have been used in studies of the mechanisms of mutagenesis in bacterial mutagenesis assays. The present studies were designed to investigate the mutagenic activity of CCHO and TCPO in Salmonella typhimurium employing the Ames histidine-reversion assays (TA98, TA100, TA1535, TA1537, TA1538) and a forward mutation assay that uses 8-azaguanine resistance in TM677 as the genetic marker. In the reverse mutation assay, TCPO (10⁻³ M) produced a mutagenic response in strains TA100 and TA1535 in the absence or presence of a rat liver metabolizing system (S9), indicating that TCPO causes base-pair substitution mutations. CCHO (10⁻³ M) showed a slight but significant mutagenic effect in strain TA100, with or without S9 and, in strain TA1535, only in the absence of S9. In the forward assay, TCPO was a strong mutagen at concentrations above 5 × 10⁻⁵ M and was toxic to the bacteria. The mutagenic and toxic effects of TCPO were slightly reduced in the presence of the S9 preparation, suggesting that the epoxide may be metabolized by the microsomal enzymes. In the forward assay, CCHO showed no mutagenic activity but some toxicity at 3 × 10⁻³ M. When epoxide hydratase activity was measured under the conditions of the forward mutation assay, 85% inhibition of activity was observed at 10⁻³ M TCPO, a concentration that caused a 45-fold increase in the mutation frequency. CCHO (3 × 10⁻³ M) produced a 55% inhibition of epoxide hydratase activity without exhibiting mutagenic effects in TM677. These results indicate that CCHO should be employed in preference to TCPO when inhibition of epoxide hydratase activity is required in bacterial mutagenesis studies.     

Mutagenicity spectra in Salmonella typhimurium strains of glutathione, L-cysteine and active oxygen species

Glutathione and L-cysteine, in the presence of rat kidney post-mitochondrialsupernatant (S9) fraction, and various forms of active oxygenwere investigated for mutagenicity in seven his^– strainsof Salmonella typhimurium. Glutathione and L-cysteine showedqualitatively and quantitatively virtually identical mutagenicactivities. The number of mutants induced in strain TA97 was3–4 times higher than in TA100, the strain in which themutagenicity was originally detected. Mutagenic effects werealso observed in strains TA92, TA102 and TA104, but not in TA1535and TA1537. Hydrogen peroxide, superoxide and glucose/glucoseoxidase in the presence and absence of kidney S9 fraction showedpronounced mutagenic effects in strains TA104 and TA102. Additionally,weak mutagenic effects were observed in TA100, while the remainingstrains, including TA97, were not responsive. These mutagenicityspectra suggest that the mutagenic species formed from glutathioneand L-cysteine are similar, if not identical, and are differentfrom hydrogen peroxide, superoxide and other oxygen speciesderived from them. Further support for this notion was givenwhen it was observed that catalase did not affect the mutagenicityof glutathione and that superoxide dismutase showed a significanteffect only when used in milligram quantities. This study showsthat mutagenicity spectra may be useful in the elucidation ofactivation pathways. Furthermore, it is interesting to notethat all the compounds and preparations showing a positive responsein the Ames test in the present study occur endogenously inorganisms: glutathione, L-cysteine, hydrogen peroxide, superoxide,glucose, glucose oxidase and kidney S9 fraction (which was mutagenicin several strains).