Synthesis and mutagenicity of 1-nitropyrene 4,5-oxide and 1-nitro-pyrene 9,10-oxide, microsomal metabolites of 1-nitropyrene

[4,5,9,10-³H]1-Nitropyrene was incubated with liver microsomesprepared from guinea pigs treated with Aroclor 1254 and theproducts were examined by h.p.l.c. The previously reported metabolites,1-nitropyrene trans-4,5-diliydrodiol, 1-nitropyrene trans-9,10-dihydrodiol,and 3-, 6-, and 8-hydroxy-1-nitropyrene were detected. In addition,h.p.l.c., nuclear magnetic resonance and mass spectral analysesindicated the presence of 1-nitropyrene 4,5-oxide and 1-nitro-pyrene9,10-oxide. The epoxide hydrase inhibitor, 1,2-epoxy-3,3,3-trichloropropane,decreased the concentration of the 4,5- and 9,10-dihydrodiolsin the microsomal incubations and increased the concentrationof their corresponding oxides. Reaction of 1-nitropyrene withm-chloroperoxybenzoic acid gave a mixture of 1-nitropyrene 4,5-oxideand 1-nitropyrene 9,10-oxide, which was separated by chromatography.The mutagenicity of the oxides was determined in Salmonellatyphimurium strains TA98, TA98NR, and TA98/1,8-DNP₆, both withand without exogenous activation by a rat liver homogenate fraction(S9). In the absence of S9, both oxides showed maximum activityin TA98, slightly decreased mutagenicity in the acetylase-deficientstrain TA98/1,8-DNP₆, and much reduced activity in the nitroreductase-deficientstrain, TA98NR. When assayed in the presence of S9, 1-nitropyrene4,5-oxide had maximum mutagenicity in TA98, and was 50 and 95%less mutagenic in TA98NR and TA98/1,8-DNP₆, respectively. 1-Nitropyrene9,10-oxide had a similar strain sensitivity, except that itstotal mutagenicity was lower. Since 1-nitropyrene is metabolizedby oxidative pathways in vivo, these K-region oxides may contributeto the toxicities elicited by this compound. ⁴To 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.     

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     

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      

Bacterial genotoxicity of nitrosated famotidine

Famotidine, a histamine H2-receptor antagonist, was devoid ofmutagenic activity in seven his^- Salmonella typhimurium strains(TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102) and wasequitoxic in repair-proficient (WP2) and repair-deficient (WP2uvr,WP67, CM561, CM571, WP100 and CM871) Escherichia coli strains,both in the presence and in the absence of S9 mix containingliver S9 fractions from Aroclor-treated rats. However, aftera short pre-incubation step with nitrite in an acidic environment,the drug increased, by a direct mechanism, the number of his⁺revertants in Salmonella strains TA100, TA102 and TA97 (a decreaseof mutagenicity being conversely observed in TA1535) and oftrp+ revertants in E. coli strains WPluvrA and WP67. Moreover,it enhanced the induction of non-reparable DNA damage in E.coli strains simultaneously lacking the uvrA-dependent excisionrepair and the lexA post-replication repair pathways. The mutagenicityof acidified nitrite-famotidine mixtures was related to dosesof both precursors, with a maximum production of mutagenic derivativesin a slight molar excess of nitrite. The optimal pH of the nitrosationreaction (2.0) was intermediate between the one required forcimetidine (1.5) and ranitidine (2.5). Potency of famotidineas a precursor of mutagenic derivatives was considerably lowerthan the one of the other two H₂ blockers. The nitrosation productsof all three drugs mainly induced base-pair substitutions inSalmonella DNA, to a greater extent at sites containing G-Cbase pairs (strain TA100) in the case of famotidine and cimetidine,and at sites containing AT base pairs (TA102) in the case ofranitidine. Although these experimental findings may suggestpossible toxicological consequences in ulcer patients receivinganti-secretory drugs, various considerations tend to minimizetheir practical in vivo relevance, especially for risk-benefitevaluations. Additionally, as in the case of cimetidine andranitidine, formation of mutagenic nitrosated famotidine wasefficiently prevented by equimolar ascorbic acid.     

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.     

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.     

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.      

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     

Mutagenic evaluation of some triazino indoles using the Salmonella/mammalian microsome assay

The mutagenicity of ethyl l,2,3-triazino[5,4-b]indole-4-carb-oxylateN(3)-oxide (D3) and 2-chloroethyl 1,2,3-triazino-[5,4-b]indole-4-carboxylateN(3)-oxide (D4), heads of series of new products with considerableplatelet antiaggregating and hypotensive activity, and theirprecursors 2-ethoxy-carbonylmethyl-l-methyUndole-3-carboxylicacid (A₃) and 2-(2-chloroethoxycarbonyhnethyl)-l-methylindole-3-carb-oxylkacid (A₄) were tested in four strains of Salmonella typhimurium(TA98, TA100, TA97 and TA102) using the standard plate incorporationtechnique. A₃ and A₄ were not mutagenic whereas D3 was mutagenicto all the strains and D₄ was mutagenic to TA97, TA98 and TA100.The addition of 4 or 10% of S9 mix decreased the mutagenic activityof both compounds. This effect was independent of the concentrationof S9 in the S9 mix.     

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.     

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.      

Methylglyoxal: genotoxicity studies and its effect in vivo on the hepatic microsomal mono-oxygenase system of the mouse

The genotoxic potential of methylglyoxal (MG) was studied inSaccharomyces cerevisiae D7 and in Salmonella typhimurium TA97and TA102 in the presence and in the absence of metabolic activationsystem (S9 fraction) prepared from mouse liver induced withß-naphthoflavone (ß-NF) and sodium phenobarbital(PB). The in vivo effects on the hepatic microsomal mixed functionmono-oxygenase system induced by MG were studied in untreated,ß-NF or PB pre-treated mice. MG was a direct-actingmutagen in S. typhimurium TA97 and TA102 when tested up to amaximum concentration of 0.47 mg/plate. Mitotic gene conversionwas also induced by MG in the yeast S. cerevisiae D7. A weakbut significant effect on reverse point mutation was also foundin S. cerevisiae. Genetic activity was lower in the presenceof S9 fraction in yeast test. In the in vivo studies, MG (atthe total dose of 600 mg/kg) was shown to increase the aminopyrineN-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD)activities in uninduced mice. Cytochrome P-450 content (cytP-450) and ethoxycoumarin O-deethylase activity (ECD) were alsoweakly enhanced by MG treatment. In contrast, no significantchanges in mono oxygenase activities were seen in ß-NF-or PB-treated mice after MG injection. ¹To whom correspondence should be addressed     

Mutagenic activity of the antitumour agent homo-aza-steroidal ester of p-N,N-bis(2-chloroethyl)aminophenoxyacetic acid (NSC 294859) in the Salmonella/microsome assay

The mutagenic activity of the new antitumour agent 3ß-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-N,N-bis(2-chloroethyl)aminophenoxyacetate(NSC 294859) was studied in the Salmonella/microsome assay.It was found to induce base pair substitutions, causing dosedependentincreases in his⁺ revertants in strains TA100 and TA1535. Thealkylating moiety, p-N,N-bis(2-chloroethyl)-aminophenoxyaceticacid, was shown to be less effective than the parent compound,while the modified steroid moiety, 3ß-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam,showed no mutagenic effect in all strains used. The presenceof metabolic activation enzymes in the test system induced afurther increase in his⁺ revertants in strains TA100 and TA1535,in both the parent compound and the alkylating moiety of theparent compound, while it had no effect in the case of the steroidallactam. ⁴To whom correspondence should be addressed     

Genotoxicity of 2-halocinnamaldehydes in two bacterial assays. Induction of SOS repair and frame-shift mutation

2-Chlorocinnamaldehyde and 2-bromocinnamaldehyde, compoundsof practical interest, for example, as bacterioddes and fungicidesor for utilization in light sensitive layers, were tested inthe Ames preincubation test with various Salmonella typhimuriumstrains, and in the SOS chromotest with Escherichia coli PQ37. 2-Chlorocinnamaldehyde was clearly mutagenic in strain TA100 (6081 revertants/µmol) and in strain TA 98 (3050 revertants/µmol)without S9 mix, and was clearly positive in the SOS chromotest(SOSIP = 0.181). 2-Bromocinnamaldehyde was a strong mutagenin strain TA 100 (105, 500 revertants/µmol), in strainTA98 (41567 revertants/µmol) and in strain TA 1538 (15825revertants/ µmol), and also unambiguously mutagenic instrain TA 1535 (2110 revertants/µmol) without S9 mix.The SOSIP in the SOS chromotest was 1.5. Addition of S9 mixled to a marked decrease in the mutagenic activity of 2-bromocinnamaldehydein all strains tested. In the case of strain TA 1535, mutagenicactivity was abolished or not significant in the presence ofS9 mix. The possible primary mechanisms underlying these mutageniceffects are discussed. Frame-shift activity of these halocinnamaldehydescan be explained by their planar structure. ¹To whom correspondence should be addressed     

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).     

Tannic acid is not mutagenic in germ cells but weakly genotoxic in somatic cells of Drosophila melanogaster

Tannic acid (TA) was tested for genotoxic activity in threedifferent assays (1–3) in Drosophila melanogaster by feedingof larvae or adult flies. TA did not induce sex-linked recessivelethals (1) nor sex-chromosome loss, mosaicism or non-disjunction(2) in male germ cells. In the wing somatic mutation and recombinationtest (SMART) (3) TA was found to be toxic for larvae of thehigh bioactivation cross and produced a weak positive response.These results suggest that this compound, when administeredorally to larvae or adults of D.melanogaster, is not mutagenicand clastogenic in male germ cells, but weakly genotoxic insomatic cells of the wing imaginal disk. ³To whom correspondence should be addressed     

Metabolic activation of the genotoxic environmental contaminants 2- and 3-nitrofluoranthene in variants of Salmonella typhimurium TA98

The mutagenic environmental pollutants 2-nitrofluoran-thene(2-NFA) and 3-nitrofluoranthene (3-NFA), labelled with ³H and¹⁴C respectively, were incubated with Salmonella typhimuriumstrain TA98, its nitroreductase-deficient variant TA98NR andits 0-acetytransferase-deficient variant TA98/1,8-DNP₆, to investigatethe activity of these metabolic pathways under conditions approximatingthose of the Ames assay, hence their contribution to mutagenicpotency. 2-Aminofluoranthene (2-AFA) was the major metaboliteof 2-NFA (4 µM) in all three TA98 variants, isolated byreverse-phase HPLC and identified by UV-vis and NMR spectroscopyand mass spectrometry. 2-AFA was formed more slowly in TA98NR(65 pmol/h/ml resting phase bacterial broth, 1 to 2x10⁹ bacteria/ml)than in TA98 (295 pmol/h/ml) or TA98/1,8-DNP₆ (82 pmol/h/ml).2-Acetamidofluoranthene (2-AAFA) was also identified in incubationswith TA98 (80 pmol/h/ml), TA98NR (21 pmol/ h/ml), and TA98/1,8-DNP6(8 pmol/h/ml). 3-Aminofluoran-thene (3-AFA, confirmed by UV-visand NMR spectroscopy and mass spectrometry) was formed by allthree variants from 3-NFA (4 µM): TA98, 1.76 nmol/h/ml;TA98NR, 0.55 nmol/h/ml; TA98/1,8-DNP₆, 2.93 nmol/h/ml. 3-Acetamidofluoranthene(3-AAFA) was not detected in any of the variants. 3-AFA and3-AAFA were less mutagenic than 3-NFA, and required S9 for activation.Mutagenicity of 3-NFA relative to initial nitroreduction ratewas similar in TA98 and in TA98NR, but almost 10-fold lowerin TA98/ 1,8-DNP₆; hence 0-acetylation considerably enhancesthe mutagenicity of reduction products of 3-NFA. Mutagenicityof 2-NFA relative to initial nitroreduction rate was similarin TA98 and in TA98/1,8-DNP₆; the bacterial genotoxicity of2-NFA is therefore largely independent of O-acetyltrans-feraseactivity. Ratios of mutagenicity to nitroreduction rate weresimilar in TA98 for 2-NFA and 3-NFA; differences in the potencyof these isomers arise primarily from their respective suitabilitiesas substrates for nitroreductase enzymes. ⁴whom correspondence should be addressed     

Genotoxidty of 2-nitropropane and 1-nitropropane in Salmonella typhimurium and human lymphocytes

A 10- and 12-fold increase of revertant numbers could be demonstratedfor 2-nitropropane (2-NP of >99% purity) tested in the preincubationassay with Salmonella typhimurium strains TA 100 and TA 98 inthe presence and absence of s9 mix. In the nitroreductase-deficientstrains TA 100NR and TA 98NR, 2-NP was less mutagenic than inthe parent strains. In human lymphocytes the induction of aweak clastogenic effect and of sister chromatid exchanges requiredexogenous metabolic activation. No significant mutagenic orcytogenetic response was found with 1-nitropropane of 97% purityin S.typhimurium or human lymphocytes.