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.     

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      

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).     

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.     

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     

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.     

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.     

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     

9-Methoxytariacuripyrone, a naturally occurring nitro-aromatic compound with strong mutagenicity in Salmonella typhimurium

9-Methoxytariacuripyrone, a nitro-aromatic compound isolatedfrom Aristolochia brevipesshowed strong mutagenic activity instrain TA98, TA100 and some YG strains of Salmonella typhimuriumwithand without S9 mix. Incubation with cytosol resulted in a heavyincrease in mutagenicity. When incubated with microsomes theactivity was dramatically decreased. The results are discussedin view of the enzymes possibly involved in activation and detoxificationof the compound. The role of the basic structure on the mutagenicitymediated through the nitro group was also considered.     

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      

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.     

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 testing of rutacridone epoxide and rutacridone, alkaloids in Ruta graveolens L., using the Salmonella/microsome assay

The mutagenicity of rutacridone and rutacridone epoxide wasinvestigated using Salmonella typhimurium without as well aswith different metabolic activation systems. Rutacridone epoxidewas found to be a direct acting mutagen in S. typhimurium strainsTA98, TA100 and TA1538; addition of rat liver preparations resultedin a marked decrease of mutagenicity. In contrast, rutacridonerequired metabolic conversion to exhibit mutagenic activity.Neither of the compounds had any effect on tester strain TA1978.S9 mixes as well as microsomal and cytosolic preparations fromuntreated, phenobarbital-treated and 3-methylcholanthrene-treatedrats were used to study the activation and deactivation capacitiesof the enzyme mixtures. In addition, the influence of enzymeinhibitors on the activation and deactivation of the furoacridoneswere tested. Evidence is presented that rutacridone is metabolizedby rat liver enzymes to the corresponding epoxide as the ultimatemutagen. ^*Dedicated to Professor Dr C.-G.Arnold on the occasion of his60th birthday.      

Role of oxidative DNA damage in hydroxychavicol-induced genotoxicity

Chewing betel quid has been linked to the development of oralcancer. In Taiwan, fresh Piper betle inflorescence is uniquelyadded to betel quid, and hydroxychavicol is the major phenoliccomponents of P.betle inflorescence. In this study, we testedthe mutagenic potential of hydroxychavicol in Salmonella typhimuriumTA97, TA98, TA100 and TA102 with and without Aroclor-1254 inducedS9 fraction. The results showed that hydroxychavicol was positivein S.typhimurium TA102 without metabolic activation. This increasein revertants was partially inhibited by catalase and superoxidedismutase. In Chinese hamster ovary (CHO-K1) cells, hydroxychavicolinduced chromosome aberrations in a dose-dependent manner (10–50µM) and the majority were chromosome-type aberrations.Hydroxychavicol also significantly increased the frequency ofmicronuclei in CHO-K1 cells up to 3-fold at a concentrationof 40 µM) In addition, hydroxychavicol dose-dependently(0.1–20 [µM] induced copper-dependent strand breaksin plasmid DNA. We further tested the oxidative DNA damage potentialof hydroxychavicol by measuring 8-hydroxydeoxyguanosine (8-OH-dG)formation in CHO-K1 cells following an 18-h incubation and foundthat hydroxychavicol (6.25–100 µM) induced 8-OH-dGlevels dose-dependently. The increase of 8-OH-dG formation waspositively correlated (r = 0.79) with the hydroxychavicol-inducedcytotoxicity. In conclusion, hydroxychavicol may exert its genotoxicpotential through oxidative DNA damage. ⁵To whom correspondence should be addressed at: Department of Medical Research, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China     

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%;     

Bacterial mutagenic evaluation of Luxabendazole, a new broad spectrum anthelmintic, with the Salmonella typhimurium His- and the Escherichia coli Tryp- reversion tests

Luxabendazole is a new benzimidazole carbamate chemo-therapeuticagent, which has proved to be effective against adult and immaturestages of the major gastrointestinal nematodes, trematodes andcestodes. The mutagenic properties of Luxabendazole were investigatedin the in vitro Ames Salmonella and E.coli tests. The productwas tested at concentrations of 0.5, 5, 50, 500, 1250 and 2500µg/ plate in the TA1535, TA1538, TA98 and TA100 strainsof Salmonella typhimurium, and 0.5, 5, 50 and 500 (µg/platein the WP2, WP2 uvrA– and its pKM 101-containing derivativeCM891 (WP2 uvrA^– pKM101) strains of Escherichia coli,with and without S9 microsomal activation (postmitochondrialliver fraction from Wistar rats pretreated with Aroclor®).Positive and negative controls were included in each experiment.From the present study it can be concluded that Luxabendazole,over a dose range of 0.5–2500 µg/plate, is unlikelyto present a mutagenic hazard, as demonstrated by the Ames test. ³To whom correspondence should be addressed     

Photomutagenicity assays in bacteria: factors affecting assay design and assessment of photomutagenic potential of para-aminobenzoic acid

Photomutagenicity assays are required for regulatory submissionsof some chemicals. As yet there are no wellvalidated protocolsavailable for these assays. Critical factors which may contributeto the ability of a bacterial assay to detect photomutagens(e. g. dose of UV and test chemical, exposure conditions, lightsource, bacterial strains) were investigated using two knownphotomutagens, chlorpromazine and 8-methoxypsoralen. Salmonellatyphimurium strains TA98, TA102 and TA1537 and Escherichia colistrains WP2 and WP2(pKM101) were used and differences in theresponsiveness of these strains were observed with these substances.Both chemicals were detected using either UV exposure in suspensionor on the agar plates. On the basis of these observations andon other results reported in the literature, recommendationsare made on protocol aspects for assessing photomutagenic potentialin routine screening tests. Using these recommendations thesunscreen para-amino-benzoic acid was tested in S. typhimuriumstrains TA98, TA100, TA1535, TA1537 and E. coli strains WP2and WP2(pKM101), using both plate irradiation and suspensionexposure conditions. No evidence of mutagenic potential wasdetected.     

The effect of biotransformation of 2,4-dinitrotoluene on its mutagenic potential

Because both oxidative and reductive metabolism of the hepatocarcinogen2,4-dinitrotoluene (2,4-DNT) can occur in vivo; we have examinedthe mutagenicity of compounds which can be formed from 2,4-DNTin an attempt to establish which metabolic pathways contributeto the formation of genotoxic products. A quantitative reversionassay using Salmonella typhimurium TA98 was used to evaluatethe mutagenicity of these compounds. 2,4-Dinitrobenzyl alcohol,2-amino-4-nitro-toluene and 2-nitroso-4-nitrotoluene were foundto be more mutagenic to S. typhimurium than is 2,4-DNT and didnot require metabolic activation by post-mitochondrial super-natantsof Aroclor-induced rat liver homogenates (S9) for their effect.2-Amino-4-nitrobenzoic acid was also mutagenic to S. typhimuriumTA98 in the absence of S9, but its mutagenicity was enhancedwhen S9 was included in the incubation mixture. 2,4-Diaminotoluenerequired S9 for demonstration of mutagenicity and was approximatelyas effective, on a molar basis, as 2,4-DNT in inducing reversionto histidine prototrophy. These results suggest that both oxidativeand reductive metabolism may be involved in production of mutagenicmetabolites of 2,4-DNT. ¹Present address to which correspondence should be addressed:Department of Pharmacology and Toxicology, University of MississippiMedical Center, 2500 N. State Street, Jackson, MS 39216, USA      

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     

Assay of ptaquiloside, the carcinogenic principle of bracken, Pteridium aquilinum, by mutagenicity testing in Salmonella typhimurium

The mutagenicity of ptaquiloside, the carcinogenic principleof Pteridium aquilinum, was tested in Salmonella typhimuriumTA100 and TA98. Under weakly basic conditions (pH 8.5), ptaquilosidedecomposed into a conjugated dienone (considered to be the ultimateform), which was mutagenic in both strains. A novel bioassay,using the pre-incubation method at pH 8.5 with S. typhimuriumtester strains was developed for the assay of ptaquiloside extractedfrom plants. By this bioassay the ptaquiloside content of fernscollected at different localities during various seasons, andin various parts of the plant was determined. The ubiquitouspresence of ptaquiloside in fresh plant materials was confirmed.Bracken processed in alkali was found not to contain the carcinogen. ¹Present address: Wako Pure Chemical Industries Ltd, Kawagoe-shi,Saitama 328, Japan. ²Present address: Tochigi Health Centre, Tochigi-shi, Japan. ³To whom correspondence should be addressed