The reduced mutagenicity of aflatoxin B1 due to hydroxylation: observations on five Salmonella typhimurium tester strains.

The mutagenicity of aflatoxin M1 relative to that of aflatoxin B1, the parent compound, was studied in 5 Ames' tester strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537, TA 1538). Aflatoxins B1 and M1 are both highly mutagenic in microsome-mediated system in TA 100. The prediction of the relative carcinogenicity of aflatoxin M1 to aflatoxin B1 posed by the mutation of TA 100 is probably more authentic than by TA 98.     

Tissue-mediated mutagenicity of vinylidene chloride in Salmonella typhimurium TA1535.

Vinylidene chloride is weakly positive in the Salmonella typhimurium TA1535 test, mediated by kidney and liver post-mitochondrial supernatant (S-9 mix) from normal mice, but strongly positive with the S-9 mix from the induced animals. In the case of mediation by rat tissue, only liver S-9 mix from induced animals affords a significant positive response. These findings agree with the greater availability in treated mice than in rats of reactive vinylidene chloride metabolites, 1,1-dichloroethylene oxide and chloroacetyl chloride [5], and with the vinylidene carcinogeneicity found in mice but not in rats [9]. Exploratory tissue-mediated testing of vinylidene chloride involving liver S-9 mix from marmosets and man suggests a trend in the generation of alkylating metabolites and their reactions with bacterial DNA for these primates which resembles rats more than mice.     

Effects of secondary biliary acids on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and 2-nitrofluorene towards Salmonella typhimurium strains

The two secondary biliary acids (lithocholic and deoxycholicacids)were co-mutagenic when they were each co-incubated withdimethylhydrazine in the presence of Salmonella typhimuriumTA 100. These observations were extended to other toxic chemicals,acting as direct (N-methyl-N'nitro-N-nitrosoguanidine and 2-nitrofluorene)or indirect (2-acetyl-aminofluorene)mutagens. Lithocholic anddeoxycholic acids show a similar behavior towards genotoxicmolecules. Nevertheless two differences must be noted. Lithocholicacid was the stronger co-mutagen. When lithocholic acid inhibitedmutagenic activity of 2-nitrofluorene, deoxycholic acid didnot modify it. Interactions between the two secondary bile acidsoccurred so that the co-mutagenic activity of the mixture ofthese two bile acids depended on the ratio of their concentrations.Besides their cancer-promoting effect, biliaryacids also couldhave co-initiating effect that could depend upon the ratio oftheir concentration in the intestine. Calculating the ratioof fecal concentrations of deoxycholic/lithocholic acids thuscould be a more sensitive index for cancer risk than simplymeasuring the fecal concentration of the two biliary acids takenseparately.     

Tumor promoting potential in male F344 rats and mutagenicity in Salmonella typhimurium of dipyrone

For assessment of the carcinogenic potential and the mutagenicity of dipyrone, an antipyretic anodyne, -[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino]-methanesulfonic acid sodium salt monohydrate, three experiments were conducted using dipyrone A produced in Japan and/or dipyrone B obtained from the Federal Republic of Germany. (i) Carcinogenic potential of dipyrone A for rat liver: 8 week old male F344 rats were pretreated with 0.01% diethylnitrosamine (DEN) in drinking water for 2 weeks and, after 1 week of resting, administered 0.4% dipyrone in drinking water, 5 days a week, for 72 weeks. After an 8 week recovery period, all surviving rats were killed at 83 weeks. Hepatocellular carcinomas developed at a higher incidence in the DEN + dipyrone group (18 of 29 rats, 62%) than in the DEN alone group (9 of 29 rats, 31%), the difference being statistically significant (P less than 0.05). No carcinogenic activity of dipyrone was demonstrated in the groups given 0.4% dipyrone for 72 weeks or 0.4% dipyrone for 25 weeks, followed by 0.05% phenobarbital (PB) for 50 weeks. However, glutathione S-transferase P positive (GST-P+) preneoplastic hepatic foci in these groups were observed at a higher incidence than in the untreated control group (P less than 0.01). (ii) Effect of dipyrone A and dipyrone B on induction of DEN-initiated GST-P+ hepatic foci in a medium-term bioassay system: 0.4% dipyrone A in drinking water and 0.57% dipyrone A or dipyrone B in powdered diet after DEN initiation had similar enhancing effects on the development of GST-P+ foci (P less than 0.001). (iii) The Ames mutation test in Salmonella: both dipyrone A and dipyrone B proved weakly mutagenic for strain TA100 in the presence or absence of S9 fraction.     

In vitro mutagenicity assays of chemical carcinogens and related compounds with Salmonella typhimurium.

The mutagenic activity of 101 chemicals was studied with the use of the Salmonella typhimurium-microsome system described by Ames. The tester strains were TA1535, TA1536, TA1537, TA1538, TA98, and TA100. Assays were conducted in the presence and absence of a metabolic activation system prepared from the livers of randomly bred Sprague-Dawley rats that had been pretreated with Aroclor 1254. The test chemicals were incorporated into the agar with bacteria and the metabolic activation system. Mutagens were defined as chemicals that induced a reproducible dose-related increase in the number of histidine-independent revertants. With the use of these procedures, 65% of the organic carcinogens and 25% of the noncarcinogens were found to be mutagenic.     

Reactivity with DNA bases and mutagenicity toward Salmonella typhimurium of methylchrysene diol epoxide enantiomers

The reactions with DNA and mutagenic activities toward Salmonella typhimurium TA 100 of the R,S,S,R and S,R,R,S enantiomers of anti-1,2,-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (anti-5-MeC-1,2-diol-3,4-epoxide), anti-5-MeC-7,8-diol-9,10-epoxide, and anti-6-MeC-1,2-diol-3,4-epoxide were compared because among these compounds only the R,S,S,R enantiomer of anti-5-MeC-1,2-diol-3,4-epoxide is highly tumorigenic. The major products formed in the reaction of each racemic diol epoxide with DNA were two pairs of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts; one product in each pair was formed from the R,S,S,R enantiomer and the other from the S,R,R,S enantiomer of each racemic diol epoxide. Formation of products from R,S,S,R enantiomers exceeded formation of those from S,R,R,S enantiomers in each case. Among the R,S,S,R enantiomers, 5-MeC-1,2-diol-3,4-epoxide, which has a methyl group in the same bay region as the epoxide ring, was most reactive toward DNA, and in particular toward dGuo. The dGuo/dAdo adduct ratios were greater for the products formed from the R,S,S,R enantiomer compared to the S,R,R,S enantiomer of each diol epoxide. The dGuo/dAdo adduct ratios were also greater for the enantiomers of anti-5-MeC-1,2-diol-3,4-epoxide than for the enantiomers of either anti-5-MeC-7,8-diol-9,10-epoxide or anti-6-MeC-1,2-diol-3,4-epoxide. In S. typhimurium TA 100, the R,S,S,R enantiomer of anti-5-MeC-1,2-diol-3,4-epoxide was the most mutagenic compound (6700 revertants/nmol), followed by the R,S,S,R enantiomer of anti-5-MeC-7,8-diol-9,10-epoxide (1500 revertants/nmol). The other diol epoxide enantiomers were weakly active or inactive at the doses tested. The results of this study demonstrate that both the absolute configuration of a diol epoxide and the position of the methyl group have major effects on its reactivity with DNA. The greatest reactivity is seen in an R,S,S,R enantiomer with the methyl group and epoxide ring in the same bay region, e.g., the highly tumorigenic and mutagenic 5-MeC-1R,2S-diol-3S,4R-epoxide. Comparison of the dGuo/dAdo adduct ratios of the various diol epoxides with their tumorigenic and mutagenic activities suggests that dGuo adducts are important in the expression of biological activity of methylchrysene diol epoxides.     

Human,rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains

Exposure of S. typhimurium strains TA 1530, TA 1535 and G-46 to vinyl chloride increased the number of His⁺ revertants/plate 16, 12 or 5 times over the spontaneous mutation rate. After 6 h of exposure to vinyl chloride, the mutagenic response for TA 1530 strain was enhanced 7-, 4- or 5-fold when fortified postmitochondrial liver fractions from humans, rats or mice were added. The enzyme-mediated vinyl chloride mutagenicity was dependent on an NADPH generating system and the enzyme activity was localized in a liver microsomal fraction; 9,000 × g liver supernatant was three times more active than microsomes, while liver cytosol or alcohol dehydrogenase did not affect the mutagenicity. Phenobarbitone pretreatment of rats and mice increased the mutagenic response by up to 15–40% as compared to untreated controls. The relative mutagenic activities of VCM, taking the value from mouse liver as 100, for TA 1530 strain mediated by 9,000 × g tissue fractions were: rat liver, 80; mouse and rat kidney, 20 and 16; mouse and rat lung, less than 7; human liver (from four biopsy specimens), 170, 64, 70 and 46. Chloroacetaldehyde and chloroacetic acid, a urinary metabolite of VCM, showed toxic effects, while chloroethanol was weakly mutagenic for TA 1530 strain.     

Enhancement of DNA binding and mutagenicity of 1-nitropyrene by zinc acetate in Salmonella typhimurium TA100

The effect of zinc acetate on the mutagenicity of 1-nitropyrene(1-NP) in Salmonella typhimurium TA100 was examined. By thepre-treatment of the cells with zinc ions (0.24 mM) the revertantcolonies caused by 1.5 µM of 1-NP in the pre-incubationmixtures increased {small tilde}3-fold, while the survival coloniesdecreased to {small tilde}75% under the same conditions. Theenhancing effect of zinc ions on the mutagenicity was not dependenton the increase of 1-NP incorporation into the cells. The additionof zinc ions to whole cell preparations had no enhancing effecton the 1-NP reductase activity. The effect of zinc acetate onthe binding of [³H]1-NP to DNA in S. typhimurium TA100 was examined.The amount of [³H]-1-NP bound to DNA increased by the pre-treatmentof the cells with zinc ions. A good correlation was found betweenthe extent of binding of 1-NP to DNA and the frequency of inducedhistidine reversions in the cells treated with zinc ions underthe experimental conditions used.     

Evaluation of the mutagenicity of 1,N6-ethenoadenine- and 3,N4-ethenocytosine-nucleosides in Salmonella typhimurium

To provide supporting evidence for the hypothetical involvement of etheno-derivative formation in DNA in chloroacetaldehyde-mediated mutagenesis, etheno nucleosides were examined for their direct-acting mutagenicity in Salmonella typhimurium strain TA100. The results, however, have shown that 1,N6-ethenoadenosine, 1,N6-ethenodeoxyadenosine, 3,N4-ethenocytidine and 3,N4-ethenodeoxycytidine lack mutagenicity in this test system. A lesson learned in this study is that 3,N4-ethenocytosine nucleosides prepared synthetically or obtained from commercial sources can give false positive mutagenicity due to mutagenic contaminants.     

The effects of bay-region methyl substitution on 6-nitrochrysene mutagenicity in Salmonella typhimurium and tumorigenicity in newborn mice

The mutagenic activities in Salmonella typhimurium and tumorigenicactivities in newborn mice of 6-nitrochrvsene (6-NC), 5-methyl-6-nitrochrysene(5-Me-6-NC), 11-methyl-6-nttrochrysene (11-Me-6-NC) and 5-methylchrysene(5-MeC) were compared. In S.typhimurium TA100 in the absenceof rat liver 9000 g supernatant, 11-Me-6-NC was the most activecompound followed by 6-NC; 5-Me-6-NC and 5-MeC were inactive.In the assays conducted in the presence of rat liver 9000 gsupernatant, the order of activity was 11-Me-6-NC < 6-NC< 5-Me-6-NC {small tilde} 5-MeC. In S.typhimurium TA98 asimilar trend was observed. For the tumorigenicity studies,groups of mice were treated with the appropriate compounds inDMSO by i.p. injections on the 1st, 8th and 15th day of life.At a dose of 100 nmol/mouse 6-NC induced significantly morelung tumors than 5-MeC, which in turn was more active than 11-Me-6-NCand 5-Me-6-NC. All compounds induced significant numbers ofliver tumors in treated males compared to controls; the orderof activity was the same as that observed for lung tumor induction.The results of this study clearly indicate that bay region methylsubstitution can either inhibit (5-position) or enhance (11-position)the mutagenic activity of 6-NC. In contrast, bay region methylsubstitution (5- and 11-positions) inhibited the tumorigenicactivity of 6-NC in newborn mice. Since ring oxidation and nitroreductionare involved in the metabolic activation of 6-NC in newbornmice, bay region methyl substitution may either inhibit thenitroreduction pathway or hinder the formation of the appropriatebay region diol epoxide. Sterk factors may be important in determiningthe tumorigenicity of methylated nitrochrysenes.     

Iron-dependent formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA102 induced by crocidolite

Treatment of isolated DNA with crocidolite asbestos significantlyincreased the concentration of 8-hydroxydeoxyguanosine (8-OHdG)above background. Furthermore, incubating DNA with H₂O₂ andcrocidolite potentiated the formation of 8-OHdG above levelsobserved with crocidolite alone. In the presence of desferrioxamine,desferrioxamine and ferrozine, dimethylsulphoxide (DMSO) oro-phenanthroline, crocidolite-induced DNA oxidation was reducedby 36, 73, 74 and 70% respectively. Crocidolite, but not chrysotileasbestos, enhanced background revertants in Salmonella typhimuriumTA102, at sub-cytotoxic concentrations in a dose-dependent manner.The mutagenic effects of crocidolite were quite small and thisindicates that crocidolite was a weak mutagen in this study.The number of revertants was reduced to the spontaneous ratefor this strain after the fibres had been pretreated with desferrioxaminebefore assaying for genotoxicity in this oxygen radical-sensitivestrain. These results help to explain a mechanistic role foriron in crocidolite-induced DNA oxidation and mutagenicity inTA102.     

The influence of molecular size and partition coefficients on the predictability of tumor initiation in mouse skin from mutagenicity in Salmonella typhimurium

Initiation of tumors in mouse skin is well correlated with mutagenesis in hamster V79 cells (correlation coefficient r = 0.88), but is poorly correlated with bacterial mutagenicity in the Ames assay (r = 0.58). Efforts to understand the difference in the degrees of correlation led to the observation that if the bacterial mutagenicity data are combined with a molecular size or partition factor, the correlation of initiating activity with this combined parameter approaches that found with the hamster cell mutagenesis. The improvement in correlation (r = 0.84) leads to the suggestion that when a broad size range of compounds is considered a most important factor in accounting for the poor correlation initially observed may be a difference between mammalian and bacterial cells in permeability or target access.     

Comparative mutagenicity of dimethylaminoazobenzene and analogues in Salmonella

The mutagen and hepatocarcinogen 4-dimethylaminoazobenzene (DAB)and three of its structural analogues, N-methyl-5-phenylazoindoline,4-N-pyrrolidinylazobenzene and 4'-ethyl-4-N-pyrrolidinylazobenzenewere tested for mutagenicity in Salmonella typhimurium TA1538.DAB was used as a standard and it was found that an S-9 mixcontaining 30% Aroclor-induced rat liver S-9 produced more mutagenicproduct than did an S-9 mix containing the standard 10% S-9.All chemicals tested were mutagenic and required S-9. Both pyrrolidineanalogues produced similar responses to DAB, while the indolinewas only weakly mutagenic.     

Methylene-bridged bay region chrysene and phenanthrene derivatives and their keto-analogs: mutagenicity in Salmonella typhimurium and tumor-initiating activity on mouse skin

A series of methylene-bridged and keto-bridged bay region derivatives of chrysene and phenanthrene were prepared and evaluated for mutagenic activity in Salmonella typhimurium TA100 and for tumor-initiating activity on CD-1 mouse skin. The compounds included in this series were 4H-cyclopenta[def]phenanthrene, 4H-cyclopenta[def]phenanthrene-4-one, 1-methyl-4H-cyclopenta[def]phenanthrene, 1-methyl-4H-cyclopenta[def] phenanthren-4-one, 4H-cyclopenta[def] chrysene, and 4H-cyclopenta[def] chrysen-4-one. Among these compounds only 4H-cyclopenta[def]phenanthrene and 1-methyl-4H-cyclopenta[def]phenanthren-4-one were not significantly mutagenic when assayed with metabolic activation using Aroclor-induced rat liver homogenate. None of the compounds assayed were active without metabolic activation. 4H-Cyclopenta[def]chrysene was the most tumorigenic of the methylene-bridged bay region PAH tested on mouse skin. At a dose of 1.0 mg this compound resulted in 100% of the animals bearing papillomas with 5.63 papillomas/animal. 4H-Cyclopenta[def]chrysen-4-one and 1-methyl-4H-cyclopenta[def]phenanthrene displayed weak tumorigenic activity at a total initiating dose of 1.0 mg.     

Fjord- and bay-region diol-epoxides investigated for stability, SOS induction in Escherichia coli, and mutagenicity in Salmonella typhimurium and mammalian cells

The fjord-region diol-epoxides of benzo(c)phenanthrene combine high mutagenic and carcinogenic activity with low chemical reactivity. To study whether this is a unique property of these compounds or a more general characteristic of fjord-region diol-epoxides, we have synthesized the anti- and syn-diastereomers of r-9,t-10-dihydroxy-11,12-oxy-9,10,11,12-tetrahydrobenzo(c)chrysene and r-11-t-12-dihydroxy-13,14-oxy-11,12,13,14-tetrahydrobenzo(g)chrysene. These compounds as well as the anti- and syn-diastereomers of the fjord-region diol-epoxides of benzo(c)phenanthrene and of the bay-region diol-epoxides of phenanthrene, chrysene, and benzo(a)pyrene were investigated for their half-lives in a physiological buffer, for their mutagenicity in Salmonella typhimurium (reversion of the his- strains TA97, TA98, TA100, and TA104), for induction of SOS response in Escherichia coli (SOS chromotest in strain PQ37) and for their mutagenicity in V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine). All six of the investigated fjord-region diol-epoxides were more stable in physiological buffer at 37 degrees C (t1/2 greater than 2 h) than the six bay-region diol-epoxides (t1/2 = 0.011 to 1.2 h). The half-lives correlated negatively with the calculated delta Edeloc values for the formation of the benzylic carbocations, and were consistently shorter for the syn- than for the corresponding anti-diastereomer. All fjord-region diol-epoxides showed extraordinarily high activity in all six genotoxicity assays used. In mammalian cells, the anti-diol-epoxide of benzo(c)chrysene was 8.6 and 12 times more active than the anti-diol-epoxides of benzo(c)phenanthrene and benzo(a)pyrene, respectively, which were the most potent mutagens among the reference compounds. The other three newly available fjord-region diol-epoxides were also markedly more mutagenic in mammalian cells than the reference compounds. Whereas the syn-diastereomers of the simple bay-region diolepoxides were clearly less mutagenic in mammalian cells than the corresponding anti-diastereomers, the differences in potency between diastereomers were small for the fjord-region diol-epoxides. In conclusion, the diol-epoxides of benzo(c)phenanthrene are not unique in their high biological activities. The two newly available diastereomeric pairs of fjord-region diol-epoxides of benzo(g)- and benzo(c)chrysene proved to be even more active. For one of them, the diol-epoxides of benzo(g)chrysene, the delta Edeloc value for the formation of the benzylic carbocation is lower than for the benzo(c)phenanthrene diol-epoxides, for the other it is higher.     

Therapeutic advantage of genetically engineered Salmonella typhimurium carrying short hairpin RNA against inhibin alpha subunit in cancer treatment

BackgroundIn contrast to its well-known endocrine function, the role of inhibin in cancer development and therapeutic response is unclear. Salmonella, particularly less toxic attenuated Salmonella strains, are used to treat cancer in two ways. First, Salmonella accumulate around tumors, penetrate the cell barrier, and replicate inside the tumors. Second, Salmonella can act as a vehicle for delivering anticancer agents or proapoptotic genes to attack tumors. In this study, we aimed to develop a suitable cancer therapeutic strategy by genetically modifying attenuated Salmonella typhimurium to harbor short hairpin RNA (shRNA) expression plasmids targeting alpha subunit of inhibin (sh-INHA).MethodsWe analyzed the expression of human INHA in normal and cancer cells and tissues. We developed genetically engineered attenuated S. typhimurium harboring sh-INHA (S. typhimurium/sh-INHA) and assessed its cancer therapeutic effects by using cell culture models and syngeneic mouse tumor models.ResultsINHA expression levels were markedly higher in colon cancer and melanoma cells and tissues than in their normal counterparts. Suppression of INHA expression mildly reduced cancer cell survival and induced caspase activation and downregulation of anti-apoptotic Bcl-2 and Bcl-xL expressions. Although the genetically engineered S. typhimurium mildly interfered with the invasion of S. typhimurium into host colon cancer and melanoma cells, S. typhimurium/sh-INHA caused remarkable cytotoxicity in cancer compared with unmodified S. typhimurium or S. typhimurium expressing a control scrambled shRNA (S. typhimurium/sh-Cont). Salmonella typhimurium/sh-INHA-treated mice also showed a significantly inhibited growth of colon cancers and melanomas, with a survival advantage.ConclusionOur results suggest that tumor-targeted therapy using S. typhimurium/sh-INHA may provide a novel cancer treatment option.     

SHORT COMMUNICATION: Glutathione modulates the formation of 8-hydroxydeoxyguannosine in isolated DNA and mutagenicity in Salmonella typhimurium TA100 induced by mineral fibres

Treatment of isolated DNA with crocidolite and man-made vitreousfibre-21 (MMVF-21) significantly increased the concentrationof 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA above background.levels and co-treatment with glutathione (GSH) eliminated thiseffect. Crocidolite, MMVF-21 and chrysolitle fibres increasedthe number of revertants in Salmonella typhimurium TA100 andGSH-deficient strains, TA100/NG-54 and TA100/NG-57, over backgroundlevels. This increase was small in TA100 but was greater inthe GSH-deficient strains. When these bacterial strains werefurther depleted of GSH by co-culture with buthuonine sulfoximine,all fibres tested caused a significant increase in the numberof revertants over the parent strains, Pre-treatment with theGSH precursor N-acetyl-L-cysteine reduced the number of revertantsto below that of the parent strain. Previous studies have showna mechanistic role for iron-catalyzed production of oxygen radicalsin the mutagenicity of fibres and this study suggests a protectiverole for GSH against such oxidative damage possibly by actingas a radical scavenger.     

Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines.

Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl. Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized. Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant. However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase. The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities. In this paper we report the construction and use of new tester strains of S. typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme. These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines.     

The 32P-post-labeling method in quantitative DNA adduct dosimetry of 2-acetylaminofluorene-induced mutagenicity in Chinese hamster ovary cells and Salmonella typhimurium TA1538

The usefulness of the ³²P-post-labeling/t.l.c. method for quantitativeDNA adduct dosimetry was evaluated. 2-Acetylaminofluorene (2-AAF)-DNAadducts from three systems were characterized qualitativelyand quantitatively by the ³H-radiolabeled technique with subsequentanalysis by h.p.l.c. (pre-labeling method) and by the ³²-post-labelingmethod. Both methods showed N-acetoxyacetylaminofluorene (N-OAc-AAF)reaction products with calf thymus DNA were predominantly N-(deoxyguanosm-8-yl)-2-acetylaminofhiorene(dG-C8-AAF) with some N-(deoxyguanosin-8-yl)-2-amino-fluorene(dG-C8-AF) and N-(deoxyguanosin-N²-yl)-2-acetyl-aminofluorene(dG-N2-AAF). In contrast, Chinese hamster ovary (CHO) cellstreated with [³H]N-OAc-AAF gave 80 or 90% dG-C8-AF adducts and20 or 10% dG-C8-AAF adducts with the post- or pre-labeling method,respectively. Likewise in CHO cells treated with 2-AAF in thepresence of rat liver homogenate, {small tilde}90% dG-C8-AFand 10% dG-C8-AAF adducts were detected using the ³²P-post-labelingmethod. In Salmonella typhimurium strain TA1538 treated with2-AAF or [³H]2-AAF in the presence of a rat liver homogenate,one adduct, dG-C8-AF, was identified. Similar quantitative resultswere also obtained with the two methods. However, the ³²P-post-labelingmethod was more sensitive and also eliminated the use of radiolabeled-mutagentreatments. Quantitative DNA adduct dosimetry was applied toAAF-induced mutagenesis in the S. typhimurium and CHO/HPRT mutationassays. A linear and reproducible relationship existed betweendG-C8-AF levels and AAF-induced mutants in both systems.