Modifying role of dietary factors on the mutagenicity of aflatoxin B1: in vitro effect of plant flavonoids

Eighteen flavonoids have been tested for their ability to inhibit the mutagenicity of aflatoxin B1 (AFB1) towards strains TA100 and TA98 of Salmonella typhimurium provided with a rat liver activation system. These flavonoids belong to 5 different groups: flavone, isoflavone, flavanone, flavanol and flavonol, and many individual members are natural products present in edible portions of a variety of food plants. Several flavonoids exhibited significant inhibitory ability in both strains. Flavonols in general are more active in this regard, while flavanones show a strain-specific response. The flavanol group of compounds did not display any activity. Among the most effective flavonoids are kaempferol, morin, fisetin, biochanin A and the glycoside rutin, all of which exhibit a dose-dependent inhibition pattern. Kaempferol and rutin, in particular, show exceptional activity inasmuch as, on a molar basis, only a 10-fold excess dose of each can inhibit the mutagenic activity of AFB1 in strain TA98 by 50%. The action of flavonoids is possibly mediated through interaction with microsomal activating enzymes. Previous evidence from this laboratory about their inhibitory action on DNA-adduct formation and metabolic activation together with the present results suggests that certain flavonoids, notably polyhydroxylated flavonols, may have potential anticarcinogenic activity against AFB1.     

Antimutagenic activity of green tea polyphenols

For centuries green tea has been a widely consumed beverage throughout the world. It is known to contain a number of pharmacologically active compounds. In this study water extracts of green tea (WEGT) and their major constituents, green tea polyphenols (GTP), were examined for antimutagenic activity. WEGT and GTP were found to significantly inhibit the reverse mutation induced by benzo[alpha]pyrene (BP), aflatoxin B1 (AFB1), 2-aminofluorene, and methanol extracts of coal tar pitch in Salmonella typhimurium TA100 and/or TA98 in the presence of a rat-liver microsomal activation system. GTP also inhibited gene forward mutation in V79 cells treated with AFB1 and BP, and also decreased the frequency of sister-chromatid exchanges and chromosomal aberrations in V79 cells treated with AFB1. The addition of GTP during and after nitrosation of methylurea resulted in a dose-dependent inhibition of mutagenicity. Studies to define the mechanism of the antimutagenic activity of GTP suggest that it may affect carcinogen metabolism, DNA adduct formation, the interaction of ultimate carcinogen or the scavenging of free radicals.     

Effect of phenolic antioxidants on the mutagenicity of aflatoxin B1.

The Ames assay employing Salmonella typhimurium TA100 and TA98 was used to investigate potential interactions between aflatoxin B1 (AFB1) and the phenolic antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate. AFB1 doses were within the linear response range, and the antioxidants were used at levels of 0 to 50 micrograms per plate. All three antioxidants were nonmutagenic in either bacterial tester strain, with or without the hepatic S-9 enzyme preparation; toxic effects were observed at doses higher than 20 micrograms per plate. Butylated hydroxytoluene and butylated hydroxyanisole substantially increased AFB1-induced mutagenesis in the two tester strains with microsomal activation. The addition of 5 to 20 micrograms of butylated hydroxytoluene or hydroxyanisole to 5 to 20 ng of AFB1 per plate caused more than a twofold increase in the number of His+ revertants. Addition of propyl gallate resulted in only a moderate increase in the number of revertants. Whereas several anticarcinogenic and antimutagenic effects by phenolic antioxidants have been reported, particularly in studies with polycyclic aromatic hydrocarbons, the enhancement of mutagenic potency of AFB1 by these compounds suggests a specificity with respect to the chemical nature of AFB1.     

Hormonal carcinogenesis: separation of estrogenicity from carcinogenicity

Forty compounds belonging to various chemical groups have been tested for their ability to suppress metabolic activation of aflatoxin B1 (AFB1) mediated by rat liver microsome. Microsomal activation has been carried out in an in vitro system containing Tris-buffer at pH 7.2. Production of the reactive metabolite, AFB1 8,9-epoxide, has been measured by separation and detection of its hydrolysis product AFB1 8,9-dihydrodiol as the Tris-diol complex. The complex is separated using simple procedures of biphasic extraction and deproteinization, and detected by its characteristic fluorescence. Quantitation is made by direct comparison of its fluorescence with that of an authentic Tris-diol prepared synthetically. The method is rapid and proved to be highly sensitive and reproducible. A large number of compounds have been observed to modulate at varying degrees the activation of AFB1 in this in vitro system. Many compounds have been tested at several concentration ranges and inhibition curve is constructed in each case from which ID50 values, i.e., the dose needed to bring about 50% inhibition can be obtained. These values expressed as nmol afford a direct and realistic comparison of the inhibitory potential of various modulators. Factors having great inhibitory potential have been identified as retinoids (retinol, retinal, retinoic acid, retinyl acetate), beta-carotene, riboflavin, ascorbic acid, copper, zinc, linoleic acid, p-hydroxy benzoic acid, butylated hydroxyanisole, butylated hydroxytoluene, disulfiram, and phenothiazine. Several other compounds have shown moderate inhibitory potential. The strong inhibition on Tris-diol formation by several vitamins, antioxidants and trace metals shows similarity with their effect on AFB1-DNA adduct formation. It is suggested that these agents may have potential anticarcinogenic activity against AFB1.     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.     

Modifying role of dietary factors on the mutagenicity of aflatoxin B1: in vitro effect of sulphur-containing amino acids

Sulphur-containing amino acids including some derivatives have been tested for their effectiveness in suppressing the mutagenic activity of aflatoxin B1 in Salmonella typhimurium strains provided with a rat liver activation system. Cysteine and N-acetylcysteine have been found to be most effective in the 2 strains tested (TA100 and TA98). Glutathione (oxidised and reduced forms) has shown partial activity, while cystine and methionine are found to be partially effective only in strain TA100. Inhibition of mutagenicity may be due to interaction of these substances with microsomal enzymes resulting in interference with the formation of ultimate mutagenic species.     

Antimutagenic effect of eight natural foods on moldy foods in a high liver cancer incidence area.

Ames test procedures were used to test 8 natural food extracts for their antimutagenic activity against the mutagenic activity induced in S. typhimurium strains TA98 and TA100 by aflatoxin B1 (AFB1) or metabolic extracts from A. versicolor or A. ochraceus. The tested substances were extracted repeatedly with acetone. The revertants induced by AFB1, metabolic extracts of A. versicolor or A. ochraceus were significantly decreased when extracts of the 8 natural foods were added to the media. The results showed that these extracts had marked inhibitory effects on the mutagenic activity induced by AFB1 or metabolic extracts of the two molds and also suggested that antimutagenic substances were present in these natural foods. These experiments provide a scientific basis for the study of food substances for the prevention of carcinogenesis. It is considered that these 8 natural food extracts produce marked antimutagenic effects and are practically valuable in the field of chemoprophylaxis of liver cancer in humans.     

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H(2)O(2)), sodium azide (NaN(3)), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H(2)O(2), BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN(3) in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames' test, as well as that of the cytosolic enzyme GST. A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Chemical investigation of different extracts and essential oil from the tubers of (Tunisian)Cyperus rotundus. Correlation with their antiradical and antimutagenic properties

The mutagenic potential of aqueous, Total Oligomers Flavonoids (TOF), ethyl acetate, and methanol extracts as well as essential oil (EO) obtained from tubers ofCyperus rotundus L. was assessed by “Ames assay”, usingSalmonella tester strains TA98 and TA100, and “SOS chromotest” usingEscherichia coli PQ37 strain with and without an exogenous metabolic activation system (S9). None of the different extracts showed a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed thatC. rotundus extracts have antimutagenic effects withSalmonella typhimurium TA98 and TA100 strains towards the mutagen Aflatoxin B1 (AFB1), as well as withE. coli PQ37 strain against AFB1 and nifuroxazide mutagens. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers ofC. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. These extracts showed IC50 values of respectively 5, 20 and 65 μg/ml. The beneficial effects of TOF, ethyl acetate, methanol and essential oil extracts ofC. rotundus have been assessed by antioxidant and antimutagenic activities.     

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.     

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1.

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors while Astragalus membranaceus (AM) and Ligustrum lucidum (LL) are often used as an adjunct in cancer therapy. In this study, we determined the effects of aqueous extracts of these four herbs on aflatoxin B1 (AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant as the activation system. The effects of these herbs on [3H]AFB1 binding to calf-thymus DNA were assessed. Organosoluble and water-soluble metabolites of AFB1 were extracted and analyzed by high-performance liquid chromatography (HPLC). Mutagenesis assays revealed that all of these herbs produced a concentration-dependent inhibition of histidine-independent revertant (His+) colonies induced by AFB1. At a concentration of 1.5 mg/plate, SB and OD in combination exhibited an additive effect. The trend of inhibition of these four herbs on AFB1-induced mutagenesis was: SB greater than LL greater than AM. LL, OD and SB significantly inhibited AFB1 binding to DNA, reduced AFB1-DNA adduct formation, and also significantly decreased the formation of organosoluble metabolites of AFB1. Our data suggest that these Chinese medicinal herbs possess cancer chemopreventive properties.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H₂O₂), sodium azide (NaN₃), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H₂O₂, BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN₃ in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames’ test, as well as that of the cytosolic enzyme GST.A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3 mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3 mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Comparison of the prevention of aflatoxin b(1)-induced genotoxicity by quercetin and quercetin pentaacetate

Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing the genotoxicity of aflatoxin B(1) (AFB(1)). We have in this report extended the observations to quercetin pentaacetate (QPA), which unlike quercetin (Q) has demonstrated time-dependent inhibition of liver microsome catalysed AFB(1) epoxidation as measured by AFB(1) binding to DNA. The action of QPA is similar to that of the acetoxy 4-methylcoumarins in that they are acted upon by microsomal transacetylase leading to modulation of catalytic activities of certain enzymes (such as P-450 enzymes, NADPH cytochrome C reductase and glutathione S-transferase) possibly by way of protein acetylation. In the present work, we have documented the transacetylase-mediated action of QPA in preventing genotoxicity due to AFB(1).     

The interaction of divalent copper and the microsomal electron transport system. A re-examination of the effects of copper chelates on the function of cytochrome P-450.

Lipophilic chelates of divalent copper, possessing superoxide dismutase-like activity, have been proposed to enhance the decay of oxycytochrome P-450 to explain their inhibitory effect on microsomal mixed-function oxidation reactions (Richter, C., Azzi, A., Weser, U., and Wendel, A. (1977) J. Biol. Chem. 252, 5061-5066). The present investigation, however, failed to provide evidence in favor of this hypothesis. In particular, it was found that the reported inhibition of cytochrome P-450-catalyzed hydroxylation reactions by copper-tyrosine is associated with an inhibition rather than a stimulation of the formation of hydrogen peroxide, the product of the dismutation of the superoxide radicals generated as a result of the decay of oxycytochrome P-450. The attenuation of both these reactions was shown to be the consequence of an impaired function of the NADPH-cytochrome P-450 reductase. Additional sites of interaction of copper chelates and the microsomal electron transport system appear to exist since divalent copper was found to undergo reduction reactions with NADPH and NADH as electron donors. These reduction reactions do not involve superoxide radicals and, therefore, are unrelated to the ability of copper chelates to function in a superoxide dismutase-like manner.     

Mutagenic activity of furfural in Salmonella typhimurium TA100.

The mutagenic activity of furfural was tested in Salmonella typhimurium strains TA98 and TA100. Furfural produced mutations in the TA100 strain, but not in the TA98 strain. A rat-liver microsomal fraction did not increase the mutagenic activity of furfural in either strain. Mutagenic activity of furfural in the TA100 strain was not increased by benzo[alpha]pyrene in the presence of metabolic activation.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Mutagenicity of aminophenyl and nitrophenyl ethers, sulfides, and disulfides.

The mutagenic activity of several aromatic amines and aromatic nitro compounds related to 4,4'-methylenedianiline towards Salmonella typhymurium tester strains TA100 and TA98 was evaluated. The heteroatomic analogs of 4,4'-methylenedianiline which include aminophenyl and nitrophenyl ethers, sulfides and disulfides were assayed in the presence of rat-liver homogenate. The relative mutagenic response of these analogs indicated the following order of activity, --S-- greater than --O-- greater than --CH2--CH2-- greater than or equal to --S--S--. In both tester strains 4-aminophenylsulfone was inactive with and without microsomal activation. The p-nitrophenyl ethers, sulfides and disulfides were relatively strong mutagens without microsomal activation towards TA100. While 4-nitrophenyldisulfide was found to possess significantly different mutagenic activity than 4-nitrothiophenol in TA98, 4-AMINOPHENYl disulfide has similar mutagenic properties to 4-aminothiophenol in both tester straains TA100 and TA98.