The mutagenic activity of 4-chloromethylbiphenyl (4CMB) and Benzyl Chloride (BC) in the bacterial/microsome assay

The mutagenic activity of 4CMB was investigated in agar layer cultures of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, and Escherichia coli WP2 and WP2 uvrA. The mutagenic activity of BC was investigated in the Salmonella strains only. Assays were performed both in the absence and in the presence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.     

Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

Arylamine activation following chronic ethanol ingestion by rats: studies on the liver S9, microsomal and cytosolic fractions and comparison with Aroclor 1254 pretreatment

That enzyme fractions derived from animals chronically fed alcohol can alter the metabolism of carcinogenic xenobiotic compounds has been documented. To further understand this relationship the mutagenicity of 3 aromatic amines was determined in the Ames test, employing activation systems derived from rats maintained on an alcohol-containing liquid diet, an isocaloric control liquid diet or Aroclor 1254-pretreated animals fed standard laboratory chow. Depending upon protein and substrate concentrations, S9 from ethanol-fed rats was 30-50% less efficient than S9 from pair-fed rats in activating arylamines (2-aminofluorene, 2-aminoanthracene and 2-acetylaminofluorene) to mutagens in Salmonella typhimurium TA98 and TA100. Cytosolic fractions from ethanol-fed animals always resulted in greater arylamine activation than that of controls whereas the opposite was true of the microsomal compartment in which the ethanol-treated group was consistently less active than the controls. The cytosolic N-acetyltransferase activities with respect to 2 different substrates, isoniazid and 2-aminofluorene, were unaffected by ethanol consumption, indicating that this activity probably does not account for the different activation profiles exhibited by the ethanol and pair-fed cytosolic systems. Both the cytosolic and microsomal compartments are required for maximal expression of the mutagenicity of each arylamine however, each compartment can activate arylamines independently of the other. Reconstituting cytosol with microsomes from ethanol- and pair-fed rats, but not Aroclor-pretreated rats, resulted in a synergistic activation of the aromatic amines and displayed an effect similar to that of S9. Compared to Aroclor pretreatment and pair-fed controls, microsomes from ethanol-fed rats displayed the least capacity for activating any of the arylamines to mutagens. Microsomes from Aroclor-pretreated rats accounted for at least 80% of the S9-mediated activation of each of the arylamines to mutagenic metabolites which was in marked contrast to the contribution of the microsomal fractions to the S9 activity in the ethanol- (5-20% of S9 activity) and pair-fed systems (22-30% of S9 activity). The data indicate that 2 opposing reactions occur in S9, a cytosolic activity that augments and a microsomal activity that attenuates the mutagenicity of arylamines. Both activities are modified by ethanol consumption and Aroclor pretreatment.     

Mutagenicity of diphenylhydantoin and some of its metabolites towards Salmonella typhimurium strains

The mutagenicities of 5,5-diphenylhydantoin (DPH) and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were tested in vitro using different Salmonella strains (TA1535, TA100, TA1537, TA1538, TA98). Experiments were carried out at various concentrations in the absence and in the presence of an activating system consisting of hepatic S9 fraction from control rats and from rats pretreated with phenobarbital (PB), β-napthoflavone (BNF), 3-methylcholanthrene (3-MC) and Aroclor 1254 (PCB).DPH slightly increased the number of revertants per plate only after incubations with TA1538 in the presence of the S9 fraction from the liver of 3-MC- and PCB-pretreated animals. A similar but more significant frameshift mutation was observed for HPPH on both TA98 and TA1538 strains and in conditions of metabolic activation by the liver microsomal fractions of rats after pretreatment with BNF, 3-MC and especially PCB.Parallel experiments on the metabolism of DPH to HPPH and of HPPH to the catechol derivative in vitro support the hypothesis of an involvement of epoxide intermediates in the mutagenic activity of DPH.     

Non-mutagenicity of toluene,o-, m- and p-xylene,o-methylbenzylalcohol and o-methylbenzylsulfate in the Ames assay

Toluene, o-, m- and p-xylene, o-methylbenzylalcohol and o-methylbenzylsulfate were assayed for mutagenicity in the Ames assay. These compounds were unable to revert Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, either with or without metabolic activation by S9 mix derived from livers of rats either untreated or induced with Aroclor 1254.     

delta 1-Tetrahydrocannabinol and 1 alpha, 2 alpha-epoxyhexahydrocannabinol: mutagenicity investigation in the Ames test.

1,2-Epoxyhexahydrocannabinol is a metabolite of delta 1-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as delta 1-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by delta 1-tetrahydrocannabinol were found.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.     

Cytosolic activation of 2-aminoanthracene: implications in its use as diagnostic mutagen in the Ames test.

The metabolic activation of 2-aminoanthracene to mutagens in the Ames test was investigated using hepatic S9, microsomal and cytosolic fractions from control and Aroclor 1254-treated rats as activation systems. Microsomal and S9 preparations from control animals could activate 2-aminoanthracene, but the efficiency of activation was suppressed by pretreatment of animals with Aroclor 1254. Cytosolic fractions from Aroclor 1254-treated rats could readily activate the promutagen more readily than microsomes. The cytosolic activation of 2-aminoanthracene required NADPH and could not be accounted for by possible microsomal contamination. The molybdenum oxygenases appear not to contribute to the cytosolic activation of this promutagen. It is concluded that (a) the microsomal activation of 2-aminoanthracene is catalysed more effectively by enzyme systems other than the P450 I family and (b) an enzyme system capable of activating this carcinogen in vitro is present in the hepatic cytosol. The implications of these findings in the use of 2-aminoanthracene as a positive control in the Ames test are discussed.     

Genotoxic activity of m-nitrobenzaldehyde

m-Nitrobenzaldehyde (MNB) was evaluated for mutagenic activity using the Ames microbial mutagenicity test and for its ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. MNB was tested in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without pretreatment with liver microsomes (S9) isolated from rats pretreated with Aroclor 1254. MNB produced 2-fold or greater increases in revertants in TA1538, both with and without S9, and in TA100 with S9 only. A 2-fold increase in revertants was seen in TA98, but only at the highest dose tested which did not produce inhibition of background growth. MNB caused a greater than 3-fold increase in elution slope, with DNA alkaline elution assay, but only at highly cytotoxic doses and, therefore, is not considered genotoxic in this system. It is concluded that MNB possesses weak genotoxic activity.     

Preferential activation of 6-aminochrysene and 2-aminoanthracene to mutagenic moieties by different forms of cytochrome P450 in hepatic 9000 X g supernatants from the rat

6-Aminochrysene and 2-aminoanthracene were activated to metabolites which were mutagenic to Salmonella typhimurium TA98 by hepatocytes or hepatic 9000 X g supernatants (S9s) from control or xenobiotic-treated rats. Hepatocytes from Aroclor-1254-treated rats were more efficient than hepatocytes from untreated rats at activating these aromatic amines. When plate-incorporation and liquid-incubation bacterial mutagenesis assays were performed in the presence of limiting amounts of rat hepatic S9, 2-aminoanthracene was activated to a greater extent in both cases, as judged by his+ revertant formation, by 3-methylcholanthrene-induced hepatic S9 than by phenobarbital-induced or control S9s. In contrast, 6-aminochrysene was activated more efficiently by phenobarbital-induced S9 than by 3-methylcholanthrene-induced or control S9s. This unexpected finding was confirmed employing polyclonal antibodies directed against specific forms of rat cytochrome P450. Thus, when employing Aroclor-1254-induced S9 as a source of metabolic activation, antibody directed against cytochrome P450IA1 inhibited the activation of 2-aminoanthracene but not of 6-aminochrysene. In contrast, antibody directed against cytochrome P450IIB1 inhibited the activation of 6-aminochrysene but not of 2-aminoanthracene. These results suggest that under conditions in which the amounts of S9 added are rate-limiting, the two aromatic amines are preferentially activated by different induced forms of cytochrome P-450.     

Modulation of the mutagenicity of three dinitropyrene isomers in vitro by rat-liver S9, cytosolic, and microsomal fractions following chronic ethanol ingestion.

The effects of chronic ethanol feeding of rats on the ability of liver fractions to modulate the bacterial mutagenicity of three dinitropyrene isomers (1,3-, 1,6- and 1,8-DNP), which require bacterial enzymes but not an exogenous enzyme source for activation, were studied. The mutagenicity of the DNP isomers toward S. typhimurium TA98 and TA100 was attenuated in the presence of post-mitochondrial supernatants (S9) from both ethanol-fed and pair-fed rats albeit, that from the ethanol-fed group was more efficient in lowering the mutagenicity. The cytosolic fraction from ethanol-fed rats enhanced the mutagenicity of all of the DNP isomers in TA100. The most notable enhancement was with 1,3-DNP in which a more than 4-fold enhancement was obtained. Cytosol from pair-fed rats enhanced only the mutagenicity of 1,3-DNP, this by 2.9-fold. Cytosolic NADPH-nitroreductase activity from ethanol-treated rats toward 1,6-, 1,8- and 1,3-DNP was increased 2.8-, 1.7- and 1.3-fold, respectively over pair-fed controls. Cytosolic NADH-nitroreductase from ethanol-fed rats was increased with 1,3-DNP (1.7-fold) and 1,8-DNP (1.4-fold) as substrates, but not with 1,6-DNP. Microsomes decreased the mutagenicity of DNP similarly to S9, i.e., fractions from ethanol-fed rats were more efficient than those of pair-fed rats in deactivating all the DNP isomers. Per mg of protein, detoxification of DNP by S9 was more efficient than with microsomes, thus both cytosolic and microsomal enzymes are required for maximal detoxification. In summary, ethanol feeding modulates both the augmented cytosolic activation of DNP to mutagens and the deactivation of the direct-acting mutagenicity of DNP by microsomes. In combination, as is the case with S9, the microsomal detoxifying activity outcompetes the cytosolic activation.     

Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources.

Over 70 naturally occurring and synthetic flavonoids were screened for mutagenicity with 5 tester strains in the Salmonella/mammalian microsome assay: TA1535, TA100, TA1537, TA1538 and TA98. Frameshift mutagenicity was confined to the flavonols (flavon-3-ols) in strain TA98, TA1537 and TA100. The two most mutagenic falvonols, namely, quercetin (3,3',4',5,7-pentahydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone), exhibiting 12 and 7 revertants/nmol in TA98 respectively, are also the most common flavonols occurring in plants. Other flavonols exhibited less activity (revertants/nmol): galangin (2.0), rhamnetin (0.45), kaempferide (0.24), fisetin (0.14), myricetin (0.12), robinetin (0.06) and morin (0.05). All of these flavonols apparently exhibited significant activation by Aroclor 1254 induced rat-liver microsome preparations (S9). However, subsequent study revealed that only those flavonols either lacking or possessing one B ring hydroxyl group had an absolute requirement for microsomal activation. Alternatively, quercetin with two B-ring OH groups is not activated by microsomal enzymes, but by soluble (S100) enzymes from liver which are apparently constitutive and not subject to the usual chemical induction. 3 flavonol glycosides, namely, quercetrin (quercetin-3-O-rhamnoside), rutin (quercetin-3-O-rutinoside) and robinin (kaempferol-3-O-galactosido-rhamnoside-7-O-rhamnoside), were found to be nonmutagenic. They could, however, be activated by a variety of mixed glycosidases incorporated in the usual pour plate procedure. The most effective enzyme mixtures were obtained from rat cecal bacteria and from the snail Helix pomatia.     

The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetate and methyl tertiary-butyl ether in Salmonella typhimurium

Tertiary-Butyl alcohol (TBA), tertiary-butyl acetate (TBAc) and methyl tertiary-butyl ether (MTBE) are chemicals to which the general public may be exposed either directly or as a result of their metabolism. There is little evidence that they are genotoxic; however, an earlier publication reported that significant results were obtained in Salmonella typhimurium TA102 mutagenicity tests with both TBA and MTBE. We now present results of testing these chemicals and TBAc against S. typhimurium strains in two laboratories. The emphasis was placed on testing with S. typhimurium TA102 and the use of both dimethyl sulphoxide and water as vehicles. Dose levels up to 5000 microg/plate were used and incubations were conducted in both the presence and absence of liver S9 prepared from male rats treated with either Arochlor 1254 or phenobarbital-beta-naphthoflavone. The experiments were replicated, but in none of them was a significant mutagenic response observed, thus the current evidence indicates the TBA, TBAc and MTBE are not mutagenic in bacteria.     

The induction of mitotic gene conversion in the yeast,Saccharomyces cerevisiae JD1 by 4-chloromethylbiphenyl (4CMB), Benzyl Chloride (BC) and 4-hydroxymethylbiphenyl (4HMB)

The induction of mitotic gene conversion by 4CMB, BC and 4HMB was studied in both log-phase and stationary-phase cultures of the yeast, Saccharomyces cerevisiae JD1. Assays were performed both in the presence and in the absence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.Exposure of both stationary-phase and log-phase cultures to 4CMB and BC resulted in an increase in mitotic gene conversion, both in the presence and in the absence of a microsomal activation system; the magnitude of response was greater in stationary-phase cultures. 4HMB did not increase the gene conversion frequency in log-phase or stationary-phase cultures.     

Activation of the food mutagens IQ and MeIQ by hepatic S9 fractions derived from various species

The ability of hepatic S9 mixes derived from different rodent species (rat, mouse, Syrian and Chinese hamster) to activate the mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was investigated using Salmonella typhimurium strain TA98. In general, the mutagenicity of IQ and MeIQ was greatest in the presence of S9 fractions from Swiss albino mice and least from fractions derived from Chinese hamsters. However, treatment of rats or hamsters with Aroclor 1254 had little or no effect on the activation of IQ or MeIQ to mutagens.     

Ex vivo modulation of chemical-induced mutagenesis by subcellular liver fractions of rats treated with rooibos (Aspalathus linearis) tea, honeybush (Cyclopia intermedia) tea, as well as green and black (Camellia sinensis) teas

Male Fischer rats were given unprocessed (not oxidized) and processed (oxidized) rooibos and honeybush teas as well as green and black teas as a sole source of drinking fluid for 10 weeks, and sub cellular liver fractions were prepared. Cytosolic fractions of rats consuming the unprocessed herbal teas, green and black teas significantly (P < 0.05) protected against 2-acetylaminofluorene (2-AAF)-induced mutagenesis in the Salmonella mutagenicity test with strain TA 98, using Aroclor 1254-induced microsomes. A marginal or no protection was obtained with the processed herbal teas. The mutagenic response of aflatoxin B1 (AFB1) against Salmonella strain TA 100 was significantly (P < 0.05) inhibited by cytosolic fractions from rats treated with processed and unprocessed herbal teas, while no effect was obtained with the green and black teas. Microsomal fractions prepared from livers of rats treated with both the processed and unprocessed rooibos teas and the unprocessed honeybush tea, significantly (P < 0.05) reduced the activation of AFB1 while no protection was observed against 2-AAF-induced mutagenesis. In contrast, microsomal fractions from rats treated with the green, black and unprocessed honeybush teas significantly (P < 0.05) enhanced the mutagenic response of 2-AAF. None of the tea treatments significantly affected the concentration of the microsomal liver cytochrome P450.     

Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by Aroclor 1254 or phenobarbital

The constitutive and Aroclor 1254-induced activities of hepatic microsomal benzo[a]pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11-day-old noninduced male rats, benzo[a]pyrenediones and 9-hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21-day-old males benzo[a]pyrene-diones and benzo[a]pyrene-9,10-dihydrodiol were predominant. In 60- and 120-day-old animals 3-hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5-dihydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21-day-old immature male rats, in which there was a 330- and 4.5-fold increase in the formation of 3-hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3- to 10.1-fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 mumol/kg) or Aroclor 1254 (100 mumol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120-day-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphenyl and fluorinated derivatives: liver enzyme-mediated mutagenicity detected in Salmonella typhimurium and Chinese hamster V79 cells.

Hepatocarcinogenic polychlorinated and polybrominated biphenyls usually show negative results in in vitro mutagenicity assays. Problems in their testing result from their low water solubility and their slow rate of metabolism. We therefore investigated better soluble model compounds, namely biphenyl and its 3 possible monofluorinated derivatives. In the direct test, these compounds proved to be nonmutagenic in Salmonella typhimurium TA98 and TA100 (reversion to histidine prototrophy) and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). However, when the exposure was carried out in the presence of NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats, all 4 compounds showed mutagenic activity in V79 cells. 3-Fluorobiphenyl produced strong mutagenic effects in S. typhimurium TA100 as well, whereas the other biphenyls were inactive. In strain TA98, 3- and 4-fluorobiphenyl showed mutagenic activity. This mutagenicity was enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase, thus suggesting that epoxides may be active metabolites.     

Studies on the mutagenicity of p-phenylenediamine in Salmonella typhimurium. Presence of PCB's in rat-liver microsomal fraction induced by Aroclor.

The mutagenicity of fresh solutions of p-phenylenediamine (PPD) and Aroclor 1254 was investigated. The histidine-requiring strains of Salmonella typhimurium were used in the absence and presence of uninduced and/or Aroclor-induced rat-liver homogenate. The presence of polychlorinated biphenyls (PCBs) was also examined by chromatographic methods in Aroclor-induced rat-liver homogenate. In the absence of metabolic activation, as well as in the presence of uninduced rat-liver homogenate, PPD was not mutagenic in the strains used. In the presence of Aroclor-induced S9 a twofold increase (or less) was observed in the number of revertant colonies over those of the controls in TA1538 and TA98. There was no increase in the number of revertant colonies over those of the controls when PPD was dissolved in NH4OH solution and the solution mixed with H2O2 before the addition of S9 mix. Aroclor 1254 was not mutagenic in TA1538 or TA98. However, the presence of PCBs in Aroclor-induced rat-liver homogenate (induced S9) was identified by gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) and gas--liquid chromatography/mass spectrometry (GC/MS).     

1-Hydroxypyrene as an indicator of the mutagenicity of coal tar after activation with human liver preparations

Liver S9 fractions were prepared from male Wistar rats, either non-induced or induced with Aroclor 1254 and from 5 human kidney transplant donors. The preparations were compared for their ability to metabolize the premutagens present in coal tar to mutagenic metabolites in the Salmonella mutagenicity assay towards strain TA98. Low levels of mutagenicity of coal tar were seen with human S9 preparations. The differences between the S9 mix of the 5 donors in capacity to activate premutagens were approximately 6-fold. The activation of coal tar by rat liver S9 preparations was higher than by the human S9 preparations. The metabolic conversion of pyrene in coal tar to 1-hydroxypyrene by the same human S9 preparations was determined in a parallel assay. 3 human preparations showed a high correlation between the formation of 1-hydroxypyrene and bioactivation of coal tar to mutagenic metabolites. The slope values of the individual regression lines were equal, suggesting that 1-hydroxypyrene is a good indicator for the activation of premutagens present in coal tar.