Mutagenicity of the phenacetin metabolites: N-hydroxy-p-phenetidine and nitrosophenetol in S. typhimurium TA100 and derivatives deficient in nitroreductase or O-acetylase: probes for testing intrabacterial metabolic activation

Two mutagenic metabolites of phenacetin, p-nitrosophenetol and N-hydroxy-p-phenetidine, were tested in S. typhimurium strains TA100, its nitroreductase-deficient derivative TA100NR, and O-acetylase-deficient strains TA100 Tn5-1-8-DNP1011 and -DNP1012 in the presence or absence of an exogenous metabolic activation system. The results indicate that bacterial nitroreductase(s) and O-acetylase(s), shown to be involved in the conversion of certain nitroarenes, are not required for the intrabacterial activation of the two phenacetin metabolites to bacterial mutagens. In view of the low reactivity of nitrosoarenes towards nucleophiles at neutrality, the mechanism by which they exert such a high mutagenic effect in S. typhimurium strains remains to be clarified, but is discussed.     

Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated 'classical' nitroreductase or acetyl-CoA:N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.     

Mutagenicity in Salmonella typhimurium of some angelicin derivatives proposed as new monofunctional agents for the photochemotherapy of psoriasis

A series of angelicin derivatives were tested for their mutagenic activity with and without near-ultraviolet irradiation (NUV) in Salmonella typhimurium strains. After irradiation with NUV, the tested compounds induced different numbers of revertants in strain TA100, indicating that the mutational events involved are base substitutions. In the dark, 3 chemicals behaved as frame-shift mutagens causing reversion in strain TA98.     

Intrasanguine host-mediated assay with Salmonella typhimurium.

A host-mediated assay in the mouse was tested, in which strains of S. typhimurium (TA 98, TA 1535) were used as indicator organisms and administered intrasanguinally. The bacterial suspension was injected intravenously at a cell density of 10¹¹/ml in a volume of 0.2 ml. The test substances were administered three times at intervals of one hour, orally, intraperitoneally or subcutaneously, the last dose being given immediately before the injection of the indicator organisms. The bacteria were re-isolated one hour later from the liver, and the total bacterial counts and mutation rates were determined. The mutagenic activity of the substances was assessed by reference to the quotients of the mutation rates in the various dosage groups over the control rate. The compounds tested were diethylnitrosamine, cyclophosphamide, dimethylaminoazobenzene, thiotepa and EMS.The bacterial recovery rates in the controls and treated groups ranged from 2.72 to 23.5%, which proved entirely adequate. All the known mutagens tested caused a measurable mutagenic effect in this assay.Comparison of the results with already published data reveals that the intrasanguine host-mediated assay is more sensitive than the intraperitoneal assay system, and that the chosen strains of S. typhimurium are well suited for this method.     

Further mutagenicity studies on pesticides in bacterial reversion assay systems

A total of 228 pesticides (88 insecticides, 60 fungicides, 62 herbicides, 12 plant-growth regulators, 3 metabolites and 3 other compounds) was tested for mutagenicity in bacterial reversion-assay systems with 5 strains (TA100, TA98, TA1535, TA1537 and TA1538) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli. 50 pesticides (25 insecticides, 20 fungicides, 3 herbicides, 1 plant-growth regulator and 1 other compound) were found to be mutagenic. 5 of them required metabolic activation (S9 mix) for their activities. Among various chemical groups, organic phosphates, halogenated alkanes and dithiocarbamates showed higher ratios of mutagens. Although 22 of the pesticides tested have been reported to be carcinogenic, 7 of them, i.e., captain, DBCP, EDB, EDC, ETU, HEH and nitrofen, were detected as mutagens in the present assay. Most of the other 15 non-mutagenic carcinogens were organochlorine pesticides such as alpha-BHC, chlorobenzilate, p,p'-DDT, dieldrin and quintozene.     

Bacterial beta-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.     

Mutagenicity of aliphatic epoxides

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity. trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food

Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate. In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S. typhimurium. However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC. More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens. Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate. These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food.     

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.     

The bacterial mutagenicity of nitropolychlorinated dibenzo-p-dioxins

The bacterial mutagenicity of 2-nitrodibenzo-p-dioxin, a mixture of 2-nitro-7-chloro- and 2-nitro-8-chlorodibenzo-p-dioxin, 7-nitro-2,3-dichloro-, 8-nitro-2,3,7-trichloro-, 2-nitro-1,3,7,8-tetrachloro- and 3-nitro-1,2,4,7,8-pentachlorodibenzo-p-dioxin was determined using Salmonella typhimurium tester strains TA98 and TA100 with and without rat hepatic S9 for metabolic activation. All the nitro-PCDDs exhibited some direct-acting mutagenicity with both tester strains, however, the activity was significantly lowered in the presence of exogenous S9 and the compounds were more mutagenic to tester strain TA98. The mutagenicity of the nitro-PCDDs was also dependent on structure because there was a marked decrease in activity with increasing chlorine content. Because nitro-PCDDs have recently been identified as incomplete combustion products of municipal waste, this study confirms that this new class of compounds contains some bacterial mutagens.     

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.     

The anti-mutagenic and antioxidant effects of bile pigments in the Ames Salmonella test

The aim of this study was to explore the potential pro- and anti-mutagenic effects of endogenous bile pigments unconjugated bilirubin (BR), biliverdin (BV) and a synthetic, water soluble conjugate, bilirubin ditaurate (BRT) in the Ames Salmonella test. The bile pigments were tested over a wide concentration range (0.01-2 micromol/plate) in the presence of three bacterial strains (TA98, TA100, TA102). A variety of mutagens including benzo[alpha]pyrene (B[alpha]P), 2,4,7 trinitrofluorenone (TNFone), 2-aminofluorene (2-AF), sodium azide (NaN(3)) and tertiary-butyl hydroperoxide (t-BuOOH), were used to promote the formation of mutant revertants. Tests were conducted with (B[alpha]P, 2-AF, t-BuOOH) and without (TNFone, NaN(3), t-BuOOH) metabolic activation incorporating the addition of the microsomal liver preparation, S9. The bile pigments alone did not induce mutagenicity in any of the strains tested (p>0.05). Anti-mutagenic effects of the bile pigments were observed in the presence of all mutagens except for NaN(3) and the anti-mutagenic effects appeared independent of the strain tested. For TNFone induced genotoxicity, the order of effectiveness was BR> or =BRT>BV. However, the order was BV> or =BRT> or =BR for 2-AF. Antioxidant testing in the TA102 strain revealed bile pigments could effectively inhibit the genotoxic effect of t-BuOOH induced oxidative stress. The apparent antioxidant and anti-mutagenic behaviour of bile pigments further suggests their presence in biological systems is of possible physiological importance.     

Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the "Ames" tester strains

The standard Ames tester strains of Salmonella typhimurium contain a number of genetic differences at loci other than his. The fact that these strains contain independently isolated uvrB-bio-gal deletions and rfa mutations implies that these are likely to vary from strain to strain. Since the strains were isolated from different parental stocks of S. typhimurium LT-2, they differ in their ability to metabolize arabinose. Other, unknown differences may exist because the isolation of some of the strains involved ultraviolet and chemical mutagenesis. We have isolated a set of isogenic S. typhimurium strains that contain the relevant genetic markers of the standard Ames tester strains. These strains all contain the same uvrB-bio-gal deletion and the same rfa mutation; they differ only in the nature of their his mutations and in the presence or absence of the plasmid pKM101. We have assessed the responsiveness of these strains to a number of mutagens and conclude that their mutagenic specificities are the same as those of the corresponding Ames strains: TA98, TA100, TA1535, TA1537 and TA1538. Therefore, the specificity of the standard Ames strains with respect to these mutagens is a result solely of the differences in the nature of their his mutations and the effects of pKM101.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.     

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: Passive dosing versus solvent spiking

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source. It compensates for any losses and results in stable freely dissolved concentrations. Passive dosing using silicone O-rings was applied in the Ames II assay to measure PAH mutagenicity in strains TA98 and TAMix – a mixture of six different bacterial strains detecting six different base-pair substitutions – after metabolic activation by S9. Initially, 10 PAHs were tested with passive dosing from saturated O-rings, aiming at levels in the test medium close to aqueous solubility. Fluoranthene, pyrene and benzo(a)pyrene were mutagenic in both TA98 and TAMix, whereas benz(a)anthracene was mutagenic in TA98 only. The concentration-dependent mutagenic activity of benzo(a)pyrene was then compared for passive dosing and solvent spiking. With spiking, nominal concentrations greatly exceeded aqueous solubility before mutagenicity was observed, due to sorptive losses and limiting dissolution kinetics. In contrast, the passive dosing concentration-response curves were more reproducible, and shifted towards lower concentrations by several orders of magnitude. This study raises fundamental questions about how to introduce hydrophobic test substances in the Ames II assay with biotransformation, since the measured mutagenicity not only depends on the compound potency but also on its supply, sorption and consumption during the assay.     

The Drosophila wing test: a comparison of the sensitivity of different strains

A range of 5 chemical mutagens were tested in the Drosophila wing test using 2 different strains and carrying out the experiments in parallel under standardised conditions. The mutagens chosen for the study were the 2 alkylating agents MMS and ENU and the anti-cancer drugs methotrexate, cytosine-arabinoside and adriamycin. As a result of the different genetic backgrounds there was a marked variation in the response of the 2 strains to the mutagens.     

Comparison of mutagen accumulation in 3 estuarine species using the Salmonella/microsome activation system

3 Estuarine organisms—oyster (Crassostea virginica), sea squirts (Mogulla sp.), and shrimp (Peneaus sp.)—were examined for Ames test detectable levels of mutagens. Whole-tissue extract of these organisms were made and tested using S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, with and without S9 activation. Positive results were obtained with sea squirts and shrimp extracts. Activation was not necessary to show activity. Toxicity was encountered with oyster extracts. Histidine, a possible source of false positives, was eliminated from shrimp extracts using XAD-2 resin and thick-layer chromatography.     

Mutagenic activity of chloramines

Mutagenesis by chloramines and hypochlorous acid (HOCl) was studied to determine whether these agents could contribute to the mutagenic and potentially carcinogenic activity of stimulated leukocytes and whether environmental exposure to these agents is a cause for concern. Mutagenic activity was measured using the S. typhimurium TA97a, TA100 and TA102 tester strains. Because chloramines and HOCl are bactericidal, react rapidly with cell components, and can destroy the histidine and biotin required for the mutagenesis assay, activity can't be compared directly with that of less toxic or reactive agents. Nevertheless, chloramines were mutagenic when tested under appropriate conditions. TA100 was the most sensitive strain, and the most active mutagens were lipophilic dichloramines (RNCl2) including derivatives of histamine, ethanolamine and putrescine. Lipophilic monochloramines (RNHCl) such as histamine-monochloramine and NH2Cl were less active. Hydrophilic chloramines such as taurine-chloramines had low activity, and HOCl was inactive. The metabolic state of the bacteria was critical. Chloramines were mutagenic when added to bacteria with glucose at 37 degrees C, but killing predominated when chloramines were added at 4 degrees C or 25 degrees C, or at 37 degrees C without glucose. Production of chloramines and HOCl by leukocytes in vivo could contribute to the association of chronic inflammation and cancer as a result of: (1) the entry of membrane-permeable chloramines into normal cells followed by attack on intracellular components including DNA, and (2) the production of secondary mutagens such as compounds with carbonyl groups or carbon-chlorine bonds. On the other hand, chlorination of water supplies is perhaps more likely to destroy than create mutagens, and chloramines from the environment are unlikely to penetrate the skin and mucous membranes.     

Mutagenic action of a series of epoxides

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the addition of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinyl cyclohexene diepoxide, glycidol, glycidaldehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose—response curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20–50 times more potent in producing mutation that were the other 3 epoxides in the dose—response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Absence of mutagenic and antimutagenic activities of fluoride in Ames salmonella assays

The mutagenicity of fluoride (as sodium fluoride, NaF) was investigated with Ames Salmonella/microsome assays in strains of TA97a, TA98, TA100, TA102 and TA1535. The concentrations of NaF tested ranged from 0.44 to 4421 micrograms/plate (0.1 to 1000 ppm F), both with and without microsome activation. In addition, the suggested antimutagenic effect of fluoride was evaluated with known mutagens at various concentrations of NaF (0.44-442.2 micrograms/plate, 0.1-100 ppm F). The data showed that NaF, in amounts from 0.44 to 442.2 micrograms/plate (0.1-100 ppm F), failed to significantly increase the number of the revertants over the number observed in the solvent (distilled deionized water) controls. Increases of NaF to, and beyond, 1100 micrograms/plate (250 ppm F) resulted in a toxic effect and a reduction of the revertants to various degrees among the strains. NaF in the presence of known mutagens did not significantly decrease the number of the revertants. The results of this study indicate that NaF does not have mutagenic or antimutagenic effects in the strains tested with Ames Salmonella assays.