Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants

The active pure compounds of 4 pesticides were tested for DNA-damaging and mutagenic activity in Bacillus subtilis and Salmonella typhimurium tester strains. Included were zinc ethylenebisdithiocarbamate (dithane), 1,2-dihydropyridazine-3,6-dione (maleic hydrazide), O,O-dimethylphosphorodithioate (malathion), and 1,2-dibromoethane (fumazone). These agents gave either weak or negative mutagenic responses with the Salmonella/microsome tests for mutagenicity, but were all positive when the tester was B. subtilis strain TKJ6321. Of the 4 chemicals, only fumazone required metabolic activation with rat-liver S9 mix. Upon activation, it produced a volatile mutagenic product. Dithane, maleic hydrazide, and malathion were all mutagenic and did not require metabolic activation. Among these agents, dithane was strongly mutagenic while fumazone, maleic hydrazide and malathion were moderately mutagenic. Only dithane gave significant DNA-damaging activity when applied to a battery of repair-deficient B. subtilis mutants. For the chemicals reported, it is concluded that B. subtilis is superior to S. typhimurium in the detection of mutagenic activity. We strongly recommend its use for prescreening procedures in combination with the S. typhimurium testers.     

Genetic toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.     

Urinary mutagenicity tests in lead-exposed workers

Urinary mutagenic activity detected by the bacterial fluctuation assay, using Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA with and without metabolic activation (S9 mix), was studied in a group of 21 workers exposed to inorganic lead and a control group of 22 non-occupationally exposed subjects. Occupational exposure to inorganic lead had no effect on urinary mutagenicity in the strains considered, with or without metabolic activation. In smokers (exposed and non-exposed), urinary mutagenic activity appeared to increase compared to non-smokers (exposed and non-exposed), only with Salmonella typhimurium TA98 in the presence of S9 mix.     

Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay

This paper describes the screening studies of 104 commercial crude drugs for mutagenicity by the rec-assay with Bacillus subtilis as well as the reversion assay with Ames strains TA98 and TA100 of Salmonella typhimurium. The rec-assays showed that 13 water extracts and 27 methanol extracts of the crude drugs were positive. The Ames assays with or without metabolic activation showed that 24 water extracts and 16 methanol extracts were mutagenic. In total, mutagenic activities were found in 45 samples among the 104 crude drugs tested.     

Mutagenicity of the Alternaria metabolites altertoxins I, II, and III

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.     

Evaluation of the mutagenicity of 'pan masala', a chewing substitute widely used in India

Mutagenicity of polar and non-polar extracts of a popular brand of 'pan masala' was examined using the Salmonella/mammalian microsome test (Ames assay) and 2 tester strains of Salmonella typhimurium, TA98 and TA100. These extracts were also subjected to pretreatment with sodium nitrite at acidic pH, to simulate conditions for endogenous nitrosation. The aqueous, aqueous:ethanolic and chloroform extracts as well as their nitrosated mixtures were non-mutagenic in the Ames assay, in the presence and absence of metabolic activation. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation demonstrating the presence of direct-acting frameshift mutagens in 'pan masala'.     

Mutagenicity of the Alternaria metabolites altertoxins I, II, and III.

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.     

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.     

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins.

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B1, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR2-toxin. A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.     

Mutagenicity and carcinogenicity of tea, Camellia sinensis

Aqueous, caffeine free and tannin fractions of commercial tea and tannic acid were tested for mutagenicity in Ames test. Tea fractions of tannic acid were non mutagenic in strains TA 100, TA 98, TA 1535 and TA 1538 of Salmonella typhimurium with or without metabolic activation (rat-S9 mix) at different doses tested. In strain TA 98 the above tea fractions and tannic acid inhibited the S9 mix mediated mutagenicity of tobacco in a dose dependent manner. The different tea fractions at 60 degrees C, did not increase the tumor incidence in Swiss mice by gavage feeding. They also failed to produce tumors when injected subcutaneously. Caffeine free tea extract decreased the tobacco induced liver tumors but had no effect on lung tumors. The same fraction was ineffective in hexachlorocyclohexane induced liver tumors in Swiss mice.     

The effects of 4'-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium

Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4'-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic potential in the order iso-propylethyl>iso-propyl>sec-butyl>tert-butyl). These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.     

Isolation of mutagenic beta-carboline derivatives after nitrite treatment of maillard reaction mixtures and analysis of these compounds from foodstuffs and human urine

Mixtures of carbohydrate decomposition products and L-tryptophan were incubated at pH 7.0 and 37 degrees C for 4 weeks. These mixtures exhibited mutagenic activity toward S. typhimurium TA 100 without metabolic activation after a nitrite treatment at pH 4.0. Four beta-carboline derivatives were isolated as premutagens from mixtures of methylglyoxal and furfural. These premutagens were also found to be contained in daily foodstuffs and human urine samples.     

Structure-mutagenicity relationship among aminoquinolines, aza-analogues of naphthylamine, and their N-acetyl derivatives

The mutagenicity of 7 positional isomers of aminoquinolines (AQ) and their N-acetyl derivatives (AcAQ) was tested in Salmonella typhimurium TA100 and TA98 in the presence and absence of S9 mix. In a series of aminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 5-AQ greater than 8-AQ greater than 7-AQ greater than 3-AQ greater than 2-AQ much greater than 4-AQ, 6-AQ. The alpha-positional isomers, 5-AQ and 8-AQ, are more mutagenic than the beta-isomer, 2-, 3-, 6-, 7-AQ's. These results are in contrast to the finding that beta-naphthylamine is more mutagenic than alpha-naphthylamine. In a series of N-acetylaminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 7-AcAQ greater than 6-AcAQ greater than 8-AcAQ much greater than all the others. It is suggested that the AQ and AcAQ series might exert their mutagenicity through different molecular mechanisms (i.e., metabolic activation) from each other. The rate of metabolic activation does not seem to be correlated with the mutagenic potency of the compounds. It is noteworthy that 7-AQ and 8-AQ are mutagenic in both the strains tested in the absence of S9 mix.     

Genetic effects of isoniazid and the relationship to in vivo and in vitro biotransformation

The mutagenic activity of isoniazid, N-acetyl-isoniazid and hydrazine dihydrochloride was investigated in S. typhimurium. Isoniazid was found to possess a weak mutagenic activity only in repair-deficient strains TA1535 and TA100 as well as in the plasmid-containing strain TA92 (10-30 mg/plate) in the Ames test without metabolic activation. Addition of microsomal enzymes by S9 mix decreased this direct mutagenic activity. In contrast, preincubation of isoniazid with crude liver homogenate from mice, rats or Syrian golden hamsters for 4 h prior to plating with bacteria liberated a mutagenic compound which is equally active in both repair-deficient and repair wild-type strains (0.5-5 mg/plate). This activation pathway is independent of NADPH, is heat-sensitive and is operative only in a total liver homogenate in suspension. The highest capacity for mutagenic activation was achieved with liver homogenate from hamsters, followed by that from mice and rats. Furthermore, this mutagenic activation is paralleled by formation of hydrazine, as demonstrated in colorimetric measurements with p-dimethylaminobenzaldehyde. N-Acetyl-isoniazid is without mutagenic activity under similar conditions, and liberation of hydrazine was never detected. This means that, besides having a weak direct genetic activity, isoniazid is a promutagen, and formation of hydrazine is the first step in metabolic activation. It is concluded that the genotoxic properties of isoniazid in mammals are primarily determined by the pharmacokinetic behavior of the ultimate reactive metabolite. This result must be taken into consideration in risk assessment performed for mutagenic and carcinogenic properties of isoniazid in man.     

Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB

We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene. The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass. Introduction of these plasmids into S. typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP). Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds. Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98. In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers.     

Genotoxic and mutagenic activity of environmental air samples in Flanders, Belgium

Atmospheric pollution is assumed to play a role in the incidence of respiratory diseases and cancers. Airborne particles are able to penetrate deep into the lung and are composed of complex chemical mixtures, including mutagens and carcinogens such as polycyclic aromatic compounds (PACs). The present study reports mutagenic and genotoxic activities associated with ambient air collected near a busy street in Borgerhout, at an industrial site in Hoboken and in Peer, a rural community 70 km east of Antwerp in Flanders, Belgium. Airborne particulates (PM10) and semi-volatile organic compounds were sampled during winter and summer. Samples were collected with a high-volume sampler using quartz filters (QF) and polyurethane foam (PUF) cartridges. The mutagenic and genotoxic activity of the organic extracts was determined using the Salmonella test/standard plate-incorporation assay and the Vitotox assay. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). The mutagenicity assay, using Salmonella typhimurium strain TA98, demonstrated direct mutagenicity of up to 58 revertants/m3 for the QF extracts and low or no mutagenic activity in the PUF extracts. Metabolic activation of the samples resulted in high indirect mutagenicity for both QF and PUF extracts: up to 96 revertants/m3 were found in QF samples and 62 revertants/m3 in PUF samples. Genotoxic effects of the filter extracts were assessed with the Vitotox assay: some direct genotoxic effects were noted, i.e. without metabolic activation, but almost no effects were observed after metabolic activation. Without activation, most PUF extracts were bacteriotoxic. With metabolic activation this toxicity disappeared, but genotoxic effects were not observed. Statistical analysis showed that the observed biological effects correlated well with the PAH concentrations.     

Mutagenicity of benzo[b]phenanthro[2,3-d]thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT

Benzo[b]phenanthro[2,3-d]thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S. typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006. Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9. In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9. Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9. BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9. In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation. In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9. Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide. The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S. typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products. Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer.     

Toxic genetic effects of flunitrazepam

The mutagenic activity of Flunitrazepam, the active ingredient of the drug Rohypnol, has been investigated by using the Salmonella/microsome mutagenicity test. A dose-related mutagenic effect was observed on Salmonella typhimurium strain TA 100 either in the absence or in the presence of a rat liver microsomal fraction (S9) as in vitro metabolic activation system. By adopting a modification of the Salmonella test, the mutagenicity of urines from rats or patients treated with the drug was evaluated. In these cases mutagenic activity was detected toward the Salmonella strains TA 98 and TA 100 both in presence and in absence of the metabolic activation system. The data indicate that Flunitrazepam and/or its urinary metabolites can induce both base-pair substitutions or frame-shift point mutations.     

Absence of mutagenic activity of fodder proteins in the Ames Salmonella/microsome test

The mutagenicities of fodder proteins (Pekilo, L-lysine and Orsan) were tested towards Salmonella typhimurium in the plate-incorporation assay in the presence or absence of metabolic activation with a rat-liver S9 preparation. Filtrates and 2-, 5- and 10-fold-concentrated filtrates of saline- or ethanol-soluble fodder proteins were tested. No mutagenic activity was observed.     

Mutagenicity of some derivatives of dipyrido[1,2-a:3',2'-d]imidazoles in Salmonella typhimurium with metabolic activation by rat liver and small intestine subcellular fractions

The mutagenic effect of 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) was compared with that of the 3-amino, 3-nitro, or 3-N-hydroxylated derivatives of the same base ring with methyl groups at positions 4 and 6 of the molecule. The compounds were tested in Salmonella typhimurium strain TA98 without metabolic activation and in the presence of different concentrations of subcellular fractions from livers or small intestines of rats pretreated with different P448/P450 inducers. The 4,6-dimethyl compounds are always more mutagenic than Glu-P-2. Pretreatment with Aroclor 1254 (ARO) is the most effective inducer in the activation of the 2- and 3-amino compounds by liver S9, whereas the same fraction decreases the mutagenicity of the 3-nitro derivative. S9 from small intestine increased the mutagenic effect of the 3-nitro and 3-N-hydroxylated compounds, but it was unable to activate the amino compounds.