Studies on the oxidation products from 2,4-diaminotoluene by hydrogen peroxide and their mutagenicities. II

2,4-Diaminotoluene (DAT) was reacted with hydrogen peroxide at room temperature for 2 days, and the resulting red precipitates were separated into 5 fractions on silica gel column chromatography. On the gas chromatographic (GC) study, the first fraction (Fr. 1), which is mutagenic (1425 and 1391 revertants/micrograms in the absence and presence of S9 respectively) in Salmonella typhimurium TA98, contained several peaks. Fr. 1 was further separated into 4 subfractions (Fr. 1-I-Fr. 1-IV) by silica gel column chromatography. The red crystals were separated from Fr. 1-III and the structure of the compound was determined to be 1,8-diamino-2,7-dimethylphenazine from physicochemical and chemical evidence. Further, o-nitro-p-toluidine, p-nitro-o-toluidine, 3,3'-diamino-4,4'-dimethylazobenzene and 3,3'-diamino-4,4'-dimethylazoxybenzene were identified with authentic and synthesized samples by gas chromatography/mass spectrometry. These compounds without nitrotoluidines were mutagenic, and phenazine, azo and azoxy compounds induced 49, 301 and 245 revertants/nmole in Salmonella typhimurium TA98 with 25 microliters S9 per plate, respectively.     

Structural determination of a directly mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2′,4′-tetranitrobiphenyl in Salmonella typhimuriumTA98

In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2′,4′-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO₂ and Al₂O₃ column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2′ ,4′-tetranitrobiphenyl with S9 mix at 37°C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4′-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4′-diamino-2′,4-dinitrobiphenyl by its chemical and physico-chemical properties.     

Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.     

Chlorination of harman and norharman with sodium hypochlorite and co-mutagenicity of the chlorinated products

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix. When these β-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.     

Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test

Soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea inhibited the mutagenicity of the directly-acting mutagens: 4-nitro-o-phenylenediamine on Salmonella typhimurium strain TA 98 and NaN₃ on S. typhimurium strain TA 100; and indirectly-acting mutagens, 2-aminofluorene using strain TA 98 and benzo[a]pyrene using strain TA 100, in the presence of a supernatant solution from mammalian hepatic microsomes.     

Mutagenicity of dipyridyls and their methylated derivatives in Salmonella typhimurium/rat liver microsome system

The Salmonella/microsome assay with strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 was used to examine the potential mutagenicity of 5 dipyridyls, 1 tripyridyl, 3 dipyridinium diiodides and 2 pyridinium monoiodides. The widely used herbicide paraquat (1,1-dimethyl-4,4'-dipyridinium diiodide) and its precursor 4,4'-dipyridyl gave weak and marginal mutagenic activity to Salmonella typhimurium TA1535 and TA1538 in the presence of S9-mix. Significantly high mutagenicity was obtained with 2,2'-, 3,3'-, 2,3'-, and 2,4'-dipyridyls, 2,2',2"-tripyridyl, and 5 pyridinium salts under the same conditions. The positive mutagenic response of 2,2',2"-tripyridyl suggests that higher polymers of pyridine contaminating paraquat preparations might be mutagenic. The dose-response curves of 1,1-dimethyl-3,3'-dipyridinium diiodide and 1,1'-dimethyl-2,2'-dipyridinium diiodide revealed an exponential relationship between the number of induced revertants and the compound concentrations. The results suggested that the mechanism of mutation induced by these two compounds might be attributed to the chain reactions of their free-radicals with molecular oxygen.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

2,7-Diamino-3,8-dimethylphenazine as the major mutagenic product from the reaction of 2,4-diaminotoluene with hydrogen peroxide

The mutagenicity of 2,4-diaminotoluene (DAT) in Ames's Salmonella/microsome test was remarkably enhanced by treatment with hydrogen peroxide. Therefore, identification of the major mutagenic reaction product of 2,4-DAT with hydrogen peroxide at room temperature has been performed. Red precipitates were produced in a 2-day reaction mixture and were column chromatographed on silica gel. 5 fractions having mutagenic potency were obtained. The red crystalline needles, obtained as the major reaction product, were separated from fraction 2 and were subjected to high resolution mass spectrometry, 1H- and 13C-NMR spectrometry. The structure of the compound was determined to be 2,7-diamino-3,8-dimethylphenazine from physicochemical and chemical evidence. The compound induced 212 revertants/nmole in Salmonella typhimurium TA98 with 25 microliters S9 per plate.     

Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Chemical purity and mutagenicity: Case study of a drug in development

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be > 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01 – 0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Mutagenicity of five food additives in Ames/Salmonella/microsome test

The mutagenic activity of five food additives (K₂S₂O₅: potassium metabisulphite, KMB; K₂SO₄: potassium sulphate, KS; Na₂SO₃: sodium sulphite, SS; KNO₃: potassium nitrate, KN; NaNO₃: sodium nitrate, SN) were investigated using histidin auxotrophs TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix. The test substance were investigated for their mutagenic effects at non toxic concentrations of 0.83, 1.66, 3.33 and 5.00 mg/plate with and without S9 mix. All the test substances were not mutagenic on TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix except KS and SN. KS and SN showed a weak mutagenic effect on TA100 strain in the absence of S9 mix.     

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes

Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamineO-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAt activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxyacetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyridol[1,2-α:3′,2′-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.     

7-Amino-2,4,6-trimethylquinoline as a mutagenic pyrolysate compound of polyurethane foam

Commercial polyurethane foam was pyrolyzed by gas burners at 600-700 degrees C for 2 h with introduction of air (200 ml/min). Gaseous pyrolysate was trapped in water and 10% hydrochloric acid. Basic and neutral pyrolysates have a mutagenic activity (447 revertants/10 micrograms) in Salmonella typhimurium TA98 in the presence of a mammalian metabolic activation system. These pyrolysates contained 12.32 mg of amino compounds as diaminotoluene per g polyurethane foam, amounts which are 120 times higher than those in unpyrolysed polyurethane foam. Basic and neutral pyrolysates were subjected to silica gel column chromatography, and 6 fractions having mutagenic potency were obtained. The colorless needles (m.p. 200.5-202 degrees C) were separated from fraction 4. These needles have the most potent mutagenicity (678 revertants/2 micrograms) in basic and neutral pyrolysates in Salmonella typhimurium TA98 with 10% S9 mix. From the physicochemical data, the structure of the compound was estimated to be an aminoquinoline derivative, and was identified using synthesized 7-amino-2,4,6-trimethylquinoline by mixed melting point, thin-layer co-chromatography and gas chromatography-mass spectrometry.     

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His⁺ revertants to a maximum of 4 times over background at a GTN dose of 5 μmol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 μmol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C → T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Differential responses of N-nitrosoamines and aromatic amines in the plant cell/microbe coincubation assay

The plant cell/microbe coincubation assay is based on employing living tobacco cells in suspension culture as the activating system for promutagens and the Ames/Salmonella cells as the genetic indicator system. In contrast to aromatic amines(e.g. 2-aminofluorene andm-phenylenediamine) that were previously reported to be activated to products mutagenic in theS. typhimurium strains TA98 or YG1024 by tobacco cells, promutagenic N-nitrosoamines (N-nitrosodimethylamine, N-nitroso-morpholine, N-nitrosopiperidine, N-nitrosomethyl-2-hydroxypropylamine) were not activated to product(s) mutagenic inS. typhimurium TA 100.     

Mutagenicity of alkylhydrazine oxalates in Salmonella typhimurium TA100 and TA102 demonstrated by modifying the growth conditions of the bacteria

Alkylhydrazines are important carcinogens. However, they show generally only weak mutagenicity and the activities reported from different laboratories are contradictory. We have developed a sensitive method to detect the mutagenicity of alkylhydrazines. The method is based on a modified preculturing procedures in the Ames test, the emphasis in the modification being a change in the growth period of tester strains. The optimal growth periods were found to be 11 h in Salmonella typhimurium TA100 and 5 h in Salmonella typhimurium TA102. We tested the mutagenic activity of 12 alkylhydrazines; 1,2-dimetehylhydrazine, 1,2-diethylhydrazine, 1,2-dipropylhydrazine. 1,2-dibutylhydrazine, 1,1-dimethylhydrazine, 1,1-diethylhydrazine, 1,1-dipropylhydrazine, 1,1-dibutylhydrazine, methylhydrazine, ethylhydrazine, propylhydrazine, and butylhdyrazine. All 12 alkylhydrazines were clearly mutagenic in Salmonella typhimurium TA102, and 10 hydrazines were mutagenic in Salmonella typhimurium TA100, both in the absence of S9 mix. The mutagenicity was inhibited by the addition of S9 mix or bovine serum albumin. This suggests deactivation of the mutagens by proteins.     

The clastogenic and mutagenic effects of ascorbigen and 1′-methylascorbigen

Ascorbingen, which occurs naturally in the human diet, and a synthetic analogue (1′-methylascorbigen), were assayed for cytotoxic and clastogenic activities in a SV₄₀-transformed Indian Muntjac cell line (SVM), and for mutagenic activity in the Ames test using Salmonella typhimurium strains TA98 and TA100. Ascorbigen had no effect upon the clonal survival of SVM at concentrations below 0.21 mg/ml and did not induce either chromosome aberrations or sister-chromatid exchanges (SCEs) at any concentration tested up to the maximum compatible with the assay conditions; nor did it induce mutations in either Salmonella strain. In contrast, 1′-methylascorbigen was an order of magnitude more cytotoxic, demonstrating a D_q of 0.03 mg/ml, and whilst it too was not found to induce chromosome aberrations it did induce SCEs in SVM (although only at higly cytotoxic doses) and mutations in the Ames test.     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.