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.     

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.     

Evaluation of the mutagenic potential of mycotoxins using Salmonella typhimurium and Saccharomyces cerevisiae

The mutagenic effects of fifteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B₁ and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Mutagenicity of marijuana and Transkei tobacco smoke condensates in the salmonella/microsome assay

Extracts and smoke condensates of marijuana, Transkei home-grown tobacco and also commercial cigarette tobaccos were assayed for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation. No mutagenic activity was detected in dichloromethane extracts of marijuana and tobacco per se, but all the smoke condensates exhibited mutagenicity with metabolic activation. The only strain not mutated by any of the pyrolyzates was TA1535. Transkei tobacco pyrolyzate proved to be the most mutagenic, followed by marijuana, pipe and cigarette tobacco. Mutagenicity was positively associated with the nitrogen content of the various products. The potent mutagenic action of marijuana smoke condensate, coupled with a condensate yield of more than 50% higher than that of cigarette and pipe tobacco, indicates a high carcinogenic risk associated with marijuana smoking.     

Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538

A series of ten azo dyes as well as various single ring aromatic amines substituted on the benzene ring were tested for bacterial mutagenicity with Salmonella typhimurium TA 1538 using a soft-agar overlay method. Two dyes, sudan 2 and chrysoidin induced mutation but only in the presence of a rat liver preparation. Chrysoidin was the more active. Testing of its reduction products, aniline and 1,2,4-triaminobenzene showed a liver metabolite of the latter compound could be responsible for the mutagenic effect, having a comparable mutagenicity with 1,2-diamino-4-nitro-benzene, one of the mutagenic constituents of hair dyes. Structure-activity studies on a series of ring-substituted anilines indicated that mutagenic activity required at least two positions to be substituted with either amino or nitro groups, or one of each. The bacteria as well as the liver enzyme preparation may partake in the activation of these chemicals. The correlation between mutagenicity and carcinogenicity for this group of compounds is discussed.     

Mutagenic activity of some platinum and palladium complexes

The mutagenic properties of 16 platinum compounds were studied using Salmonella typhimurium TA98 and TA100. Eight of the compounds were considered direct mutagens, as their mutagenicity was not dependent on metabolic activation by liver extracts. Potent mutagenicity and high toxicity were exhibited by cis-Pt(NH₃)₂Br₂, cis-Pt(NH₃)₂Cl₂, Pt(C₅H₁₂N₂)Cl₂ and Pt(en)Cl₂ for both bacterial strains. When distilled water was used as the carrier solvent, these compounds were strongly mutagenic and toxic, but much less so when dimethyl sulfoxide was the solvent.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

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

Absence of mutagenic activity of benorylate,paracetamol and aspirin in the Salmonella//mammalian microsome test

Benorylate and its two major hydroyssis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Inverse correlation between combined mutagenicity in Salmonella typhimurium and strength of coordinate bond in mixtures of cobalt(II) chloride and 4-substituted pyridines

Cobalt(II) chloride (CoCl₂), non-mutagenic by itself, has been tested for mutagenic activity in the presence of 4-substituted pyridines in the test strains of Salmonella typhimurium. CoCl₂ was found to be mutagenic in strains TA1537 and TA2637, when combined pyridine, with methyl isonicotinate, 4-methylpyridine, 4-ethylpyridine, 4-chloropyridine or 4-bromopyridine. Mixtures of CoCl₂ and isonicotinic acid, 4-cyanopyridine, 4-aminopyridine, or 4-dimethylaminopyridine exhibited no mutagenicity. Judging from the spectral observations, such combined mutagenicity may be due to the formation of moderate to weak complexes between these compounds and the Co(II) cation.     

Potential cytotoxic and mutagenic effect of Pinus wallichiana,Daphne oleiodes and Bidens chinensis

The Pinus wallichiana, Daphne oleiodes and Bidens chinensis have a long history of being used traditionally for treatment of various types of disorders. Most of the uses have been without any scientific evidence and toxicological assessment. We evaluated the mutagenic and cytotoxic capabilities of various parts of P. wallichiana, D. oleoides and B. chinensis. Ames Salmonella mutagenicity assay determined the mutagenicity activity against TA 98 and TA 100 bacterial strains of Salmonella typhimurium without metabolic activator S9 system. The number of mutant colonies in negative control was considered as limit to determine the mutagenicity effects of every extract. Brine shrimps lethality bioassay was used to determine the cytotoxic capabilities of the selected plants. The P. wallichiana, D. oleiodes and B. chinensis did not showed any mutagenic activity both for frameshift mutation (TA98) and base-pair substitution (TA100) without S9 mixture. The crude methanolic extract of P. wallichiana stem showed moderate cytotoxicity (53.33%) at 1000 μg/ml with LD₅₀ value 599.634. The D. oleoides fruit showed a toxicity of 60% at 1000 μg/ml with LD₅₀ value 367.730. The B. chinensis (whole plant) showed lethality of 63.3% at 1000 μg/ml, with LD₅₀ 204.833. The absence of any mutagenic activity of crude extract of the tested plants in both bacteria strains, TA 98 and TA 100 without the S9 mix confirms the safety of these plants to the consumers.     

Mutagenic activity of furfural in Salmonella typhimurium TA100

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

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O: 7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents.There was a positive relationship between carcinogenicity and the initial ratios of 7-: O⁶-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O⁶ (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.     

Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium — a series of chloro- or fluoro-nitrobenzene derivatives

The mutagenicity of 21 chloro- or fluoronitrobenzene compounds and 9 chloro- or fluorobenzene compounds in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined. The tests were carried out under the conditions of absence and presence of liver microsomal activation.15 nitro-group compounds had mutagenic activity; above all, compounds of fluoronitrobenzene were mutagenic for both types of strain. On the other hand, chloronitrobenzene compounds were mutagenic for base-pair substitution strains only. Mutagenic activity was exhibited by all compounds having a chloro or fluoro substituent at the para and ortho position in the nitrobenzene nucleus. All compounds without a nitro substituent showed no mutagenic activity.     

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.     

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.     

Mutagenicity of methylated fluorenes and benzofluorenes

Methylated fluorenes were assayed for mutagenic activity towards Salmonella typhimurium TA98 and TA100. None of these methylfluorenes were mutagenic in the absence of metabolic activation. In the presence of 900 × g supernatant from Aroclor-induced rats, 9-methylfluorene and 1,9-dimethylfluorene were active towards TA98 and TA100. The structural requirement for mutagenic activity within this series was the presence of a single methyl substituent at the 9-position. Enhanced mutagenic activity was also observed for benzofluorenes similarly methylated at their benzylic positions.     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

Absence of mutagenic action of 5β-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5β-cholanoic acids including 2 of the biie acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5β-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.     

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.