UKEMS trial: Bacterial mutation tests of 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl,and benzyl chloride,using E. coli WP2uvrA(pKM101) and S. typhimurium TA98 and TA100

4CMB, 4HMB and BC were assayed in plate tests, using E. coli WP2uvrA(pKM101), and S. typhimurium TA98 and TA100, in the presence or absence of microsomal activation. 4CMB was also assayed in fluctuation tests using E. coli WP2uvrA(pKM101). 4HMB was uniformly negative, and 4CMB was mutagenic to all 3 strains. BC was negative in TA98 and positive in TA100 and WP2uvrA(pKM101). The presence or absence of S9 made no substantial difference to the mutagenicity of 4CMB or BC.     

Mutagenicity of nitrofuran drugs in bacterial systems

The mutagenic activity of 5 nitrofuran drugs (furadantin, furoxon, furacin, benzazon VII and lampit) was tested on strainsSalmonella typhimurium TA 100, TA 1535, TA 1538 andEscherichia coli WP2uvra ⁺ and WP2uvr A. All nitrofurans tested had a marked mutagenic effect on strain TA 100 and, partially, on strain TA 1535 except for furoxon which was strongly toxic for this strain. No significant mutagenic effects of the drugs were observed with strain TA 1538. With the exception of lympit, all drugs exerted a mutagenie action onE.coli WP2uvrA but no on WP2 uvrA⁺ which has an intact excision repair system. The only drug exerting a mutagenie effect on the latter strain was furoxon. All five nitrofurans exhibited a positive repair rest. The results support the notion that the nitrofuran mutagens under study induce single base substitutions.     

4-chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethylbiphenyl (4HMB): Reverse mutation tests with Salmonella typhimurium

In this study 4CMB was shown to be a strong, direct-acting, mutagen for S. typhimurium strains TA1538, TA1537, TA98 and TA100. However, for strain TA1535 the compound was only weakly mutagenic. No conclusive evidence of mutagenic activity was seen in tests with BC or 4HMB.     

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.     

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 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.     

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5′,8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA⁻(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5′,8-trimethylpsoralen (4,5′,8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors).The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5′,8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted.Both 8-MOP and 4,5′,8-TMP were mutagenic in WP2uvrA⁻(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5′,8-TMP to psoriatic patients may present reduced risk of malignant disease.     

In vitro production of azide mutagenic metabolite in Arabidopsis,Drosophila and Neurospora

The ability of Arabidopsis, Drosophila and Neurospora to convert azide to its mutagenic metabolite was investigated. Cultures of these organisms all contained significant levels of O-acetylserine sulfhydrylase activity. Extracts from each organism produced a product from O-acetylserine and azide in vitro which was mutagenic in Salmonella typhimurium TA1530.     

Mutagenic evaluation of the monocyclic aromatic amine N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene in the Salmonella typhimurium/mammalian microsome test

The hair-dye component N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene was investigated for mutagenic activity in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The testing was performed in the absence and in the presence of a rat-liver microsomal activation system induced by Aroclor 1254. Our results indicate that N-methylamino-2-nitro-4-N′,N′-bis(2-hydroxyethyl)aminobenzene does not induce mutations in Salmonella typhimurium strains, either in the absence or in the presence of the metabolic activation system. The purity of the compound was controlled by utilizing high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC).     

Fluctuation test data on 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) using Salmonella typhimurium TA98 and TA100

Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the ‘Ames’ Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC.4CMB and 4HMB were tested on the same occasion. However, 4CMB was only compared to BC in one assay. The results also show an independent test of BC.     

Mutagenic activities of gentisin and isogentisin from Gentianae radix (Gentianaceae)

The mutagenic activities of 2 hydroxyxanthones, gentisin and isogentisin, obtained from the methanol extract of Gentianae radix (Gentianacea) were investigated.The methanol extract of Gentianae radix, which showed mutagenicity in the Ames test in Salmonella typhimurium strain TA100 with S9 mix, was fractionated by column chromatography on Sephadex LH-20, and the fractions were purified by preparative TLC and column chromatography on polyamide. 2 mutagenic materials thus obtained, S1 and S2, each gave a single band on TLC.Identification of S1 and S2 was accomplished by comparing the analytical (mps, elementary analyses) and spectral (UV, IR, mass, NMR) results for S1 and S2 with literature data for gentisin and isogentisin.At doses below 10 μg, S1 (gentisin) and S2 (isogentisin) had similar specific mutagenic activities. At doses of over 10 to 50 μg, the mutagenic activities of S2 and S1 were 19.1 and 6.94 revertants per μg respectively. This much lower activity of S1 than S2 may be a result of its poor solubility owing to the presence of the OMe group at C-3.The combined yield of S1 and S2 was about 76 mg (40 mg as S1 and 36 mg as S2), which accounted for 76% of the content of mutagenic compounds (100 mg) estimated roughly from the total mutagenic activity in the extract of the starting materials (100 g).     

Evaluation of the mutagenic properties of the coccolithophorePleurochrysis carterae (Haptophyceae) as a potential human food supplement

The mutagenicity of the algaPleurochrysis carterae for use as human food was tested by the Ames method with the modification of pre-incubation, by usingSalmonella typhimurium TA98, TA100, TA1535, TA1537 andEscherichia coli WP2uvrA. The freeze-dried powder ofP. carterae was not mutagenic to any strain either with or without S9 mix. In view of the absence of adverse effects ofP. carterae in this mutagenicity study, it is suggested thatP. carterae is safe for human consumption as a human food supplement.Author for correspondence     

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.     

Mutagenic activity of the methyl and phenyl derivatives of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) in the Ames test

The mutagenic activity of 15 different mono-, di-, tri-, and tetramethyl derivatives of the food mutagen IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline), one diphenyl derivative of IQx and two phenyl derivatives of 5-MeIQx (2-amino-3,5-dimethylimidazo[4,5-f]quinoxaline) were studied in the Ames test with Salmonella typhimurium TA98 and enzymatic activation (S9). The number and positioning of the methyl groups strongly affected the mutagenic activity. The phenylated compounds showed weak mutagenic potency. It seems that both resonance stabilization of the nitrenium ion and steric effects are important in determining mutagenic potency.     

Mutagenicity of benzotrichloride and related compounds.

Benzotrichloride (BTC), benzal chloride (BDC), benzyl chloride (BC) and benzoyl chloride (BOC) were surveyed for their mutagenicity in microbial systems such as rec-assay using Bacillus subtilis and reversion assays using E. coli WP2 and Ames Salmonella TA strains with or without metabolic activation in vitro. BTC and BDC required metabolic activation for their mutagenic activities in several strains of E. coli and Salmonella. The mutagenic metabolites of these compounds may not have been produced by hydrolysis. BC was weakly mutagenic without metabolic activation. Only BOC exhibited no mutagenic activity in the detection procedures used. The mutagenic metabolite of BTC might be very unstable under our experimental conditions. The strain E. coli WP2 try hcr was more sensitive than E. coli B/r WP2 try (hcr+) with regard to the mutagenicity of BTC.     

Mutagenic action of phaseolinone, a mycotoxin isolated from Macrophomina phaseolina

Phaseolinone was mutagenic to excision-repair-deficient strains of Escherichia coli WP2 and also to Salmonella typhimurium TA 100. The repair test was indicative of covalent binding of the toxin to DNA. The side-chain epoxide and the hydroxy groups of the molecule were found to be essential for mutagenic activity.     

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 micrograms/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.     

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.     

Synthesis and enantioselective mutagenicity of azidoalanine in Salmonella typhimurium

Azide mutagenicity involves the requisite formation of the putative novel aminoacid metabolite, beta-azidoalanine. The role of this metabolite, however, is unclear. In order to confirm the identity of this metabolite and provide additional information on possible stereochemical requirements for mutagenicity, authentic racemic and L-azidoalanine were synthesized by an unambiguous route and tested for mutagenicity in Salmonella typhimurium TA100, TA1535, hisG46 and Escherichia coli WP2-. A marked antipodal potency ratio was observed in strains TA100 and TA1535 when racemic and L-azidoalanine were compared. The mutagenic activity resided primarily in the L-isomer. The molar potency of L-azidoalanine in TA100 and TA1535 was nearly identical to that of azide. The lack of mutagenic response for racemic or L-azidoalanine in hisG46 and E. coli WP2- was like that reported for azide and is consistent with similar modes of action for these agents.     

Mutagenic activity of benzofurans and naphthofurans in the Salmonella/microsome assay: 2-nitro-7-methoxynaphtho[2,1-b]furan (R7000), a new highly potent mutagenic agent

A series ¹ of benzofurans and naphthofurans was examined through the Salmonella/microsome assay. (i) With one possible exception, only 2-nitro derivatives give a mutagenic response. However, it appears that the mutagenic potency depends notably on the nature and the position of the other substituents in the molecule. (ii) The mutagenic response occurs in strains TA1537, TA1538, TA98 and TA100 but not in strain TA1535. Reversion of the missense mutation of TA1535 is thus induced only in presence of the plasmid pKM101. (iii) This mutagenic response is at least partially dependent on the bacterial nitroreductase activities and is usually lower in presence of activating mixture from rat liver. (iv) One of the compounds tested, 2-nitro-7-methoxynaphtho-[2,1-b]furan (R7000), may be the most potent mutagen examined so far in the Salmonella/microsome assay. It yields about 200 000 revertants/nanomole on strain TA100 in the standard plate test.The relation between structure, mutagenic potency and other biological activities of the compounds are briefly discussed.