Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.     

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.     

The mutagenicity of electrocautery smoke.

Careful analysis of electrocautery smoke produced during breast surgery has found organic compounds that are unidentifiable with current analytical techniques. The purpose of this study was to determine the potential mutagenicity of the smoke produced by the electrocautery knife during reduction mammaplasty. Multiple air samples were collected in the operating room during two reduction mammaplasty procedures. Airborne smoke particles were tested for mutagenic potential in both tester strains of Salmonella typhimurium (TA98 and TA100) using the standard Salmonella microsomal test (Ames test). All testing was performed by the Hazard Evaluations and Technical Assistance Branch of the National Institute of Occupational Safety and Health. The smoke produced with the electrocautery knife during reduction mammaplasty was found to be mutagenic to the TA98 strain. The Ames test, an established technique for evaluating the mutagenicity of a substance, was convincingly positive for the smoke collected during the breast surgery. Whether the smoke represents a serious health risk to operating room personnel is not known. Development of techniques to limit electrocautery smoke exposure in the operating room appears to be needed, and surgeons should attempt to minimize their exposure.     

Benzidine: mechanisms of oxidative activation and mutagenesis

Benzidine oxidative activation may proceed by peroxidase-catalyzed one-electron oxidation via free radical intermediates, or by N-acetylation followed by monooxygenase-catalyzed N-hydroxylation. The peroxidase route has been examined by using horseradish peroxidase or prostaglandin H synthase in vitro. In the presence of nucleophiles such as phenols, thiols, or nucleic acids, isolable adducts are formed. The structures of these adducts have been elucidated by spectroscopic methods. The Ames test provides a useful system for studying benzidine bioactivation to mutagenic intermediates. An endogenous bacterial acetylase plays an important auxiliary role in the hepatic S9-dependent activation of benzidine. Bacterial peroxidases may also support benzidine oxidation in the Ames test.     

Structure-activity relationships of aromatic diamines in the Ames Salmonella typhimurium assay. Part II.

Structure-activity relationships in the case of aromatic monoamines, diversely substituted on the ring, using the mutagenic activity in the Ames test were studied in part I. This part II is based on the same general principles but applied to phenylene diamines (ortho, para and meta) diversely substituted on the ring.     

Reduction and partial degradation mechanisms of naphthylaminesulfonic azo dye amaranth by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Reduction and biodegradation mechanisms of naphthylaminesulfonic azo dye amaranth using a newly isolated Shewanella decolorationis strain S12 were investigated. Under anaerobic conditions, amaranth was reduced by strain S12, and a stoichiometric amount of two reduction products RP-1 and RP-2 were generated. UV/visible spectrophotometric and high performance liquid chromatography (HPLC) analysis indicated that RP-1 and RP-2 were 1-aminenaphthylene -4-sulfonic acid and 1-aminenaphthylene-2-hydroxy-3, 6-disulfonic acid. The result strongly supports a mechanism of azo dye reduction by the process via the reductive cleavage of the azo bond to form corresponding aromatic amines. The result of HPLC analyses revealed that these aromatic amines were not able to be mineralized by strain S12 under anaerobic conditions. But after re-aeration of the decolorized culture, RP-2 was mineralized completely by this microorganism, but the consumption of RP-1 was not observed. Ames test showed that amaranth had mutagenic but no cytotoxic potential. The mutagenic potential was relieved after the anaerobic treatment with strain S12 as the mutagenic effect of the two reduction products from amaranth was not detected by Ames test. Thus, the ability of strain S12 to reduce and partially mineralize the naphthylaminesulfonic azo dye efficiently was demonstrated, which can potentially be used to biodegrade and detoxify wastewater containing azo dyes using an alternating anaerobic/aerobic treatment procedure.     

Mutagenicity testing of imidazole and related compounds.

Ames tests have been performed with imidazole and its principal metabolites, hydantoin and hydantoic acid. N-Acetyl-imidazole, a potential metabolite resulting from the action of intestinal bacteria, and histamine, a structurally related compound which is widely distributed in mammalian tissues, have also been tested. Imidazole and histamine were also tested in the UDS assay in primary rat hepatocytes, while imidazole alone was tested in the M2-C3H mouse fibroblast malignant transformation assay. Imidazole gave consistently negative results in the Ames test, the UDS assay and the transformation assay. The three metabolites of imidazole, namely hydantoin, hydantoic acid and N-acetyl-imidazole, all gave negative results in the Ames test. Histamine gave no evidence of mutagenic activity in the Ames test or of genotoxicity in the UDS assay. These results indicate that imidazole and its metabolites are unlikely to present a mutagenic or carcinogenic hazard.     

Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.     

Azo reduction of trypan blue to a known carcinogen by a cell-free extract of a human intestinal anaerobe.

The azo reductase activity of a cell-free extract of Fusobacterium sp. 2 is characterized using trypan blue as a substrate. Either chemical reduction of this dye with sodium hydrosulfite or reduction by the cell-free extract produces a mutagenic product, o-tolidine. The o-tolidine is mutagenic in the Ames Salmonella/mammalian-microsome mutagenicity test when activated by a rat liver S9 preparation.     

Pathways of reductive fragmentation of heterocyclic nitroarylmethyl quaternary ammonium prodrugs of mechlorethamine

Nitroarylmethyl quaternary (NMQ) ammonium salts have potential as prodrugs for enzymatic or radiolytic reduction to release amine effectors under hypoxia. Earlier studies demonstrated one-electron release of the cytotoxic amine mechlorethamine (HN2) from 4-nitroimidazolyl and 2-nitropyrrolyl NMQ prodrugs (but not from nitrobenzyl analogs) through intramolecular electron transfer. In this study we determined whether this is a general feature of heterocyclic NMQ prodrugs of HN2 and examined the reductive pathways in detail using pulse and steady-state radiolysis. The kinetics of radical fragmentation varied by more than four orders of magnitude, independently of the one-electron reduction potential, within the series of eight nitroheterocycles examined. In addition to the compounds identified previously, new 2-nitropyrrole and 3-nitrothiophene NMQ prodrugs were found to provide efficient HN2 release (G > 0.5 micromol/J in anoxic formate buffer). However, the nitrothiophene was sensitive to nucleophilic displacement of HN2, making it less promising. Product analysis by HPLC/mass spectrometry identified symmetrical dimers arising from benzyl-type radical intermediates but also demonstrated that these dimers are not reliable markers for the intramolecular fragmentation of the initial nitro radical anion. This study elucidated multiple competing pathways for reductive fragmentation of NMQ prodrugs and identified the preferred electron acceptors for use in the development of analogs that release more potent cytotoxins.     

Validation of the Salmonella (SV50)/arabinose-resistant forward mutation assay system with 26 compounds

Mutagenic sensitivity of the Salmonella/arabinose-resistant (Ara^r) assay system using the tester strain SV50 was evaluated with 26 compounds both by the preincubation and the standard plate incorporation tests. The mutagenic activity of all 26 compounds was also tested with TA98 and/or TA100 of the Ames Salmonella/microsome assay system. The results indicate that 13 and 10 of 26 compounds were mutagenic and nonmutagenic, respectively, in both assay systems. PR toxin and hydrogen peroxide were mutagenic only in the Ara^r assay, while 2-nitrofluorene was mutagenic only in the Ames assay. The results also show that the mutagenic response of SV50 to 13 of 15 mutagenic compounds was much higher (2.1–154-fold) if the compounds were tested with the preincubation rather than the plate incorporation test. The mutagenic activity of 4 compounds (diethyl sulfate, niridazole, PR toxin and hydrogen peroxide) in the Ara^r assay was detected only with the preincubation test.Since the Ara^r assay using tester strain SV50 has similar mutagenic sensitivity as the Ames assay to chemicals with different modes of action and since it requires only one tester strain, we find this assay system to be useful for screening environmental mutagens. Based on the effectiveness of the preincubation test in this study, it is recommended that the preincubation test instead of the plate incorporation test be used for the Ara^r assay system with tester strain SV50.     

Mutagenicity of benzyl chloride in the Salmonella/microsome mutagenesis assay depends on exposure conditions

Benzyl chloride (BCl) is a clear yellowish, volatile liquid that is widely used as an intermediate for the production of benzyl alcohol and benzyl compounds used in perfumery, dyes and pharmaceuticals. In previous studies BCl has shown weak and inconsistent mutagenicity in the Salmonella/microsome mutagenesis assay (Ames test). The aim of the present study was to investigate the potential mutagenic activity of BCl using modifications of the standard Ames test in order to adapt the method to the volatile nature of the test compound. Tests were performed using (a) the standard plate-incorporation method, (b) incubation of the treated plates in closed containers, (c) a vaporization-diffusion method to expose Ames test plates to volatilised BCl and (d) the pre-incubation method. Using the standard plate-incorporation method, BCl showed no (in the absence of metabolic activation) or very weak (in the presence of metabolic activation) mutagenic activity in Salmonella typhimurium tester strain TA 100. The use of the pre-incubation method did not improve detection of mutagenic activity of BCl. The use of closed containers significantly increased the response, but the most marked response was obtained by testing BCl in volatilised form in the vaporization-diffusion method. Using the latter approach there appeared to be less toxicity of the BCl treatments to the tester bacteria. Our findings suggest that BCl may show greater mutagenic activity in the gaseous phase. This work underlines the importance of using appropriate methods for the evaluation of volatile compounds. The modifications described here are easy to realize in practice and should prove useful for the investigation of other volatile materials or atmospheric contaminants.     

Chemical structure and mutagenic activity of aminoimidazoquinolines and aminonaphthimidazoles related to 2-amino-3-methylimidazo[4,5-f]quinoline

We have synthesized 11 heterocyclic aromatic amines with chemical structures related to that of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), a potent mutagen occurring in broiled sardines, fried beef and beef extract. The mutagenic activity of these IQ analogs was studied and compared with that of IQ using the Ames test with strain TA98 of Salmonella typhimurium in presence of a metabolic activation system (S9 mix) derived from rat liver. The mutagenic activities of the IQ analogs vary over a million-fold; structure-activity comparisons indicate major contributions of the methyl substitution in the imidazole ring and of the quinoline-N, and significant contributions of methylation of the exocyclic amino group and of the geometry of the entire ring system.     

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.     

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.     

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.     

Determination of the activity of 16 hydrazine derivatives in the bioluminescence test for genotoxic agents

The activity of 16 hydrazine derivatives was determined in the bioluminescence test for genotoxic agents (BLT). Hydrazine compounds that were shown to exert mutagenic activity in the Ames test were also active in the BLT. Isoniazid and p-tolylhydrazine which reacted as weak mutagens in the Ames test were highly active in the BLT.     

Potent suppressive effect of green tea polyphenols on tobacco-induced mutagenicity

Antimutagenic activity of green tea (Camellia sinensis) was studied using Salmonella typhimurium strains (TA 102) (Ames test). Aqueous tobacco extract was found to be mutagenic to S. typhimurium TA 102 at concentration of 50 mg/plate. Green tea polyphenols was found to inhibit the mutagenicity of tobacco in a concentration-dependent manner. Concentrations needed for 50% inhibition of mutagen-induced revertant formation was found to be 5 mg/plate. Green tea polyphenols was also found to inhibit the urinary mutagenicity in rats induced by tobacco extract. Moreover green tea polyphenols were found to inhibit in vitro nitrosation reaction produced by reaction sodium nitrite and methyl urea and further inhibition of mutagenicity indicating that green tea has dual action to bring out a reduction in the mutagenic and carcinogenic potential of tobacco.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.