In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline

We have previously shown that the common antiamebic drug diiodohydroxyquinoline (DIHQ) exhibits mutagenic activity in the in vivo micronucleus test in Swiss albino mice. Results of experiments undertaken to study the influence of ascorbic acid (vitamin C) on the mutagenicity of DIHQ in this model system showed that ascorbic acid acts as an antimutagen against DIHQ. The effective antimutagenic doses of ascorbic acid themselves do not show any genotoxic effects in this in vivo system. It will be necessary, however, to elucidate the mechanism of action of ascorbic acid as well as its effects on the therapeutic properties of DIHQ before a practical use of ascorbic acid is contemplated for this purpose.     

Mutagenic activity and structure-activity relationships of short-chain dialkyl N-nitrosamines in a hamster hepatocyte V79 cell-mediated system

A series of 19 short chain dialkyl N-nitrosamines was studied for mutagenic activity in an uninduced hamster hepatocyte V79 cell-mediated mutagenesis system. Ouabain was used as the selective agent to quantitatively analyze for chemically induced mutants. None of the nitrosamines was mutagenic in the absence of hamster hepatocyte activation. The relative mutagenic activities of the nitrosamines at an equimolar dose are presented. The results of the study indicated that: (a) increasing alkyl chain length decreased mutagenic activity; (b) oxidation of the carbon position to a carbonyl group increased the mutagenic activity of symmetrical and asymmetrical nitrosamines, whereas oxidation to a hydroxyl group only increased the mutagenic activity of the asymmetrical nitrosamines tested and (c) the carbon position at which oxidation occurred was important in determining mutagenic activity. The relationships between structure, metabolic activation, and mechanisms of mutagenic activity are discussed.     

Ames test: mutagenic activity of airborne particles collected on airconditioner filters during the fire at a Sandoz storehouse in Schweizerhalle on November 1, 1986

The mutagenic activity of methanol extracts from airconditioner filters was tested using the standard plate-incorporation procedure for the Ames test and the strains Salmonella typhimurium TA97, TA98 and TA100 as test organisms. In a first set of experiments filters from 4 buildings were investigated. For each building the mutagenic activity of a filter which was in use during the fire (fire-exposed) was compared with the mutagenic activity of a filter which was exposed for a similar time span to normal urban air (non-exposed). While for 1 pair of filters the non-exposed extract was more mutagenic than the fire-exposed material, the opposite was found for 2 other filter pairs, and in 1 case there was hardly any difference in the mutagenic activities of the fire-exposed and the non-exposed filter extracts. Overall differences by factors up to 100 were observed in the mutants/m3 air values of the most and the least mutagenic filter extracts. The second group of experiments was performed to investigate the variations in mutagenic activity of filter extracts occurring due to changes in the weather conditions. Airborne particles were collected for 3 consecutive periods of 10-11 days at the 2 buildings where the extracts of the fire-exposed filters had been found to be more mutagenic than the corresponding control materials. The differences between the strongest and the weakest mutagenic filter extracts of these series were similar to the differences observed previously between the fire-exposed filters and the non-exposed filter materials. The highest mutagenic activities found in the second group of experiments was similar, at both sites, to the mutagenic activities measured in the fire-exposed extracts from these 2 buildings. Since the differences in the mutagenic activities of filters exposed to urban air during the different meteorological conditions were similar to the differences observed between fire-exposed and non-exposed materials, it is not possible to state whether the fire led to the distribution of mutagenic chemicals, although it is theoretically possible. However, based on the observation that the maximal mutagenic activities of the fire-exposed and the non-exposed extracts were similar, the present study allows the conclusion that the mutagenic burden for the population of the area of Basel was not significantly increased by the fire in Schweizerhalle on November 1, 1986.     

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.     

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.     

Mutagenicities of N-nitrosamines on Salmonella.

The mutagenic activities of 11 N-nitrosamines were tested using Salmonella typhimurium TA100 and TA98. All the carcinogenic N-nitrosamines were mutagenic on TA100 with a drug-activating system from the rat liver, whereas N,N-diphenylnitrosamine, a non-carcinogen, was not mutagenic. None of the N-nitrosamines was mutagenic on TA98, except N,N-diethylnitrosamine which was weakly mutagenic. To detect the mutagenicity of N,N-dimethylnitrosamine, the pre-incubation of bacteria and N,N-dimethylnitrosamine with S-9 Mix before if was poured onto plates was obligatorily required. Dimethyl sulfoxide inhibited the mutagenic effect of N,N-dimethylnitrosamine.     

Induction of 6-thioguanine-resistant mutants by hyperoxia and gamma-irradiation: effect of compromising cellular antioxidant systems

Hyperoxia and gamma-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10(6) survivors after 48 h of growth in 70% O2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, gamma-irradiation was 4 times more mutagenic than 70% O2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both gamma-irradiation and hyperoxia. Cells thus treated showed an 855 reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of gamma-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphere.     

Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium.

Benzoyl chloride and 53 commercially available aromatic heterocyclic and aliphatic nitro compounds were tested for mutagenicity in Salmonella typhimurium TA98 and TA100. 34 of 53 nitro compounds (64%) were mutagenic, 4 in TA100 only, 15 in TA98 only, and 15 in both strains. 13 of the heterocyclic derivatives of pyridine, indole, indazole, quinoline, and benzimidazole were mutagenic. 21 of 34 mutagenic nitro compounds were bactericidal. Nitromethane was the only aliphatic tested and was not mutagenic. Benzoyl chloride, a human carcinogen, was mutagenic for TA98.     

The release of mutagens from airborne particles in the presence of physiological fluids

Airborne particulates collected indoors in residences and outdoors were extracted by soxhlet extraction and sonication with methanol. In a comparative study in which mutagenic activity was evaluated in the Salmonella typhimurium reversion assay both soxhlet extraction and sonication proved to be suitable extraction methods. First, the residue, obtained by sonication of loaded filters with methanol followed by evaporation to dryness (tar), was sonicated with newborn calf serum and lung lavage fluid from pigs. All serum extracts of the tar were mutagenic to Salmonella typhimurium TA98, and contained direct- and indirect-acting mutagens. However, the mutagenic activity recovered by serum was only about half of the total mutagenic activity of the tar. The other part of the mutagenic activity remained in the tar. Lung lavage fluid was only able to remove 5-10% of direct-acting mutagens from the tar of all samples. About 20% of indirect-acting mutagens from indoor air were recovered in lung lavage fluid, while the lung lavage fluid extract from outdoor air did not show indirect mutagenic activity. Second, mutagenic activity recovered by direct sonication of the filters with physiological fluids was comparable with the recovery obtained by sonication of the tar. However, after sonication of the filter with lung lavage fluid hardly any mutagenic activity remained on the filter, whereas after sonication of the tar a clear mutagenic activity was observed in the non-soluble residue.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.     

Mutagenic profile of rubber dust and fume exposure in two rubber tire companies

The aim of this study was to evaluate current mutagenic activity of ambient rubber dust and fume exposure in the mixing and curing departments of two rubber tire companies situated in The Netherlands and Sweden. Salmonella typhimurium strains YG1021, YG1024 and YG1041 were used to study the possible presence of mutagenic nitroarenes and aromatic amines. A large difference in mutagenic activity was found between the two companies. While the rubber tire company situated in The Netherlands revealed overall high mutagenic activity of rubber dust and fumes in the mixing and curing departments, respectively, 430 and 279 rev/m(3) (YG1041), the Swedish company showed almost no mutagenic activity, respectively, 18 and 54 rev/m(3) (YG1041). Further identification of the mutagenic profile showed that mutagenic activity was exclusively observed in S. typhimurium strains with elevated levels of O-acetyltransferase activity (YG1041 and YG1024) in the presence of a metabolic active liver S9 fraction, possibly indicating the presence of indirect mutagenic aromatic amines. These results show that although production processes and lay-out within rubber tire companies are comparable, differences in rubber chemicals used and overall level of control measures (e.g., good housekeeping, cleanliness) are likely to result in substantial differences in mutagenic exposure levels between companies.     

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.     

The mutagenic action of nitroimidazoles. IV. A comparison of the mutagenic action of several nitroimidazoles and some imidazoles.

The mutagenic action of 51 imidazoles was investigated. The fluctuation test of Luria and Delbrück was used, with Klebsiella pneumoniae as test organism. 8 compounds, including 5 with a weak mutagenic action in the fluctuation test, were also investigated by the Ames test in which Salmonella typhimurium TA100 was used. Of the 51 imidazoles examined, 33 were nitroimidazoles. 31 of the latter appeared to be mutagenic, whereas out of the 18 other imidazoles without a nitro group only 2 were mutagenic. Several of the substances tested for mutagenicity showed an antimicrobial activity. No direct relationship between antimicrobial action, growth inhibition and mutagenicity was established. With methyl-nitroimidazoles a relationship was found between the chemical structure and mutagenic action. However, when the nitroimidazoles had a more complex chemical structure, a relationship between this structure and mutagenicity could not be established.     

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18-24 h or when S9 mix was preincubated in Fischer's medium at 37 degrees C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.     

Mutagenicity,Creatine and Nutrient Contents of Pan Fried Meat from Various Animal Species

The mutagenic activity in extracts of fried meat from 16 different animal species was studied in Salmonella typhimurium TA98. In each experiment, 1 meat sample together with a standard beef sample was fried, and the mutagenicity was expressed relative to the beef sample. All meat samples showed less mutagenic activity than beef. The contents of creatine, creatinine, water, protein, carbohydrate and fat in the meat samples were analyzed, but mutagenicity was not correlated with the concentration of any of these constituents. Beef meat treated with creatinase to remove creatine produced reduced mutagenic activity. Possibly a threshold concentration of creatine is necessary to give a high mutagenic response.     

Mutagenicity of N-arylacetohydroxamic acids and their O-glucosides derived from chlorinated 4-nitrobiphenyl ethers

The mutagenic activity of N-arylacetohydroxamic acids, their O-acetates, their O-glucosides, and N-arylhydroxylamines, derived from chlorinated 4-nitrobiphenyl ethers (CNBs), was tested in the Salmonella reversion assay. N-Arylhydroxylamines were mutagenic by themselves; however, other compounds containing an N-acetyl group showed mutagenic activity in the presence of guinea pig liver S9. The mutagenic activation of the glucosides of N-arylacetohydroxamic acids was caused by Ms but not by S10.5, whereas their aglycones, N-arylacetohydroxamic acids, were activated to mutagens by both the fractions. The mutagenic activation of these compounds was inhibited by bis(p-nitrophenyl)phosphate, which indicates that enzymatic deacetylation is a crucial step in the mutagenic activation. Analysis of metabolites of the O-glucosides of N-arylacetohydroxamic acids by h.p.l.c. indicates that the corresponding deacetylated O-glucosides are primary metabolites, which decomposed to amino and azoxy (via hydroxylamine) derivatives, and that the deacetylating activity of S9 locates exclusively in Ms.     

Mutagenicity studies of p-substituted benzyl derivatives in the Ames Salmonella plate-incorporation assay

The mutagenicity of 24 benzyl derivatives, containing a variety of substituents and leaving groups, were assayed in strain TA100 using the Ames plate-incorporation assay. p-Nitrobenzyl chloride (12 000 revertants/mumole), p-nitrobenzyl tosylate (6100 revertants/mumole), and p-acetoxybenzyl chloride (100 revertants/mumole) were mutagenic; none of the remaining 21 compounds were mutagenic. p-Nitrobenzyl chloride was also found to be mutagenic in strain TA98 (700 revertants/mumole), but not in strain TA98NR (a strain deficient in nitro reductase activity). p-Acetoxybenzyl chloride was nonenzymatically hydrolyzed to p-hydroxybenzyl alcohol and p-acetoxybenzyl alcohol. These findings suggest that nitrobenzyl derivatives were mutagenic due to nitro reductive metabolism and that p-acetoxybenzyl chloride was mutagenic due to the intermediate formation of p-hydroxybenzyl chloride during the hydrolysis of p-acetoxybenzyl chloride.     

Mutagenicity of N-nitrosopyrrolidine derivatives in Salmonella (Ames) and Escherichia coli K-12 (343/113) assays

The mutagenicity of nitrosopyrrolidine (NPYR) and its derivatives was determined by use of the Ames Salmonella assay. A clear specificity to revert the missense stain of TA1535 and a requirement for the phenobarbital-induced rat-liver activation system (S9 mix) were noted. 3,4-Dichloronitrosopyrrolidine was more mutagenic than NPYR, whereas 3-hydroxynitrosopyrrolidine was weakly mutagenic. The carcinogenic nitroso-3-pyrrolidine was not mutagenic under the test conditions. The noncarcinogenic derivatives (2,5-dimethylnitrosopyrrolidine, nitrosoproline and 4-hydroxynitrosoproline) were not mutagenic. Liquid preincubation assays were not any more effective than the pour-plate assays. Selected derivatives of NPYR were tested in the Escherichia coli K-12 (343/113) assay A specificity to revert the missense mutation at the arg locus and a dependence on phenobarbital-induced rat-liver S9 mix were noted with NPYR and its derivatives. 3,4-Dibromonitrosopyrrolidine, which was not mutagenic in Salmonella, was effective in E. coli, and the weakly carcinogenic NPRL was a weak mutagen resulting in a 2-fold enhancement in the E. coli arginine reversion assay.