Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers.

A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix. The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl. Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic. Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic. However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic. All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic. A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).     

Hycanthone and its congeners as bacterial mutagens.

Hycanthone methanesulfonate (HCT) was shown to induce "forward" and "reverse" mutations in Salmonella typhimurium and Escherichia coli. Mutational effects of HCT on S. tyhhimurium TA1532 were concentration and time dependent. A comparison of mutagenic potency for TA1532 was made between HCT and the frame-shift mutagens quinacrine and ICR-191. An investigation of structure-activity relationships revealed the substituent in the 4-position of ring A to be critical for mutagenicity. Activity was found when this group was a hydroxymethyl (i.e., HCT) or an aldehyde (Win 25,315), but the analogues having a carboxyl group (Win 25,850) or methyl group (lucanthone) in this positionwere inactive. Removal of a single ethyl group from the side chain did not affect mutagenic activity inasmuch as the potency of desethyl HCT (Win 27,262) equaled that of HCT on a molar basis. A marginal activity was found with a sample of HCT sulfoxide (win 27,266), but this sample was found to contain traces of HCT. The HCT analogue with a terminal N-oxide in the side chain (Win 29,329) was inactive at the concentration tested.     

Metabolic epoxidation of trans-4-acetylaminostilbene: a protective mechanism against its activation to a mutagen

Trans-4-acetylaminostilbene is activated by liver preparations to mutagens for Salmonella typhimurium. Since this compound is metabolized to the trans-alpha,beta-epoxide and since many epoxides are ultimate mutagens, this epoxide was tested for direct mutagenicity. It was, however, found to be non-mutagenic, and, in contrast to the parent compound, the epoxide was no longer activated by liver preparations to mutagens. The same was found for the beta-ketone and for the threo-alpha,beta-dihydrodiol, which are formed metabolically from trans-4-acetylaminostilbene and from its alpha,beta-epoxide. 4-Acetylaminobibenzyl showed a very weak mutagenic activity in the presence of the liver preparation. Thus, it is important to realize that where epoxides are formed from compounds which are known to be metabolized to mutagens, they are not necessarily responsible for the mutagenicity. Epoxidation may even prevent the possibility of bioactivation to mutagens.     

Evaluation of new 2,2'-dimethyl-5,5'-dipropoxybenzidine- and 3,3'-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure-activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.     

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.     

Verapamil as a co-mutagen in the Salmonella/mammalian microsome mutagenicity test

Verapamil is a calcium-channel blocking agent, commonly used for chronic treatment of heart conditions. We have previously demonstrated that verapamil acts as a co-mutagen in a bacterial mutagenicity test for some experimental anilinoacridine antitumour drugs. Within the anilinoacridines series there are several compounds which are apparently non-mutagenic (or very weak mutagens) in the absence of verapamil, but strong mutagens in its presence. We have now tested a wider range of materials for verapamil enhancement of mutagenicity, to include some of those to which persons on verapamil therapy might be exposed through life-style or occupation. Some verapamil enhancement of mutagenicity was seen with most mutagenic compounds including anticancer drugs, antiparasitic agents, one biological stain and one hair dye. A number of tricyclic antidepressants and biological stains were tested and found to be non-mutagenic. If these results extrapolate to mammalian cells, long-term verapamil therapy could potentially increase the effects of certain environmental mutagens.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium.

Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens. It may therefore play a key role in the metabolic activation of xenobiotics. The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays. The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested. When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activation by this mammalian peroxidase. The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet. Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens. Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes. Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems.     

Mutagenicity and metabolism studies on 12 thiuram and dithiocarbamate compounds used as accelerators in the Swedish rubber industry.

12 thiuram and dithiocarbamate compounds used in the rubber industry as accelerators, and to some extent as sources of sulfur, were tested, as well as carbon disulfide, a metabolite found in vivo after dithiocarbamate treatment, for mutagenicity in Salmonella typhimurium. A mutagenic effect on the base-substitution-sensitive strains TA1535 and TA100 was found for 7 compounds. The most potent directly acting mutagens were: tetramethylthiuram disulfide (TMTD), zinc dimethyldithiocarbamate (ziram), cadmium diethyldithiocarbamate and zinc diethyldithiocarbamate. Tetraethylthiuram disulfide (TETD), also known as Antabus, and carbon disulfide were non-mutagenic. The relatively low direct mutagenic effect of tetramethylthiuram monosulfide (TMTM) was enhanced in the presence of a metabolizing system (S9 mix). A hypothesis is given regarding the activation process of the monosulfide TMTM.     

Evaluation of the mutagenicity of 'pan masala', a chewing substitute widely used in India

Mutagenicity of polar and non-polar extracts of a popular brand of 'pan masala' was examined using the Salmonella/mammalian microsome test (Ames assay) and 2 tester strains of Salmonella typhimurium, TA98 and TA100. These extracts were also subjected to pretreatment with sodium nitrite at acidic pH, to simulate conditions for endogenous nitrosation. The aqueous, aqueous:ethanolic and chloroform extracts as well as their nitrosated mixtures were non-mutagenic in the Ames assay, in the presence and absence of metabolic activation. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation demonstrating the presence of direct-acting frameshift mutagens in 'pan masala'.     

An association between mutagenicity and carcinogenic potency

A comparison between mutagenic and non-mutagenic rodent carcinogens studied by the U.S. National Toxicology Program revealed that as a group, rat carcinogens mutagenic in Salmonella typhimurium are more potent than their non-mutagenic counterparts.     

Mutagenicity of anthraquinones in the Salmonella preincubation test

The mutagenicities of 15 naturally occurring anthraquinones were examined in Salmonella typhimurium strains TA98, TA100 and TA2637 by the preincubation method. 7 of the 15 compounds tested, i.e., chrysazin, emodin, islandicin, alizarin, chrysophanol, 2-hydroxyanthraquinone and emodic acid, were strong mutagens in strain TA2637 with metabolic activation. All of these compounds contain 1-3 hydroxyl groups, and some also have methyl groups. Cynodontin, an anthraquinone with 4 hydroxyl groups and 1 methyl group, was only slightly mutagenic in strain TA2637. 2-Hydroxyanthraquinone, alizarin, emodin, islandicin and chrysazin were also mutagenic in strain TA100 with S9 mix. All the bisanthraquinones tested, i.e., skyrin, (+)rugulosin, (-)luteoskyrin, (-)rubroskyrin and sennoside A, were non-mutagenic in this test system with or without metabolic activation. Unsubstituted anthraquinone and anthrone were also non-mutagenic. These results show that hydroxyl substituents are necessary for the mutagenicity of anthraquinones, the optimal substitutions being 1-3 hydroxyl groups per molecule. The 4th hydroxyl group, in the compound cynodontin reduces the mutagenicity considerably.     

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver (= 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.     

Further mutagenicity studies on pesticides in bacterial reversion assay systems

A total of 228 pesticides (88 insecticides, 60 fungicides, 62 herbicides, 12 plant-growth regulators, 3 metabolites and 3 other compounds) was tested for mutagenicity in bacterial reversion-assay systems with 5 strains (TA100, TA98, TA1535, TA1537 and TA1538) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli. 50 pesticides (25 insecticides, 20 fungicides, 3 herbicides, 1 plant-growth regulator and 1 other compound) were found to be mutagenic. 5 of them required metabolic activation (S9 mix) for their activities. Among various chemical groups, organic phosphates, halogenated alkanes and dithiocarbamates showed higher ratios of mutagens. Although 22 of the pesticides tested have been reported to be carcinogenic, 7 of them, i.e., captain, DBCP, EDB, EDC, ETU, HEH and nitrofen, were detected as mutagens in the present assay. Most of the other 15 non-mutagenic carcinogens were organochlorine pesticides such as alpha-BHC, chlorobenzilate, p,p'-DDT, dieldrin and quintozene.     

The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity

Mutagens in urban air pollution come from anthropogenic sources (especially combustion sources) and are products of airborne chemical reactions. Bacterial mutation tests have been used for large, multi-site, and/or time series studies, for bioassay-directed fractionation studies, for identifying the presence of specific classes of mutagens, and for doing site- or source-comparisons for relative levels of airborne mutagens. Early research recognized that although carcinogenic PAHs were present in air samples they could not account for the majority of the mutagenic activity detected. The mutagenicity of airborne particulate organics is due to at least 500 identified compounds from varying chemical classes. Bioassay-directed fractionation studies for identifying toxicants are difficult to compare because they do not identify all of the mutagens present, and both the analytical and bioassay protocols vary from study to study. However, these studies show that the majority of mutagenicity is usually associated with moderately polar/highly polar classes of compounds that tend to contain nitroaromatic compounds, aromatic amines, and aromatic ketones. Smog chamber studies have shown that mutagenic aliphatic and aromatic nitrogen-containing compounds are produced in the atmosphere when organic compounds (even non-mutagenic compounds) are exposed to nitrogen oxides and sunlight. Reactions that occur in the atmosphere, therefore, can have a profound effect on the genotoxic burden of ambient air. This review illustrates that the mutagenesis protocol and tester strains should be selected based on the design and purpose of the study and that the correlation with animal cancer bioassay results depends upon chemical class. Future emphasis needs to be placed on volatile and semi-volatile genotoxicants, and on multi-national studies that identify, quantify, and apportion mutagenicity. Initial efforts at replacing the Salmonella assay for ambient air studies with some emerging technology should be initiated.     

Study of metabolic activation of chemical compounds by using microorganisms. I. Effect of inducers of microsomal systems]

The effect of psychotropic drugs phenobarbital, benzonal, hexamidine and steroid hormone hydrocortizon acetate on the process of metabolic activation of mutagenicity of nitrosomorpholine, cyclophosphamide and benzidine was examines using tester strains TA 1950 and TA 1538 of Salmonella typhimurium (by B. N. Ames). The listed above activators did not modify essentially the mutagenic effect of benzidine. The mutagenic action of nitrosomorpholine was increased by the presence of hydrocortizon acetate. Psychotropic drugs phenobarbital and its structural analogues increased the mutagenic effect of cyclophosphamide and nitrosomorpholine. Phenobarbital was the most potent as an inducer. Benzonal occupied the intermediate position according to the including activity of mutagens examined. Phenobarbital has shown to increase both the content of rat liver microsomal proteins and the specific activity of those. A possible role of microsomal enzymatic inducers as modifiers of the effects of environmental mutagens is discussed.     

Detection of mutagenic derivatives of cyclophosphamide and a variety of other mutagens in a “microtitre”R fluctuation test,without microsomal activation

The fluctuation test recently introduced by Green et al. has been used on a ‘microtitre’ scale, to detect mutagenic activity in the breakdown product or products of CP formed during its storage in phosphate buffer.The mutagenic product(s) accumulated over a period of 72 h at 37°C, however after 7 days further degradation into non-mutagenic forms seemed to occur. At 23°C although a slower breakdown into mutagenic product(s) was observed, these persisted even after 7 days at this temperature.The breakdown product or products produced base-pair mutations in E. coli strains, WP2, WP2 uvrA, which appeared to be subject to the excision-repair system in this bacterium. Frameshift mutations were not detected using S. typhimurium strain TA98.The ‘microtitre’ fluctuation test was also used to detect a variety of other mutagens, including proflavin (an acridine); NG (alkylating agent); Nifuroxime (nitrofuran); Hycanthone (anti-schistosomal drug): AZ (azo-dye) and MMC (anti-neoplastic antibiotic). It appears to be as sensitive as the macroscale test, and it can be used with both the E. coli strains and Salmonella strains currently available. Because of its potential automation the use of a ‘microtitre’ system greatly extends the application of fluctuation testing as a general screen for mutagenic compounds. These tests involve chronic treatment of the test strains with low doses of mutagen, which corresponds more closely to the environmental situation.     

Mutagenicity of trialkyltriazenes: Mutagenic potency of alkyldiazonium ions,the putative ultimate carcinogens from dialkylnitrosamines

Although aryldialkyltriazenes have been known for many years and their mutagenic, carcinogenic and carcinostatic properties have been investigated, almost nothing is known of the related trialkyltriazenes. Our recently developed general preparative route to these substances has allowed the examination of the mutagenic properties of several representative examples of this class of compounds. This, 1-benzyl-3,3-dimethyl-, 3-benzyl-1,3-dimethyl-, 3-benzyl-3-ethyl-1-n-butyl- and 1,3-di-n-butyl-3-methyltriazenes are direct acting mutagens in the TA1535 strain of Salmonella typhimurium. The respective mutagenic potencies of these substances can be accounted for by the in situ generation of alkyldiazonium ions. These ions are considered to be strong candidates for the ultimate mutagens/carcinogens derived from some dialkylnitrosamines.     

Occurrence, identification, and bacterial mutagenicity of heterocyclic amines in cooked food

Potent mutagenic activity in Salmonella bacteria has been reported in cooked foods in numerous laboratories worldwide. Determining the human risk from exposure to these biologically active compounds in our diet requires genotoxic and carcinogenic evaluation of the chemicals coupled with determination of the dose consumed. Thus, knowledge of the exact structure of the mutagens present in the food and enough synthesized material for biological assessment are essential for this evaluation. To reach this goal, isolation of these compounds requires the Ames/Salmonella assay to guide the purification and identification process. Mass and NMR spectrometry are used to identify the isolated compounds. Finally, these findings are followed by synthesis of the exact isomer. The predominant class of mutagens found in cooked foods of the western diet are amino-imidazo-quinoxalines, amino-imidazo-pyridines and amino-imidazo-quinolines, collectively called amino-imidazoazaarenes (AIAs). Mass amounts of these specific compounds range from less than 1 to 70 ng/g of meat. The mutagens are formed from the heating of natural precursors (creatinine, amino acids, and possibly sugars) in the food. These AIAs are some of the most potent mutagens ever tested in Salmonella bacteria with the number and position of methyl groups having an important influence on the mutagenic activity.     

The role of bacterial metabolism in transformation of non-mutagenic compounds into mutagens. I. Participation of Escherichia coli nitroreductase in creation of mutagens from non-mutagenic new pesticides]

Investigations concerned Escherichia coli nitroreductase in creation of mutagens from non-mutagenic pesticides-derivatives of urea. Three new compounds were studied: N-phenyl-N'-methylurea (IPO 4328), N-methyl,N-(2-hydroxyethyl)-N'phenylurea (IPO 2363), N-(2-hydroxyethyl), N-methyl-N'-(3,4 dichloroethyl) urea, and diurone-3-(3,4 dichlorophenyl)-1,1 dimethylurea. These compounds were incubated in anaerobic conditions with cells of E. coli K-12 (KF) strain and nitrate or nitrite. Using Ames test, mutagenicity of resulting metabolites was investigated. It was found that during incubation of herbicide IPO 4328 with cells of E. coli K-12 (KF) and nitrate, mutagenic product for strain of S. typhimurium TA 1537 is created. Very weak mutagenic metabolite for the same strain was appearing during incubation of herbicide IPO 2363 with cells of E. coli K-12 (KF) in presence of nitrite. Incubation of investigated compounds with E. coli K-12 (KF) cells alone did not result in appearance of mutagenic substances. Thus, role of Escherichia coli in creation of mutagenic compounds from non-mutagenic derivatives of urea consisted of nitrite from nitrate production with participation of nitroreductase, which afterwards in absence of bacteria or action of their enzymes reacted with investigated pesticides.