The role of steric effects in the direct mutagenicity of N-acyloxy-N-alkoxyamides

Electrophilic N-acyloxy-N-alkoxyamides are mutagenic in Salmonella typhimurium TA100 without the need for S9 metabolic activation and they react with DNA at guanine-N7 at physiological pH. Since these are direct-acting mutagens, structural factors influence binding and reactivity with DNA. Mutagenicity in TA100 can be predicted by a QSAR incorporating hydrophobicity (logP), stability to substitution reactions at nitrogen (pK(a) of the leaving acid) and steric effects of para-aryl substituents (E(s)). A number of mutagens exhibit activities that deviate markedly from the predicted values and they fall into two classes: di-tert-butylated N-benzoyloxy-N-benzyloxybenzamides, which - because of their size - are most probably excluded from the major groove or are unable to achieve a transition state for reaction with DNA, and N-benzoyloxy-N-butoxyalkylamides with branching alpha-to the amide carbonyl, which are resistant to S(N)2 reactions at the amide nitrogen.     

Steric effects on the direct mutagenicity of N-acyloxy-N-alkoxyamides-probes for drug-DNA interactions

N-Acyloxy-N-alkoxyamides (see structure 1, below) are direct-acting mutagens for which a QSAR has been established that predicts with accuracy their activity in the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium TA100. Steric bulk next to oxygen on the alkoxyl side-chain in structure 4 has no impact on activity, but branching at the position adjacent (alpha) to the ester-carbonyl of the leaving group in structure 5 strongly inhibits mutagenicity. Both results reflect the manner in which these molecules interact with DNA. The alkoxyl group has greater flexibility, which minimises steric effects within the major groove. Bulk adjacent to the carbonyl of the ester group must impose conformational constraints that impede reaction at the N7 position of guanine. A new, expanded QSAR shows a clear dependence of activity on logP, although with a smaller coefficient relative to indirect-acting mutagens.     

Urinary and faecal mutagenicity in car mechanics exposed to diesel exhaust and in unexposed office workers

The occurrence of mutagens in the urine and faeces of a group of car mechanics (n = 8) exposed to high concentrations of diesel exhaust in their working place and of a group of office workers (n = 9) not exposed to diesel exhaust during working hours was compared. The aim of the study was to investigate whether the specific diesel exposure and/or other, more lifestyle-related, factors such as diet had any influence on the mutagenicity of excreta. Faeces were collected and pooled for a consecutive period of 48 h, urine was collected in the same period, but in 4 separate portions representing the urine produced during the day and at night on the 2 collection days. Information about food intake was collected by a 2-day dietary record method. Smoking habits and medicinal drug use were recorded as well. Air particulates were collected in and outside the garage during working hours. The mutagenicity of extracts of air particulates (methanol extracts), urine (XAD-2 and XAD-7 extracts) and faeces (acetone, ether and ether-NaOH extracts) was examined in the Ames test. The results did not suggest that exposure to diesel exhaust mutagens enhanced the incidence and/or degree of either faecal or urinary mutagenicity. Urine of 2 mechanics appeared to contain rather high levels of XAD-7 mutagens, but in view of the uneven distribution over the different collection periods any relationship with the exposure to diesel exhaust mutagens seems improbable. Degree and frequency of faecal mutagenicity was higher in office workers than in mechanics. The pattern of faecal mutagenicity was characteristic of that of faecapentaenes. Statistical analysis did not reveal any consistent relationships between urinary and faecal mutagenicity and the various dietary variables.     

Mutagenicity of aliphatic epoxides.

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity, trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

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.     

‘Petite’ mutagenesis and mitotic crossing-over in yeast by DNA-targeted alkylating agents

Although the biological properties (cytotoxicity, mutagenicity and carcinogenicity) of alkylating agents result from their bonding interactions with DNA, such compounds generally do not show any special binding affinity for DNA. A series of acridine-linked aniline mustards of widely-varying alkylator reactivity have been designed as DNA-directed alkylating agents. We have considered whether such DNA targeting has an effect on mutagenic properties by evaluating this series of drugs in comparison with their untargeted counterparts for toxic, recombinogenic and mutagenic properties in Saccharomyces cerevisae strain D5. The simple untargeted aniline mustards are effective inducers of mitotic crossing-over in this strain, but resemble other reported alkylators in being rather inefficient inducers of the “petite” or mitochondrial mutation in yeast. However, the majority of the DNA-targeted mustards were very efficient petite, mutagens, while showing little evidence of mitotic crossing-over or other nuclear events. The 100% conversion of cells into petites and the lack of a differential between growing and non-growing cells are similar to the effects of the well characterised mitochondrial mutagen ethidium bromide. These data suggest very different modes of action between the DNA-targeted alkylators and their non-targeted counterparts.     

Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemicals were not created equal

The nitrated polycyclic aromatic hydrocarbons constitute a group of chemicals of environmental concern which display a broad spectrum of mutagenic, genotoxic and carcinogenic properties. Some members of the group are the most potent direct-acting bacterial mutagens while others exhibit low levels of potencies which require metabolic activation mixtures. Bacterial mutagenicity is dependent upon reduction of the nitro function. In mammalian cell systems the genetic and genotoxic effects of these nitrated chemicals include the induction of unscheduled DNA synthesis, sister-chromatid exchanges, chromosomal aberrations, gene mutations and cell transformation. The qualitative as well as quantitative expression of these effects is dependent upon the species and tissue of origin as well as culture history of the cell which in turn determine their enzymic capabilities and the conversion of these nitroarenes to ultimate mutagens and genotoxicants. In eukaryotic cells the following bioactivation pathways have been recognized: (a) reduction of the nitro moiety, (b) ring oxidation (the nature of which is influenced by the nitro function) followed by reduction of the nitro group, and (c) ring oxidation without concomitant reduction of the nitro moiety.     

Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

Inhibitors of topoisomerase II enzymes: a unique group of environmental mutagens and carcinogens

Many inhibitors of topoisomerase II enzymes are potent mutagens, leading to major chromosomal deletions, illegitimate recombination and aneuploidy. There is increasing evidence that they are also human carcinogens. However, their lack of chemical reactivity means that they may give weak or negative results in commonly used mutagenicity tests, or may give data with characteristics quite distinct from chemicals that alkylate DNA. They do not form DNA adducts and assays such as ³²P-postlabelling will not detect their presence in the body. They are generally not point mutagens and may fail to provide distinctive fingerprints in mutation spectra. These characteristics may be limiting a realistic evaluation of their role in human carcinogenesis using current methodologies.     

Toxicity, mutagenicity, intracellular drug concentration and DNA binding in Escherichia coli treated with cis-platinum(II) complexes

The genotoxic effects of six cis-platinum(II)chloramine complexes with different alkyl substituents on their amine ligands have been measured using Escherichia coli. The toxicity and mutagenicity of these compounds were compared, after exposure of bacteria, to drug concentrations which gave known quantities of platinum-DNA lesions. The results permit several observations concerning structure-activity relations of platinum(II) complexes. Firstly, methyl substitution on the amine ligands of cis-diamminedichloro-platinum(II) (DDP) is reported to reduce its antitumor activity. The methyl group did not exert an effect in bacteria where the toxicity and mutagenicity of cis-bis(methylamine)dichloroplatinum(II) and DDP were equivalent. In fact, at equal levels of DNA binding, complexes with substituted amines were generally more toxic toward bacteria than DDP. Secondly, replacement of the chloro groups of DDP by nitrato ligands increased its toxicity and mutagenicity at a given level of DNA binding. Hence, although DDP and its dinitrato derivative have identical ammine ligands, they may form different platinum-DNA lesions in bacteria. Finally, cis-bis(cyclohexylamine)-dichloroplatinum(II) was unique among the compounds studied since it did not cause bacterial filamentation or mutagenesis. These results suggest that, although this compound binds to the bacterial genome, it may not induce the SOS response.     

Mutagens in feces from vegetarians and non-vegetarians

Mutagens in water extracts from feces of persons in 3 different diet groups were measured with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. The 3 diet groups were ovo-lacto vegetarians (N = 6), strict vegetarians (N = 11) and non-vegetarians (N = 12). All subjects were from the urban area of Vancouver, British Columbia, Canada. On TA100 ovo-lacto vegetarians and strict vegetarians had significantly lower levels of fecal mutagens than non-vegetarians (P less than or equal to 0.025 and P less than 0.010, resp.). The same pattern, although less significant, was obtained with TA98. Correlation studies between mutagenicity on TA100 and TA98 and between the pH of the fecal homogenate and mutagenicity indicate the presence of 2 or more major fecal mutagens.     

Genotoxicity studies with mineral oils; effects of oils on the microbial mutagenicity of precursor mutagens and genotoxic metabolites

In vitro genotoxicity assays are extensively used to predict carcinogenic activity in vivo. The standard microbial mutagenicity assays however often fail to yield positive results with mineral oils which are carcinogenic to mice in long-term skin-cancer studies. A comprehensive programme of studies has therefore investigated the basis of this apparently anomalous behaviour. This investigation has addressed the possible effects of oils on the bioactivation of precursor mutagens and the disposition of mutagenic metabolites by studying the microbial mutagenicity of selected precursor mutagens (benzo[a]pyrene, benzo[a]anthracene, 2-aminoanthracene and 2-naphthylamine) and intrinsically reactive mutagens [+/- )-benzo[a]pyrene-4,5-oxide and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) in the presence and absence of mineral oils. Notably the mutagenicity associated with the deliberate additions of these mutagens or precursor mutagens to oils was readily detected by the microbial assays. The mutagenicity of only one of the precursor mutagens, benzo[a]pyrene, was significantly reduced by the oils, and then only in the standard plate-incorporation assay. Interestingly the degree of suppression appeared to be related to the polycyclic aromatic hydrocarbon content of the oils. In the case of 2-aminoanthracene large enhancements in its mutagenicity were observed in the presence of oils. These latter findings appear to be due to effects of oils on the bioactivation of precursor mutagens rather than on the disposition of their bioactivation products. The mutagenicity of intrinsically reactive mutagens, of a type generated by bioactivation of polycyclic aromatic hydrocarbons, was not significantly reduced in the presence of mineral oils. This indicates that it is unlikely that components in oils trap or facilitate the deactivation of ultimate mutagens whether these pre-exist in the oil or are formed from precursors by bioactivation in the in vitro test system. Viewed overall these results suggest that mineral oils judged to be carcinogenic on the basis of in vivo studies in mouse skin may possess only very weak genotoxic potential. While this potential is likely to be a prerequisite for carcinogenic action, the current results cause attention to be focussed on other factors, e.g. promotion, as potentially important determinants of the carcinogenic potencies of mineral oils in mouse skin.     

Mammalian cell mutagenicity and metabolism of heterocyclic aromatic amines

Heterocyclic aromatic amines are bacterial mutagens which also induce DNA damage in mammalian cells. Damage has been demonstrated using a number of endpoints, including gene mutation, chromosome aberrations, sister-chromatid exchange, DNA-strand breaks, DNA repair and oncogene activation. Although the responses in mammalian cells are weak when compared to bacterial mutagenicity, heterocyclic aromatic amines are rodent carcinogens. Metabolic N-oxidation by cytochrome P450 is an initial activation step with subsequent transformation of the N-hydroxy metabolites to the ultimate mutagenic species by O-acetyltransferase or sulfotransferase. Major routes of detoxification include cytochrome P450-mediated ring oxidation followed by conjugation to glucuronic or sulfuric acid. Direct conjugation to the exocyclic amine group also occurs. Major reactions include N-glucuronidation and sulfamate formation.     

Inverse correlation between bacterial frameshift mutagenicity and yeast mitochondrial effects of antitumour anilinoacridines

The mutagenicity of a series of derivatives of 9-anilinoacridine, including the clinical antitumour agent amsacrine, has been assessed using a bacterial frameshift tester strain (Salmonella typhimurium TA1537) and a yeast petite colony assay (Saccharomyces cerevisiae 5178B). The results have been compared with microbial mammalian cell cytotoxicity, DNA binding affinity and acridine base strength (pKa). Compounds containing strong electron donor substituents on the acridine ring, and which have a high acridine pKa, show minimal frameshift mutagenicity but are strong inducers of petite yeast mutants. Conversely, some compounds which have a high DNA binding constant but a significant proportion of uncharged form at neutral pH, show high frameshift mutagenicity but minimal induction of petite mutants. It is hypothesised that this inverse relationship arises from the presence of trans-membrane drug transport mechanisms which act to exclude some compounds, particularly strongly basic compounds from the cytoplasm and to concentrate them in mitochondria.     

Mutagenicity of K-region epoxides of polycyclic aromatic compounds: Structure-activity relationship

The mutagenicity of several K-region arene oxides waas tested in histidine-dependent mutants of Salmonella typhimurium. Benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide as well as some substituted phenanthrene oxides were mutagenic in strains TA 1538 and TA 98 which detect frame-shift mutagens.Structure-activity relationships are discussed from the standpoint of chemical reactivity. The absence of direct correlation between electrophilic reactivity and mutagenicity may suggest that primilarily physical properties, such as relative position of the epoxide group and molecular shape of arene oxides, are important for the emergence of mutagenicity of arene oxides.     

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467. 8+/-211.0 nmoles-24 h (range 94.6-890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693+/-30 867 vs. 6877+/-6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0. 6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null (n=6) were on average higher than those of GSTM1-positive subjects (n=9) (55 498+/-45 957 vs. 34 156+/-11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136. 8+/-34.7 vs. 70.8+/-23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann-Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.     

On the mutagenicity of nitroimidazoles

Regarding mutagenicity, metronidazole is one of the best-investigated compounds of the nitroimidazoles. This drug is mutagenic on bacteria, especially if base-pair tester strains are used and bacterial nitroreductases are present. The serum levels attained in man after intake of this drug are sufficient to cause mutations in bacteria. Furthermore, interaction with and binding to DNA occurs under anaerobic conditions and sometimes DNA breaks are observed. However, metronidazole does not show mutagenic activity in mammalian cells in vitro; the micronucleus test is negative and chromosome aberrations are only found under anaerobic conditions. With microbial systems the mutagenicity of 47 nitroimidazoles has been investigated. Only 4 compounds were always negative in the applied test systems. Because with base-pair tester strains mutagenicity was assessed, this class of compounds should be regarded as a base-pair mutagen. In fungi, some compounds (e.g. ZK 26173 and azathioprine) are potent mutagens, whilst with most investigated nitroimidazoles only a weak or no mutagenic activity could be detected. Somewhat similar observations have been made in tests with Drosophila melanogaster, a test for gene mutations in mammalian cells, the micronucleus test, cytogenic tests and the dominant lethal test. The reduction products of metronidazole, misonidazole and 1-methyl-2-nitro-5-vinylimidazole, cause DNA damage if the nitro group is reduced in the presence of DNA. Reduction products are formed by microbes in the gut or by mammalian cells under anaerobic conditions. No teratological effect due to metronidazole or most other nitroimidazoles has been observed. Metronidazole is carcinogenic in mice and rats, and dimetridazole in rats. Up to the present, no carcinogenic effects have been observed in man. Azathioprine is probably carcinogenic for man. It is unlikely that the therapeutic applications of the presently used nitroimidazoles, except for azathioprine, will cause an increase in the tumor incidence in man or will cause other genotoxic effects, although such effects cannot be excluded with certainty.     

Testing for mutagens in an aluminium plant: The results of Salmonella typhimurium tests on urine from exposed workers

Urine concentrates from workers in a Söderberg potroom and an anode paste plant were tested for mutagenicity by the Salmonella reversion assay. The study is aimed at group exposure as an indicator of the effect of the work atmosphere.Urine from exposed smokers showed mutagenic activity, whereas urine from exposed non-smokers did not. The mutagenicity of exposed smokers' urine was not significantly different from the urine from non-exposed smokers. Significant mutagenicity of smokers' urine was evident only in the presence of a rat-liver metabolic system.Since an earlier expectorate analysis has shown that mutagens from the work atmosphere are deposited in the workers' respiration system and the urine analysis does not show any effect of occupational exposure, it is likely that the mutagens are eliminated from the body via other routes than renal excretion.     

Testing for mutagens in an aluminium plant. The results of Salmonella typhimurium tests on urine from exposed workers

Urine concentrates from workers in a S?derberg potroom and an anode paste plant were tested for mutagenicity by the Salmonella reversion assay. The study is aimed at group exposure as an indicator of the effect of the work atmosphere. Urine from exposed smokers showed mutagenic activity, whereas urine from exposed non-smokers did not. The mutagenicity of exposed smokers' urine was not significantly different from the urine from non-exposed smokers. Significant mutagenicity of smokers' urine was evident only in the presence of a rat-liver metabolic system. Since an earlier expectorate analysis has shown that mutagens from the work atmosphere are deposited in the workers' respiration system and the urine analysis does not show any effect of occupational exposure, it is likely that the mutagens are eliminated from the body via other routes than renal excretion.     

DNA-biding of IQ, Me-IQ and Me-IQx, strong mutagens found in broiled foods

Non-covalent DNA-binding has been studied of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (Me-IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (Me-IQx), strong mutagens found in broiled foods. These mutagens are intercalated into DNA, as found by ultraviolet absorption gel electrophoresis. The binding of IQ is stronger with GC pairs than AT pairs in DNA. The binding constants with calf thymus DNA are 1.6 × 10⁶ (Me-IQ), 0.9 × 10⁶ (IQ) and 0.7 × 10⁶ M⁻¹ (Me-IQx) at pH 6.0. This order of DNA affinity agrees with the order of mutagenicity towards Salmonella typhimurium TA98.