Mutagens that are not carcinogens: faulty theory or faulty tests?

In the National Toxicology Program database of 172 chemicals that were judged non-carcinogenic or equivocal in 2 year rodent studies in both sexes of rats and mice, there are 38 chemicals that were mutagenic in Salmonella. All but two of the chemicals had structural alerts for mutagenicity. The largest proportion of the mutagenic non-carcinogens were benzeneamines and substituted benzeneamines. In all, 12 of the mutagenic non-carcinogens had mutagenic carcinogen analogues, and for two chemicals, the carcinogenic analogues were not mutagenic. Non-carcinogens that were mutagenic in Salmonella also tended to be mutagenic and clastogenic in mammalian in vitro tests. The mutagenic responses are discussed and explanations offered for the mutagenicity and lack of carcinogenic activity of these chemicals.     

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.     

Evolution of the P450 gene superfamily: animal-plant 'warfare', molecular drive and human genetic differences in drug oxidation

Drug-metabolizing enzymes, such as those encoded by the cytochrome P450 genes, are noted for their high degree of interspecies and intraspecies variability. We believe that much of this diversity is the result of continuous molecularly driven coevolution of plants producing phytoalexins and animals responding with new enzymes to detoxify these chemicals. One consequence of human P450 gene evolution is polymorphism in drug metabolism, leading to marked differences in the response of individuals to the toxic and carcinogenic effects of drugs and other environmental chemicals.     

Structural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants

A set of 189 chemicals tested in the National Toxicology Program Cancer Bioassay was subjected to analysis by CASE, the Computer-Automated Structure Evaluation system. In the data set, 63% of the chemicals were carcinogens, approx. 40% of the carcinogens were non-genotoxic, i.e., they possessed neither "structural alerts" for DNA reactivity as defined by Ashby and Tennant, 1988, nor were they mutagenic for Salmonella. The data base can be characterized as a "combined rodent" compilation as chemicals were characterized as "carcinogenic" if they were carcinogenic in either rats or mice or both. CASE identified 23 fragments which accounted for the carcinogenicity, or lack thereof, of most of the chemicals. The sensitivity and specificity were unexpectedly high: 1.00 and 0.86, respectively. Based upon the identified biophores and biophobes, CASE performed exceedingly well in predicting the activity of chemicals not included among the 189 in the original set. CASE predicted correctly the carcinogenicity of non-genotoxic carcinogens thereby suggesting a structural commonality in the action of this group of carcinogens. As a matter of fact biophores restricted to non-genotoxic carcinogens were identified as were "non-electrophilic" biophores shared by genotoxic and non-genotoxic carcinogens. The findings suggest that the CASE program may help in the elucidation of the basis of the action of non-genotoxic carcinogens.     

Comparison of a forward and a reverse mutation assay in Salmonella typhimurium measuring L-arabinose resistance and histidine prototrophy

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.     

Formation of mutagenic N-nitroso compounds from the pesticides prometryne, dodine and carbaryl in the presence of nitrite at pH 1

Environmental chemicals including pesticides carrying secondary and tertiary amino groups are suggested to be a health hazard to man since potentially carcinogenic nitroso compounds may be formed in the presence of nitrite at low pH values resembling conditions in the human stomach. Nitrosation of the isopropylamino-triazine Prometryne, the n-dodecyl guanidine Dodine and the N-methylcarbamate carbaryl was investigated in the presence of HCl and acetic acid at pH 1 and excess sodium nitrite for 4 h at 37 degrees C. The reaction products were extracted with CCl4 and were analyzed qualitatively and quantitatively by infrared spectroscopy, nuclear-resonance spectrometry, GC/mass spectrometry and by spectrophotometry. All compounds investigated formed N-nitroso derivatives in the following yields: carbaryl 67%, Dodine 12% and Prometryne 14%. The N-nitroso derivatives per se were not or only slightly mutagenic to Escherichia coli K12 or Salmonella typhimurium TA 1538. However, significantly increased mutation frequencies were seen after metabolic activation by mouse-liver microsomes. These results add to the observations that among environmental chemicals not only those containing methyl- or ethyl-substituted amino groups form potentially carcinogenic nitroso derivatives but also those with iso-propylamino groups as well as alkyl-substituted guanidine derivatives.     

Predicting rodent carcinogenicity of Ames test false positives by in vivo biochemical parameters

28 chemicals known to be mutagenic in the Ames test but not carconigenic in rodent bioassays were selected for study. The chemicals were administered by gavage in 2 dose levels to female Sprague-Dawley rats. The effects of these 28 chemicals on 4 biochemical assays (hepatic DNA damage by alkaline elution (DD), hepatic ornithine decarboxylase activity (ODC), serum alanine aminotransferase activity (ALT), and hepatic cytochrome P-450 content (P450)) were determined. The scientific approach taken was to either experimentally find individual cancer predictors of high specificity or to mathematically create composite predictors of high specificity.

Composite predictive parameters are defined as follows: CP = [ODC and P450], CT = [ALT and ODC] and TS = [DD or CP or CT]. The specificity (percent of rodent noncarcinogens which test negative) of DD, ODC, ALT, P450, CP, CT and TS was 100%, 46%, 89%, 86%, 93%, 93% and 86%, respectively. For these 28 mutagenic noncarcinogens, the specificity of structural alerts (SA) 13%, mutation in mouse lymphoma cells (MOLY) 0%, chromosomal aberrations in Chinese hamster ovary cells (ABS) 13%, and sister-chromatid exchange in Chinese hamster ovary cells (SCE) 0% were much lower. The k_e test, an experimental measure of electron attachment, had a specificity of 33%. DD was the only DNA related parameter to predict well the noncarcinogenic rodent bioassay result of Ames false-positive chemicals. 5 nongenotoxic parameters (ALT, P450, CP, CT and [CP or CT]) predicted the rodent bioassay result well. Depending on the prevalence of chemicals carcinogenic to humans, the problem of Ames test false positives for predicting human cancer may be either small or large.     

Mutagenicities of quinoline and its derivatives.

Quinoline, recently reported to be carcinogenic in rats [12], was mutagenic to Salmonella typhimurium tester strains TA100 and TA98 in the presence of the metabolic activation system S-9 mix. 2-Chloroquinoline, a non-carcinogen [12], was non-mutagenic with or without S-9 mix. 8-Hydroxyquinoline, which is t known to be carcinogenic, was mutagenic with S-9 mix to both bacterial strains. The mutagenicities of 17 other quinoline derivatives that are not known to be carcinogenic were tested, and 12 of these compounds were mutagenic.     

Decreased electrophilicity of chemicals carcinogenic only at the maximum tolerated dose.

While there was no significant difference between the actual or predicted mutagenicity and clastogenicity of a group of chemicals carcinogenic only at the maximum tolerated dose (MTD) and a group of chemicals carcinogenic below the MTD, as a group, the chemicals carcinogenic below the MTD exhibited a significantly decreased LUMO (Lowest Unoccupied Molecular Orbital) energy, indicative of increased electrophilicity (i.e. DNA reactivity). These findings suggest that chemicals carcinogenic only at the MTD either require increased doses of "weak" electrophiles to be carcinogenic or that they may act by a "non-genotoxic" mechanism.     

Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents

Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.     

Principles of formalizing a quantitative evaluation of the genetic danger of chemical compounds for man

Specific characteristics of the mutagenic effect of chemicals, which must be taken into account in developing the test system to assess the potential genetic risk caused by chemical substances, are considered. The organizational principles of the procedures currently available for testing and ranking chemicals by their mutagenic and carcinogenic hazard to humans are discussed. The use of selective information suggested by Wiener and Shannon as an efficiency measure of testing and estimating the potential genetic hazard of chemical substances is substantiated. The feasibility of this approach was demonstrated by testing the efficiency of the battery of two short-term in vitro tests as an example. It was shown that selective information is able to serve as an integral universal criterion of the efficiency of testing, if either one test or the test battery were used.     

Bacterial systems for carcinogenicity testing

During the past 30 years, bacterial test systems have been extensively refined in their ability to detect not only mutagenic agents but, in many cases, carcinogenic ones as well. Since many carcinogens are known to be activated within the mammalian body, major improvements in bacterial test systems were made when representative parts of mammalian metabolism were included as part of the test protocol. Presently, systems of great simplicity and convenience are available for the efficient detection of gene mutations, lysogenic induction of prophages, and differential DNA repair. These qualities render bacterial systems potentially useful in distinguishing between carcinogens and non-carcinogens, in characterizing induced mutation spectra, and possibly in quantifying mutagenic potency that may be used to predict tumor-initiating potency. Sensitive strains of Salmonella typhimurium. Escherichia coli and Bacillus subtilis with altered DNA-repair capacities have been constructed which accurately identify many carcinogens. Comparative studies have shown that techniques using these strains can be standardized to some extent and that the majority of carcinogens are active in all adequately sensitive genetic systems. Because of this redundancy, it may be sufficient to employ only one standardized set of tester strains and methodology. However, serveral classes of known carcinogens are undetected or underestimated when assayed in standard testing procedures. Some of these chemicals can be efficiently recognized as mutagens upon varying the methodology, the genetic endpoint, or the mammalian activation system. Thus, to modify and adjust the experimental protocol to the particular type of chemical under study and to calibrate the system with appropriate carcinogenic and non-carcinogenic reference compounds is advisable. It is noteworthy that chemical carcinogens which probably act by non-genotoxic mechanisms thus far remain undetected in bacterial tests. Newly developed systems which measure specific types of genetic events, such as transpositions of DNA segments and derepression of genes, presently are being tested for their ability to detect such carcinogens. A final matter of growing concern is the increasing number of environmental chemicals that are found to be mutagenic in bacteria but for which information about carcinogenic activity in vivo is insufficient. The possible use of bacteria for quantifying mutagenic potency and extrapolating this information to tumor-initiating potency can be envisaged in three ways: (i) direct extrapolation from standard in vitro tests, (ii) indirect extrapolation making use of an in vitro/in vivo comparison of induced effects (the parallelogram method) as devised by Sobels [138] on the basis of identical dose (to DNA), and (iii) host-mediated assays to assess mutagenic potency of carcinogens in selected organs of mammals...     

The lack of ability of carcinogens and non-carcinogenic analogs to induce sex-linked recessive lethals in Drosophila melanogaster

The mutagenic activity of two known carcinogens (benzo(a) pyrene and 2-acetylaminofluorene) and that of two structurally closely related but not carcinogenic compounds (pyrene and 4-acetylaminofluorene) was examined by the Muller-5 test for sex-linked recessive lethals (SRL). The chemicals tested were applied to the food medium for larvae of Canton-S Drosophila melanogaster. No statistically significant differences in frequencies of induced SRL were found either within pairs of chemicals or between treated and untreated animals.     

A system for assessment of chemical substances on mutagenicity in man: general priniciples, practical recommendations and further elaboration]

As a basis of the suggested test-system, the following conditions are observed: 1) the economy of fulfilment in a short time; 2) the analysis of gene and chromosome mutations in germ and somatic cells; 3) the evaluation of mutagenic effects of not only substance, but also of the products of its metabolism; 4) including in the system only the tests which give the minimal variability between separate experiments; 5) the evaluation of dose-effect relationship. The practical scheme of testing is divided into two parts: a screening and a complete one. The screening programme consists of two tests: a) a test on microorganisms with a metabolic activation in vitro; b) a cytogenetic analysis of bone marrow of mammals. The complete programme of testing includes 4 tests: a) a test on microorganisms with a metabolic activation in vitro and in vivo; b) a test of dominant lethal mutations on mammals; c) a cytogenetic analysis of bone marrow of mammals; d) a cytogenetic analysis in the culture of human lymphocytes. There are good reasons for the principles of selection of substance for testing according to the screening and complete programme: population occurence, economic (or medical) significance, information about relative chemicals showing mutagenic, carcinogenic and teratogenic effect. In the group of chemicals which are to be tested according to the screening programme, such ones can be included: industrial chemicals, phosphoorganic insecticides, drugs which are taken by a limited group of patients. The group of chemicals which are to be tested according to the complete programme consists of the following ones: pesticides, food additices, widespread drugs, the chemicals of the group 1, if during one of the tests of the screening programme a genetic effect is detected. At the genetic risk estimation it is advisable to keep to the following rule: a positive effect, identified in any object of the system must in the direct meaning extrapolate on men. The quantitative evaluation of the mutagenic danger of a chemical can be determined by the increase of the spontaneous level of mutations in the test-object on the basis of an average dose and exposition of the given chemical in the human population. Those chemicals are subject to the quantitative evaluation, which have shown a mutagenic activity during any of the test-objects; they are also widespread and because of their social or economic value can not be replaced or excluded from taking. From the point of view of genetics any substance with a mutagenic activity is dangerous and must be prohibited from using or replaced by any other non-mutagenic chemical, or limited by the contact of persons of non-reproductive age. As a temporary measure from a hygienic point of view, it is recommended to evaluate this chemical as especially mutagenic and prohibit or limit its using, when its average population dose produces 1/10 or more increase of the spontaneous level of mutations.     

Metabolic studies and the evaluation of genetic risk from the viewpoint of industrial toxicology

In recent years some important industrial chemicals, e.g. solvents, and monomers used in the production of plastics, have been found to be more dangerous than had been suspected. Some of them are mutagens and carcinogens. The active substance may be the compound itself, but more frequently it is a reactive intermediate, an alkylating agent, formed from the parent compound by oxidation under the influence of liver-microsome mixed-function oxidases. Trichloroethylene, vinyl chloride, 1,1-dichloroethylene and 2-chloro-1,3-butadiene can be mentioned as examples, where mutagenic or carcinogenic activity has been demonstrated; similar activity of some others is to be suspected. Metabolic studies may explain unexpected effects or they may allow one to predict such effects. Tests on microorganisms with a metabolic activation in vitro seem to be extremely valuable for fast and efficient screening of man-made chemicals to be introduced into the environment, because such tests combine the use of liver microsomes and bacteria for the detection and classification of the effect. Mutagenicity tests in mammals do not lose their usefulness, since they take into account the distribution of the compound in the body, its transport to the target organ, species-specific differences etc. As exposures of workers to chemicals mentioned above have been quantitatively measured regularly, epidemiological studies should be performed with regard to mutagenic and carcinogenic effects in relation to the degree of exposure. Here is a chance for better understanding of how to extrapolate experimental results to human populations and how to establish safe levels of chemicals in the environment if at all possible and acceptable.     

Genotoxicity of non-covalent interactions: DNA intercalators

This review provides an update on the mutagenicity of intercalating chemicals, as carried out over the last 17 years. The most extensively studied DNA intercalating agents are acridine and its derivatives, that bind reversibly but non-covalently to DNA. These are frameshift mutagens, especially in bacteria and bacteriophage, but do not otherwise show a wide range of mutagenic properties. Di-acridines or di-quinolines may be either mono- or bis-intercalators, depending upon the length of the alkyl chain separating the chromophores. Those which monointercalate appear as either weak frameshift mutagens in bacteria, or as non-mutagens. However, some of the bisintercalators act as "petite" mutagens in Saccharomyces cerevisiae, suggesting that they may be more likely to target mitochondrial as compared with nuclear DNA. Some of the new methodologies for detecting intercalation suggest this may be a property of a wider range of chemicals than previously recognised. For example, quite a number of flavonoids appear to intercalate into DNA. However, their mutagenic properties may be dominated by the fact that many of them are also able to inhibit topoisomerase II enzymes, and this property implies that they will be potent recombinogens and clastogens. DNA intercalation may serve to position other, chemically reactive molecules, in specific ways on the DNA, leading to a distinctive (and wider) range of mutagenic properties, and possible carcinogenic potential.     

'Our load of mutation': reappraisal of an old problem.

H. J. Muller, in a paper in 1950 entitled 'Our load of mutation', predicted the genetic decay of the human species due to increasing mutation pressure combined with relaxation of natural selection. In the meantime, much information on spontaneous and induced mutations in humans has been accumulated, and a reappraisal of Muller's conclusions gives a much less gloomy overall picture. However, a certain increase of malformation and disease can be predicted as a result of ionizing radiation and chemical mutagens. On the other hand, genetic counselling and antenatal diagnosis of genetic anomalies may help to keep the genetic risks within tolerable limits. Research on the biological conditions for the untoward effects of mutagenic chemicals considered necessary for the wellbeing of humans may also help to reduce genetic risks. The extent and kind of the risks as well as possibilities for prevention are discussed with a few examples.     

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.     

Identification of cytochrome P450 2D6 and 2C9 substrates and inhibitors by QSAR analysis

This paper presents four new QSAR models for CYP2C9 and CYP2D6 substrate recognition and inhibitor identification based on human clinical data. The models were used to screen a large data set of environmental chemicals for CYP activity, and to analyze the frequency of CYP activity among these compounds. A large fraction of these chemicals were found to be CYP active, and thus potentially capable of affecting human physiology. 20% of the compounds within applicability domain of the models were predicted to be CYP2C9 substrates, and 17% to be inhibitors. The corresponding numbers for CYP2D6 were 9% and 21%. Where the majority of CYP2C9 active compounds were predicted to be both a substrate and an inhibitor at the same time, the CYP2D6 active compounds were primarily predicted to be only inhibitors. It was demonstrated that the models could identify compound classes with a high occurrence of specific CYP activity. An overrepresentation was seen for poly-aromatic hydrocarbons (group of procarcinogens) among CYP2C9 active and mutagenic compounds compared to CYP2C9 inactive and mutagenic compounds. The mutagenicity was predicted with a QSAR model based on Ames in vitro test data.     

Mutagenic properties of a nitrofuran, 7-methoxy-2-nitronaphtho[2, 1-b]furan (R7000), in lacI transgenic mice

The in vivo mutagenic properties of a 5-nitrofuran, the 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), already well known in bacteria, was evaluated in lacI transgenic mice (Big Blue). The mutation frequency was determined in various organs of i.p. - treated mice and the nature of induced mutations was determined for the target organs in which mutation induction was significant. It was found that R7000 is mutagenic in mice, although, on the basis of the number of induced mutants per unit mass in comparison with other known mutagenic chemicals, R7000 appears to be considerably less mutagenic in mice than in bacteria. The most affected organs, small intestine, caecum and colon organs belong to the digestive apparatus. The distribution of R7000-induced mutations in the lacI gene recovered from small intestine of transgenic mice was very similar to that which had been found in E. coli. The difference between mouse and E. coli in the R7000 induced mutational spectra are mainly in the proportion of single base frameshifts versus base substitutions. Since R7000 induced mutations seemed to arise in the population of stem cells and that the stem cells are important for carcinogenesis, our results are compatible with a possible carcinogenic effect of R7000 and other nitrofurans.