The unique role of rodents in the detection of possible human carcinogens and mutagens

Some of the probable reasons underlying the observation that not all chemicals shown to be genotoxic in vitro are capable of eliciting tumours in rodents or humans are discussed using appropriate examples. It is suggested that a substantial proportion of the resources currently available for conducting rodent carcinogenicity bioassays should be employed in the short-term evaluation in vivo of some of the many hundreds of chemicals recently defined as genotoxic in vitro, rather than in the protracted evaluation of a few chemicals, often of unknown activity in vitro, for carcinogenicity. A decision tree approach to the evaluation of chemicals for human mutagenic/carcinogenic potential is presented which is at variance with the construction and philosophy of many of the current legislative guidelines. The immediate need for the adoption of one of the available short-term in vivo liver assays, and/or the development of a short-term in vivo rodent assay capable of concomitantly monitoring different genetic end-points in a range of organs or tissues is emphasized.     

The conceptual framework of monitoring mutagens/carcinogens: a critical appraisal

The main theme of this paper is an analysis of the present knowledge and conceptual framework in the field of monitoring for carcinogenic and mutagenic agents. The conclusions of the International Symposium on "Monitoring human exposure to carcinogenic and mutagenic agents" (Helsinki, 1983) were taken as a starting point for a series of considerations; methods suitable for the quantitative treatment of the information were applied to evidence the profile of current research in this area. The analysis indicated that the major underlying paradigms were related to technical considerations, such as chemical specificity, precision, reproducibility or ease of execution, as well as to the concept of mechanism of action of genotoxic agents. On the other hand, the lack of reliable information about the operational relevance of the systems for assessing health effects was dramatically evidenced. As a conclusion, the dangers of relying on a scientific background derived only from the basic research, without a critical re-examination in terms of real, operational performances of the systems, are emphasized.     

Studies of DNA-strand induced in human fibroblasts by chemical mutagens/carcinogens.

A method for the study of DNA-strand breaks using alkaline denaturation followed by hydroxylapatite chromatography has been modified and used for the detection of chemically induced DNA-strand breaks. A new procedure for the incubation of human fibroblasts with a metabolizing system and the detection of DNA-strans breaks is presented. With this method the induction and repair of DNA-strand breaks have been studied in human fibroblasts exposed to methyl methanesulphonate, melphalan, benzo[a]pyrene and cyclophosphamide. These agents all give rise to DNA-strand breaks. In cells exposed to methyl methanesulphonate, melphalan or benzo[a]pyrene these breaks disappeared within 21 h after re moval of the drug. In cells exposed to the bifunctional alkylating agent cyclophosphamide, studies of DNA-strand breaks suggest the presence of inter-strand cross links.     

Methods for analysis of the mutagenicity of indirect mutagens/carcinogens in eukaryotic cells

Summary The review discusses the variety of methods for activation of indirect mutagens/carcinogens and testing them in cell cultures, especially in mammalian cell cultures.After the necessity for including metabolizing components in mutagenicity tests has been pointed out, the enzymes that transform foreign compounds metabolically, and the factors influencing them, are described. In the main section the various methods of activating indirect mutagens/carcinogens are presented. The methods of including in vivo metabolism in mutagenicity tests are: Analysis of cells from organisms contaminated with a chemical (III.1.a); body fluid-mediated mutagenesis (III.1.b); host-mediated assay (III.1.c).The following activation systems are suitable for including in vitro metabolism of test compounds in mutagenicity tests: Liver and lung perfusion (III.2.a.); organ slices and homogenates (III.2.a.); subcellular fractions (III.2.a.); cultivated cells (cell-mediated mutagenesis) (III.2.b); nonenzymatic activation systems (III.2.c).Finally the main factors that influence the metabolism of test substances are summarized. Two figures illustrate the mutagenicity tests with regard to the metabolism of mammalian livers and the methods of performing mutagenicity tests in man.     

Epidemiology in protection and prevention against environmental mutagens/carcinogens Examples from occupational medicine

Subjects occupationally exposed to potential mutagens/carcinogens represent the most suitable groups for epidemiological studies aimed at assessing the risk for the individual or the offspring. Several cancer risks to humans have been detected by epidemiological studies performed in occupational settings. Cancer epidemiology studies have been able (a) to identify specific occupations or agents associated with the risk; (b) to verify the results of experimental studies; (c) to test the effectiveness of changes in production or preventive measures in decreasing risks. Reproductive epidemiology has suggested a risk of spontaneous abortions or of malformation in the offspring of workers exposed to some chemicals or occupations, but data are often conflicting due to methodological problems. With the aim of early assessment of risk in mind, the epidemiological use of indicators of exposure or of the early effect of exposure to genotoxic agents is increasingly applied to occupational groups. Cytological monitoring of subjects at risk of occupational cancer of lung or bladder is carried out mainly to diagnose precancerous lesions of target tissues. Cytogenetic methods (chromosome aberrations, SCE, micronuclei) in somatic cells provide a means for detecting early effects of occupational exposure to known or potential mutagens/carcinogens in selected groups of individuals, but their significance is widely debated. Monitoring of urinary mutagenicity, as applied in nurses handling cytostatic drugs, is an example of how an indicator of exposure to genotoxins can be used to evaluate the impact of preventive measures. Among the perspectives, biochemical epidemiology seems to be promising in detecting individuals genetically susceptible to cancer.     

Lycopersicon assays of chemical/radiation genotoxicity for the study of environmental mutagens

From a literature survey, 21 chemicals are tabulated that have been evaluated in 39 assays for their clastogenic effects in Lycopersicon. Nineteen of the 21 chemicals are reported as giving a positive reaction (i.e. causing chromosome aberrations). Of these, five are reported positive with a dose response. In addition, 23 assays have been recorded for six types of radiation, all of which reacted positively. The results of 102 assays with 32 chemicals and seven types of radiation tested for the induction of gene mutations are tabulated, as well as 20 chemicals and/or radiation in combined treatments. The Lycopersicon esculentum (2n=24) assay is a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis and for somatic mutations induced by chemicals and radiations. The Lycopersicon bioassay has been shown to be as sensitive and as specific an assay as other plant genotoxicity assays, such as Hordeum vulgare, Vicia faba, Crepis capillaris, Pisum sativum and Allium cepa and should be considered in further studies in assessing clastogenicity. Tests using L. esculentum can be made for a spectrum of mutant phenotypes of which many are identifiable in young seedlings.     

Zea mays assays of chemical/radiation genotoxicity for the study of environmental mutagens

From a literature survey, 86 chemicals are tabulated that have been evaluated in 121 assays for their clastogenic effects in Zea mays. Eighty-one of the 86 chemicals are reported as giving a positive reaction (i.e. causing chromosome aberrations). Of these, 36 are reported positive with a dose response. In addition, 32 assays have been recorded for 7 types of radiation, all of which reacted positively. The results of 126 assays with 63 chemicals and 12 types of radiation tested for the inductions of gene mutations are tabulated, as well as 63 chemicals and/or radiation in combined treatments. Three studies reported positive results for mutations on Zea mays seed sent on space flights. The Zea mays (2n=20) assay is a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis and for somatic mutations induced by chemicals and radiations. The carcinogenicity and Salmonella assays correlate in all cases. The maize bioassay has been shown to be as sensitive and as specific an assay as other plant genotoxicity assays, such as Hordeum vulgare, Vicia faba, Crepis capillaris, Pisum sativum, Lycopersicon esculentum and Allium cepa and should be considered in further studies in assessing clastogenicity. Tests using Zea mays can be made for a spectrum of mutant phenotypes of which many are identifiable in young seedlings.     

Use of yeast respiratory adaptation test system to detect chemical mutagens/carcinogens in mammals.

An approach to follow distribution of injected DNA-acting chemicals (mutagens/carcinogens) in animal tissues has been described. This is based on the use of respiratory adaptation (mitochondrial biogenesis) process in Saccharomyces cerevisiae during transition from anaerobic to aerobic state. By virtue of specific interaction of such chemicals with mitochondrial DNA associated with promitochondrial structures this process is extremely sensitive to DNA-acting chemicals. Solutions of berylium sulphate, aflatoxin G1, aflatoxin B1, and carbaryl (all known DNA-acting agents) were injected to rats at low concentrations and, after 24 hr, distribution of these chemicals or their metabolites was studied by determining the inhibitory action of appropriately diluted urine and tissue homogenates on respiratory adaptation in S.cerevisiae. Detectable amounts of the chemicals and their DNA-acting metabolites could be analyzed in urine, liver, lungs, kidney and spleen.     

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.     

Inhibitory effects of coffee on the genotoxicity of carcinogens in mice

The mouse bone marrow micronucleus test was carried out to evaluate the possible inhibitory effects of 3 doses (125, 250 and 500 mg/kg) of standard instant coffee on the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP), aflatoxin B1 (AFB1) and urethane (UR). Coffee was orally administered twice, 2 and 20 h before the carcinogens were injected intraperitoneally. From the results obtained, it was evident that the administration of 250 and 500 mg coffee/kg body weight could significantly inhibit the in vivo genotoxicity of these carcinogens. A linear dose response was observed for the inhibitory effect of coffee. Furthermore, inhibition of genotoxicity by coffee was observed in bone marrow cells which were sampled at 6-h intervals (48, 54, 60, 66 and 72 h) from the time of peak induction of micronuclei by DMBA.     

Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater

Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B₁ to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment.     

Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater.

Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B1 to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.