Estimates of genotoxic effects of mutagens at low and protracted doses are often based on linear extrapolation of data obtained at relatively high doses. To test the validity of such an approach, a comparison was made between the mutagenicity of N-methyl-N-nitrosourea (MNU) in T-lymphocytes of the rat following two treatment protocols, i.e. sub-chronic exposure to a low dose (15-45 repeated exposures to 1mg/kg of MNU) or acute exposure to a single high dose (15, 30 or 45 mg/kg of MNU). Mutation induction appeared dramatically lower following sub-chronic treatment compared to treatment with a single high exposure. Furthermore, DNA sequence analysis of the coding region of the hprt gene in MNU-induced mutants showed that acute high dose treatment causes mainly GC-->AT base pair changes, whereas sub-chronic treatment results in a significant contribution of AT base pair changes to mutation induction. We hypothesize that O(6)-methylguanine-DNA methyltransferase is saturated after acute treatments, while after sub-chronic treatment most O(6)-methylguanine is efficiently repaired. These data suggest (i) that risk estimations at low and protracted doses of MNU on the basis of linear extrapolation of effects measured at high dose are too high and (ii) that the protective effects of DNA repair processes are relatively strong at low sub-chronic exposure.

The usefulness of fluorescence in situ hybridization (FISH) with rat satellite I DNA was compared with immunocytochemical staining with CREST serum for the analysis of the content of micronuclei from primary rat fibroblasts. We analyzed micronuclei induced in vitro by the aneugenic compound diethylstilbestrol (DES) or the clastogenic compound mitomycin C (MMC). Since a centromeric probe was not available for the rat, we isolated rat satellite I DNA by PCR with primers designed on the basis of the known rat satellite I DNA sequence. The PCR products obtained as well as the cloned PCR products showed hybridization to the centromeric regions of a large number of chromosomes, but not of chromosome 1, 19, 20, X and Y. Clone 18-5 was further analyzed and was shown to contain at least 4 repeats of the rat satellite I family. This probe, which hybridizes in the centromeric region of 34 of the 42 chromosomes, was used throughout the study as a probe for the FISH analysis of the micronuclei. For the immunocytochemical staining, the commonly used commercial anti-centromeric antibodies could not be used because of the weakness of the fluorescent signals given. Consequently, CREST serum of a single patient was used, which showed bright and distinct signals on the kinetochores of each chromosome. After treatment of the cells with the aneugen DES an increase in centromere (FISH) and kinetochore (CREST) positive micronuclei was found, whereas after treatment with the clastogen MMC, the percentage of centromere-positive micronuclei was similar to that observed in controls. Analysis of a large number of DES-induced micronuclei showed that the immunocytochemical method is equally as or slightly less sensitive for the detection of chromosomes in micronuclei and we therefore recommend FISH with probe 18-5 for the detection of chromosome loss in rat cells.

The distribution of genotoxic factors in various organs of mice treated orally with nitro-aromatic compounds of actual or potential use as chemotherapeutic (antiprotozoal and anthelminthical) agents was investigated in the DNA-repair host-mediated assay, with mice as host animals and a pair of E. coli K12 strains differing in DNA-repair capacity as indicators of genotoxicity. The test substances were derivatives of nitroimidazole (metronidazole), nitrofuran (SQ 18 506) and nitrodiphenylamine (amoscanate).

The effect of the mixed-function oxidase inhibitor phenylimidazole (PI) and the amine oxidase inhibitors iproniazid (IPRO) and aminoacetonitrile (AAN) on the mutagenic activity of various carcinogens was determined in intrasanguineous host-mediated assays, using mice as hosts and E. coli 343/113 as an indicator of mutagenic activity. The carcinogenic compounds dimethyl-, diethyl-, methylethyl-, and diethanolnitrosamine (DMNA, DENA, MENA, and DELNA respectively) and 1,2-dimethylhydrazine (SDMH) were administered i.p. to mice pretreated or not with one of the inhibitors. After 4 h exposure to each of the carcinogens, E. coli cells recovered from the liver of non-pretreated mice showed considerable induction of VALr mutations; after pretreatment of the hosts with the three inhibitors, significant reduction of the amounts of induced mutants in vivo was observed. Particularly, PI proved a very efficient inhibitor of DENA, MENA, DELNA, and SDMH mutagenicity (93%–97% reduction), suggesting that these carcinogens are mainly activated by cytochrome P-450-dependent enzymes. However, since PI might also inhibit the NAD-mediated activation of DELNA by alcohol dehydrogenase (ADH), the present experiments do not rule out an additional role of ADH in the in vivo mutagenic activation of DELNA. AAN and IPRO were less and much less effective, respectively, in reducing the mutagenic activity of all compounds. Surprisingly, PI showed less inhibition of the mutagenic activity of DMNA (60% reduction), as compared to the other carcinogens; this indicates that metabolic routes other than the cytochrome P-450-dependent enzyme system may be important for the activation of DMNA.

The distribution of genotoxic factors in various organs of mice treated orally with methylazoxymethanol-beta-D-glycoside (cycasin) was investigated using the DNA-repair host mediated assay. Indicator of genotoxic activity was a pair of streptomycin dependent Escherichia coli strains differing vastly in DNA repair capacity; uvrB/recA vs. uvr+/rec+. The animal-mediated assays were performed by injecting mixtures of the two strains i.v. and orally into mice, which were subsequently treated with the test chemical and from which the differential survival of the indicator bacteria present in several organs was determined. The same strains and selection procedures were also used for assessing the DNA-damaging activity in vitro. In the animal-mediated assays in which cycasin was applied orally, significant effects were observed at doses of 100 and 500 mg/kg body weight. The organ distribution of genotoxic factors in the host animal was as follows: the highest genotoxic activity was observed in the liver, followed by intestine and stomach; a clear effect was also observed in the kidneys and, to a lower extent, in the blood stream and in the lungs at the highest dose administered (500 mg/kg body weight). Under in vitro conditions a marginal genotoxic effect was observed even in the absence of liver homogenate, indicating that the test compound is possible activated (hydrolysed) by the E. coli cells. Therefore the genotoxic activity of cycasin observed in the gastrointestinal tract was not unexpected, since the substance was applied orally, thereby exposing the indicator bacteria in these organs to high levels of unmetabolised compound, especially in the stomach. In the intestine members of the microbial flora probably contribute to the metabolic activation of the test compound. The occurrence of genotoxic factors remote from the gastrointestinal tract shows that the present compound or active metabolites thereof penetrate through the intestinal barrier. The extraordinarily high genotoxic activity observed in the liver suggests that the compound is additionally activated in this organ. In compliance with previous in vitro findings this second activation step might lead to the formation of the highly reactive aldehydic form of methylazoxymethanol (MAMAL) mediated by dehydrogenases. Comparison with carcinogenicity studies indicates a good correlation between the distribution of genotoxic effects as determined in the present studies and the localisation of tumors in various organs of rodents treated with cycasin.

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

The DNA repair host-mediated assay, in which repairable DNA damage is determined in E. coli cells present in various organs of mice exposed to genotoxic agents, was further developed to broaden the range of organs under study and to simplify the procedure of assessing differential bacterial cell survival. A pair of derivatives of E. coli K-12 strain 343/113 was constructed which differed vastly in DNA repair capacity (uvr+/rec+ vs uvrB/recA), as a means of assessing DNA damaging effects; furthermore, the strains differed in their ability to ferment lactose (Δ Lac vs Lac+), so that the individual survival of both strains could be determined on a single agar medium (containing neutral red as pH indicator), on which the strains had different colony colour morphology (red, Lac+ vs white, Lac− colonies). Finally, the strains were made streptomycin-dependent, to prevent uncontrolled growth of the bacterial cells within the various organs and also to inhibit contamination of the survival agar medium by representatives of the normal intestinal microflora.

Studies were performed to determine the DNA interactions of and the induction of cytotoxic effects by the radical cation (CPZ+·) formed enzymatically from chlorpromazine (CPZ): in the presence of native DNA the lifetime of CPZ+· is markedly increased. The decreased reactivity of CPZ+· in the presence of native DNA and the concomitant increased viscosity of CPZ+·-DNA complexes strongly support the assumption that CPZ+· does form intercalation complexes with DNA. The relative strong bacteriotoxicity of CPZ+· hindered the accurate determination of mutagenesis in various Salmonella indicator strains, but a test for repairable DNA damage in Escherichia coli using various repair-deficient strains indicated that the cytotoxic action of CPZ+· is in part due to DNA alterations which can be excised in wild-type DNA repair-proficient strains. After activation of CPZ with long wavelength UV light, genetic effects are observed in S. typhimurium strain TA98, as well as in the E. coli tester strains. The possible role of CPZ+· in the photosensitization of CPZ is discussed.

The introduction of foreign cells (e.g. ascites tumor cells) into laboratory mammals and their subsequent recovery after treatment of the host with exogenous chemicals to determine the induction of genetic effects (e.g. chromosomal aberrations) is a technique which has been employed for more than 20 years. The use of bacteria as indicators of induced point mutations was first described by Legator et al. (1969). In their technique which they called a host-mediated assay, the microbes (e.g. Salmonella typhimurium) were injected into the peritoneal cavity of mice and, thus, exposed to potentially mutagenic metabolites of the compound under test. Today a wide variety of genetical changes can be detected in several indicator organisms. In addition to the histidine-requiring strains of Salmonella which allow the detection of different types of back-mutations, some other enterobacteria have proven useful. Auxotrophic strains of Serratia marcescens to detect back mutations, and Escherichia coli bacteria in which both forward and back mutations can be assayed simultaneously in several different genes. With fungi or fungal spores as indicators further effects of genetical importance can be determined, e.g. the consequences of recombination processes such as mitotic gene conversion and mitotic recombination in the yeasts, and of deletions in Neurospora crassa (conidia). After the successful development of methods to measure the induction of point mutations in cultured mammalian cells, it is also possible now to use established animal cell lines (e.g. mouse lymphoma cells) in a host-mediated assay provided isogenic or compatible hosts are available.

The phototoxic potential of a number of furocoumarins is well established. On the other hand, studies have shown that bergamottin, a furocoumarin containing a bulky, hydrophobic side chain, has significantly less or is even absent of phototoxicity potential. The OECD Test Guideline 432 3T3/Neutral Red Uptake (NRU) in vitro phototoxicity test has shown to be a highly predictive test for identifying compounds that exhibit no phototoxicological potential. In this study using OECD 432, the established phototoxic furocoumarin 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen were phototoxic, whereas bergamottin showed no phototoxic potential. When compared to 5-MOP, 8-MOP and psoralen, bergamottin was clearly negative at molar-adjusted concentrations that were more than 9 times higher than those that produced phototoxicity in 8-MOP; nearly 16 times than those for psoralen and more than 36 times higher than those for 5-MOP. These data using in vitro 3T3 NRU Phototoxicity Test (OECD 432) are supportive of earlier studies showing bergamottin does not exhibit phototoxicological properties. The detection and quantification of bergamottin should therefore not contribute to the potential marker furocoumarins for risk management interventions intended to reduce the phototoxicity of natural furocoumarin containing preparations.

The repeated isolation of mutants sensitive to radiation and also showing high spontaneous mutability indicated that in corresponding wild types some spontaneously occurring premutational lesions are repaired in a way similar to that for lesions induced chemically and by radiation. The question as to whether the high sensitivity and high mutability are pleiotropic manifestations of a single mutation or the results of different but closely linked mutations was investigated. Reveriants were treated with x radiation, ethyl methanesulfonate, isopropylmethanesulfonate, methyl methanesulfonate, N-methyl-N'-nitro- Nnitrosoquanidine, N-methyl-N-nitrosourea, and 5-methyl-DLtryptophan. Figures are presented to show survival following treatment with these agents.

The biauxotrophic strain of E. coli K-12 (λ), met 1/his 7, which exhibits an 8 times higher rate of met1→met+-backmutation as compared with the parent strain met 1, was found to be also more sensitive to UV irradiation. In addition, the maximum UV induction of prophage λ occurs at lower doses, and the capacity of the strain to propagate induced prophage is reduced. The mutant strain has also lost part of the ability to repair UV-induced lesions in phage T 1.