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 (i) exposure level, (ii) organ distribution of (DNA) dose, and (iii) 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 mutagenicity 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 > ENU ⩾ DES > DEN ≅ EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations. The results also indicated that O 6-ethylguanine was the DNA adduct most probably responsible for mutagenesis both in the bacteria and the mammalian cells. Comparative mutagenesis experiments performed with the E. coli cells in vitro with and without mammalian liver preparations (mouse S9) and in animal-mediated assays, in which the E. coli indicators were present in livers of treated mice, gave the following results: The addition of mouse liver S9 allows the detection of activating (DEN) or deactivating (ENNG) effects of mammalian metabolism, but the ranking order of mutagenic potency remained the same, namely ENNG > ENU > DES > EMS ≅ DEN. In the animal-mediated assays, on the other hand, the order of mutagenic potency was quite different, namely, DEN > ENU > ENNG > EMS. This latter order of activity is similar to the order of carcinogenicity of these compounds in rodents, and probably also reflects the degree of DNA alkylation in liver cells of the treated animals. These and other earlier results indicate that the relative ranking order of mutagenic potency observed in vitro, regardless whether it was obtained with bacterial or mammalian cells, is not representative of the situation in vivo. Although the addition in in vitro assays of mammalian liver preparations allows the detection of those compounds which are likely to be activated or deactivated in vivo, it appears that the magnitude of the effect is not adequately represented in the in vitro assays. In search for rapid methods for estimating genotoxic factors in various organs in vivo, use was made of the DNA repair host-mediated assay. In this test, repairable DNA damage is measured in pairs of E. coli K12 cells which differ by their ability of repairing lethal DNA damage, and which can be recovered from stomach, intestine, colon, liver, spleen, lungs, kidneys, testes and the blood of treated rodents (mice). Preliminary experiments using a variety of differently acting compounds have indicated that this assay procedure may become useful in rapidly assessing the presence and persistence of genotoxic factors in various organs of mice; its general usefulness as genotoxic dose monitor in vivo is presently being investigated.
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Apr 1, 2011
