© 1992 Oxford University Press
research-article |
Strategies for advancement of short-term mutagenicity tests: on the optimal ionic strength for the liver microsomal assay
1Dipartimento di Farmacologica, Laboratori di Biochimica Tossicologica
2Istituto di Cancerologia, Universitá degli Studi di Bologna, Italy
3Division of Environmental Toxicology and Community Health, University of Texas Medical Branch, Galveston, TX, USA
The aim of this work was to optimize the ionic strength (
) in the liver microsomal assay (LMA) hi performing short-term genotoxicity tests, optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, UIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the range 0.06–1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), gluthathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, ß-naphthoflavone, isosafrol, ethanol and pregnenolone 16-
carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (
sp), up to a 46% increase, was found at = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of sp for post-oxidative reactions was seen. LP was not changed appreciably by varying . In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at = 0.864 (2.25-fold) compared to the usual (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at = 0.86 compared to = 0.06 (traditional). These data show that = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased sp phase-I/sp phase-II ratio), as well as DNA binding and genotoxic response.