Wide spectrum detection of precarcinogens in short-term bioassays by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes

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Abstract
The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction preocedure, using β-naphthoflavon (β-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combinaed with sodium phenobarbital (PB), was found to be equally effective. The aim of this work is to realize a novel schedule of induction for the preparation of metabolizing systems containing a wider spctrum of induced cytochrome P450s. Five inducers of different ‘classes’ such as PB (classIIB P450s), β-NF (IA), isosafrol (IA2), ethanol (IIE1) and proegnenolone 16α-carbonitrile (IIA) were injected daily both separately (to achieve maximal monooxygenase induction) and in combination (to obtain the broadest P450s induction) in male and female mice. Induction was monitored using specific P450-linked activitgies. In the optimal schedule for complete induction, the various monoxygenases were greater ( 2- to 4-fold) than those achieved by the classical scheduel. More than a 14-fold increase of total P450 and 3.3-fold increase of NADPH-cytochrome (P450) c-reductase activity, over those uninduced, account for the above increase. For example, there was a marked increase in the deethylation of ethoxyresorufin (37-fold) compared to the uninduced mice that was considerably higher than classical induction (8-fold over uninduced). On kthe contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-ttransferase, glutathione S-epoxide transferase and UDP-glucuronosyl transferase, examined to compare the phase I/ phase II ratios in the traditional and proposed procedures, were increased to a leser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of kthe induced P450s elicited a higher mutagenicity response in the presence of superinduced fractions with respect to the classical one, when tested on Salmonella typhimurium (cyclophosphamide, benzo)a]pyrene, 2-naphthylamie and dimethylnitrosamine) or Saccharomyces cerevisiase D7 strain (diethylstilbestrol). These novel metabolizing biosystems, with an enhanced spectrum of induced P450s and oxidative/post-oxidative reaction rates, are recommended for detecting unknown xenobiotics in genotoxicity studies.

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