Wide spectrum detection of precarcinogens in short-term bioassays by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes.
The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction procedure, using beta-naphthoflavone (beta-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combined 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 spectrum of induced cytochrome P450s. Five inducers of different 'classes' such as PB (class IIB P450s), beta-NF (IA), isosafrol (IA2), ethanol (IIE1) and pregnenolone 16 alpha-carbonitrile (IIIA) were injected daily both separately (to achieve maximal monooxygenase induction) in male and female mice. Induction was monitored using specific P450-linked activities. In the optimal schedule for complete induction, the various monooxygenases were greater (2- to 4-fold) than those achieved by the classical schedule. 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 the contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-transferase, 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 lesser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of the 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[alpha]pyrene, 2-naphthylamine and dimethylnitrosamine) or Saccharomyces cerevisiae 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.
Paolini
M
M
Dipartimento di Farmacologia, Universita' degli Studi, Bologna, Italy.
Sapigni
E
E
Hrelia
P
P
Scotti
M
M
Morotti
M
M
Cantelli-Forti
G
G
D000818:Animals; D001711:Biotransformation; D002273:Carcinogens / Q000493:pharmacokinetics*; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000378:metabolism; D004249:DNA Damage; D004790:Enzyme Induction; D005260:Female; D006900:Hydroxylation; D007527:Isoenzymes / Q000096:biosynthesis*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009152:Mutagenicity Tests; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism
D002273:Carcinogens; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019392:Cytochrome P-450 CYP2E1; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D009251:NADPH-Ferrihemoprotein Reductase