Stability of microsomal monooxygenases in murine liver S9 fractions derived from phenobarbital and beta-naphthoflavone induced animals under various long-term conditions of storage.
The aim of this study was to define the long-term stability of metabolizing enzymes in activating preparations for short-term genotoxicity bioassays under various storage conditions. Expressions of cytochrome P450 content, NADPH-cytochrome (P450) c-reductase activity, and of the several monooxygenases, such as aminopyrine N-demethylase (class IIIA P450), p-nitroanisole O-demethylase (mixed), dinemorphan N-demethylase (IIB1), ethoxyresorufin O-deethylase (IA1), ethoxycoumarin O-deethylase (mixed), and pentoxyresorufin O-dealkylase (IIB1), were examined in S9 fractions derived from Na-phenobarbital (PB) plus beta-naphthoflavone (beta-NF) induced male and female mice, stored at -80 degrees C, or lyophilized and stored at -20 degrees C. Lipid peroxidation was also determined. Cytochrome P450 and the associated activities were decreased by 30-82% within 9 months of storage. The pattern and degree of relative stabilities were different for the various isoforms. The IA1-like activity, for example, was much more stable (approximately 49% loss) than IIB1-like activities (up to 82% loss). In general, lyophilized enzymes were less stable than directly frozen preparations. In addition, immediately after freeze-drying (lyophilization), a marked decrease in activity of up to 35% was observed. On the contrary, demethylation of aminopyrine and p-nitroanisole remains almost constant over 6 months storage at -196 degrees C. The results obtained indicate that either fresh, daily made S9 fractions or, alternatively, fractions stored in liquid nitrogen (up to 6 months) are recommended for mutagenesis studies.
Bauer
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Dipartimento di Fisiologia e Biochimica, Laboratori di Biochimica, Pisa, Italy.
Corsi
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Paolini
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D000633:Aminopyrine N-Demethylase / Q000032:analysis; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000737:chemistry; D004790:Enzyme Induction; D004795:Enzyme Stability; D005260:Female; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis; D010634:Phenobarbital / Q000494:pharmacology; D012737:Sex Factors; D014021:Tissue Preservation; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D000633:Aminopyrine N-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital