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 metabol izing enzymes in activating preparations for short-term genotoxicity b ioassays under various storage conditions. Expressions of cytochrome P 450 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-deethyla se (mixed), and pentoxyresorufin O-dealkylase (IIB1), were examined in S9 fractions derived from Na-phenobarbital (PB) plus beta-naphthoflav one (beta-NF) induced male and female mice, stored at -80 degrees C, o r lyophilized and stored at -20 degrees C. Lipid peroxidation was also determined. Cytochrome P450 and the associated activities were decrea sed by 30-82% within 9 months of storage. The pattern and degree of re lative stabilities were different for the various isoforms. The IA1-li ke activity, for example, was much more stable (similar to 49% loss) t han IIB1-like activities (up to 82% loss). In general, lyophilized enz ymes 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 f resh, daily made S9 fractions or, alternatively, fractions stored in l iquid nitrogen (up to 6 months) are recommended for mutagenesis studie s. (C) 1994 Wiley-Liss, Inc.