Stability of microsomal monooxygenases in murine liver S9 fractions derived from phenobarbital and β‐naphthoflavone induced animals under various long‐term conditions of storage

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Abstract
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-re-ductase activity, and of the several monooxygenases, such as aminopyrine N-demethylase (class IIIA P450), p-nitroanisole O-demethylase (mixed), dinemorphan N-demethylase (HB1), ethoxyresorufin O-deethylase (IA1), ethoxycoumarin O-deethylase (mixed), and pentoxyresorufin O-dealkylase (IIB1), were examined in S9 fractions derived from Na-phenobarbital (PB) plus β-naphthoflavone (β-NF) induced male and female mice, stored at −80°C, or lyophilized and stored at −20°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 IA 1-like activity, for example, was much more stable (˜49%loss) than IIB 1-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°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.