EFFECTS OF FREEZING, THAWING, AND STORING HUMAN LIVER-MICROSOMES ON CYTOCHROME-P450 ACTIVITY

The stability of cytochrome P450 enzymes, cytochrome bg, and NADPH-cyt ochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsome s suspended in 250 mM sucrose and stored at -80 degrees C, and (C) hum an liver microsomes subjected to as many as 10 cycles of thawing and f reezing. In study A, microsomes from five human livers were prepared f rom fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b(5) and the activity of NADPH-cytochrome c reduc tase decreased 20-40% as a result of freezing the Liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases wer e observed in the activities of cytochrome P450 enzymes belonging to s everal gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP 2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'-hydro xylation), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzox azone 6-hydroxylation), CYP3A4/5 (testosterone 6 beta-hydroxylation), and CYP4A9/11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, bu t it increased the contamination of liver microsomes with hemoglobin o r other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b(5) and in the spec ific activity of all P450 enzymes. In study B, the concentration of cy tochromes P450 and bg, the activity of NADPH-cytochrome c reductase, a nd the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for ap proximately 0, I, or 2 years. The sample-to-sample variation in the co ncentration and activity of cytochrome P450, cytochrome bg, and NADPH- cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no diffe rential loss of cytochrome P450 activity, cytochrome bg concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activi ty. Overall, these results suggest that our current methods for storin g and processing human liver are well suited to preserving microsomal P450 enzyme activity. (C) 1996 Academic Press, Inc.