The preparation of hepatocytes using the two-stage perfusion technique usually involves the use of collagenase (CII) alone or in combination with dispase (C/D) or trypsin inhibitor (CA/TI) as digestion enzymes. The effect of CII, C/D and CA/TI on cell viability, yield, cytochrome P450 mediated oxidation of testosterone, glucuronidation and sulfation of 7-hydroxycoumarin, glutathione content, glutathione-S-transferase activity and glutathione-conjugation capacity of hepatocytes has been assessed. Cytochrome P450 mediated oxidation of testosterone was significantly (p < 0.05) decreased with CII isolated hepatocytes (81.7 +/- 3.3 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (96.6 +/- 1.9 nmol/10(6) cells) or C/D (95.1 +/- 2.1 nmol/10(6) cells). In contrast, glutathione conjugation of the non-specific substrate 1-chloro-2,4-dinitrobenzene was significantly (p < 0.05) increased with CII isolated hepatocytes (56.9 +/- 5.9 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (36.0 +/- 3.7 nmol/10(6) cells) or C/D (31.6 +/- 3.7 nmol/10(6) cells). These findings have significant implications for the interpretation of metabolism data derived from hepatocytes in suspension, particularly in terms of glutathione conjugation of potentially toxic reactive intermediates of xenobiotic metabolism. Indeed, data presented show that the presence of trypsin inhibitor in the preparation of isolated rat hepatocytes significantly affects the formation of glutathione conjugates of reactive intermediate products of troglitazone metabolism.
- trypsin inhibitor
- reactive intermediates