Oxygen Tension, Insulin, and Glucagon Affect the Preservation and Induction of Cytochrome P450 Isoforms in Cultured Rat Hepatocytes

Abstract

The role of oxygen tension, insulin, and glucagon on the preservation and induction of cytochrome P450 isoenzyme activities and contents was investigated in rat hepatocytes cultured for 4 days on crude liver membrane fractions at 4 or 13% O 2. At 13% O 2, three out of six immunochemically analyzed P450 isoenzymes were significantly higher than in 4% O 2. Exposure to phenobarbital (PB) from Days 1 to 4 dose dependently increased the protein content and decreased the albumin secretion in 13% O 2 cultures only. The maximal induction of P450 isoenzymes CYP2B1/2B2 (20- to 25-fold) and CYP2C6 (6-fold) were found at 0.75 mM PB at both oxygen tensions. In contrast, the highest induction of CYP1A1/1A2 (3-fold), of CYP3A (2-fold), and EROD activity were found with 3 mM PB in 4% O 2 cultures. CYP2B-dependent testosterone hydroxylation at positions 16 α/β was elevated to a greater extent in 13% O 2 cultures (96-fold at 0.75 mM PB) compared to 4% O 2 cultures (42-fold). This activity was affected by the insulin concentrations and the insulin:glucagon ratio. With decreasing insulin concentration (100 to 1 nM) or with increasing insulin:glucagon ratios (1:100-1:0.1), the enzyme activity increased preferentially in 4% O 2 cultures. The results of these investigations demonstrate that different tissue oxygen tension modulates the responsiveness of the cultured hepatocytes to the glucoregulatory hormones insulin and glucagon and this modulation results in a altered activity of cytochrome P450 isoforms.