Effect of periportal- and centrilobular-equivalent oxygen tension on liver specific functions in long-term rat hepatocyte cultures

Abstract

The influence of periportal- and centrilobular-equivalent oxygen tensions on cellular functions and xenobiotic metabolism was investigated in rat hepatocytes cultured under 20% (air/CO 2), 13% or 4% O 2 for up to 9 days on teflon membrane dishes, coated with crude membrane fractions and collagen. Protein content, total cytochrome P-450 content, 7-ethoxy-resorufin- O-deethylase (EROD) and the profile of hydroxytestosterone metabolites were not significantly affected by the oxygen tension, whereas intracellular lactate dehydrogenase activity and albumin secretion were increased at 4% O 2 in comparison with 13 and 20% O 2 cultures. The induction of P-450 isoenzymes and corresponding catalytic activity was determined on days 4 and 9, following a 3-day exposure to phenobarbital (PB, 0.75 m m) or 3-methylcholanthrene (3-MC, 6.25 μ m). On day 4, PB increased CYP2B1/B2 content 19-fold (4% O 2) or 27-fold (13% O 2), CYP2C6 six-fold at both oxygen tensions, and CYP3A2 two-three-fold only in 4% O 2 cultures. On day 9, CYP2B1/B2 was still significantly increased. A clear correlation was found between P-450 isoenzyme content and corresponding isoenzyme activities, as assayed by testosterone hydroxylation and EROD activity on day 4. 3-MC increased CYP1A1/2 11-fold and eight-fold in 4 and 13% O 2 cultures, respectively, and the EROD activity 37-fold (4% O 2) and 30-fold (13% O 2) on day 4. Activities were still measurable on day 9, whereas the corresponding apoproteins CYP1A1 was not detectable. These results suggest that hepatocytes are able to adapt their xenobiotic metabolism to different tissue oxygen tensions and confirm the likelihood that oxygen tension is an important modulator of the region-specific expression of xenobiotic metabolism in liver.