Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Abstract

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[ a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P- 450 b e IgG established that low levels of “b-like” P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450′s b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti- P- 450 b e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (>2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (<1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (~40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti- P- 450 b e . A protein comigrating with P-450e was well above detection (6–7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity. PB treatment, therefore, increases both P-450's b and e in each of the extrahepatic tissues examined, consistent with hepatic induction although far less effectively. 3-Methylcholanthrene induced P-450c (>50-fold) in all tissues except the adrenal (only 3-fold) but with the exception of the latter, the associated activities were typically 10-fold lower than anticipated. These studies demonstrate that very small and even undetectable levels of individual forms of cytochrome P-450 play a major role in PAH metabolism in extrahepatic tissues.