Cytochrome P-450 dependent monooxygenases in the mammalian adrenal cortex : properties and role in steroidogenesis and metabolism of polycyclic aromatic hydrocarbons

Sammanfattning: A cytochrome P-450-dependent hydroxylase system (llß-hydroxy- lase) participating in the steroidogenic pathway in bovine adrenal cortex mitochondria was investigated. Deoxycorticosterone and de- oxycorticosterone-21-sulphate bind to and are llß-hydroxylated by the same cytochrome P-450 species, i.e., cytochrome P-450^g. Sul- phoconjugation of deoxycorticosterone results in decreased velocity of transport across the mitochondrial inner membrane as well as a lower affinity for the cytochrome P-450^g. By the use of the slower penetrating sulphoconjugated steroid and the membrane impermeable protein reagent diazoniunbenzenesulphonate, it was concluded that the steroid binding site of the cytochrome P-450..g faces the matrix side of the mitochondrial inner membrane. In the presence of 0.3% of the non-ionic detergent Triton X-100, the cytochrome P-450^g was in a monomeric form and catalytically active in both NADPH and ortho-nitroiodosobenzene-supported hydroxylation reactions. On the basis of these results it was suggested that the monomeric form of the cytochrome is the active form of the protein. Homogeneous NADPH-adrenodoxin reductase was purified with high specific activity and in good yields by a procedure involving affinity chromatography on 2',5'-ADP-Sepharose 4B. The reductase was active in reconstitution experiments with adrenodoxin and partially purified or membraneous P-450^g. The cytochrome P-4 50-dependent metabolism of polycyclic aromatic hydrocarbons, i.e., 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP), in rat adrenal was also investigated. DMBA and BP, of which DMBA is adrenocorticolytic whereas BP is not, were shown to be metabolized by rat adrenal microsomes at specific activities exceeding those of uninduced rat liver microsomes; with both substrates reactive products were formed that bind covalently to macroraolecules. DMBA and BP appear to be metabolized by a common cytochrome P-450 species which is unrelated to various known steroid hydroxylases but may be related to the metabolism of estradiol. Attempts to correlate the adrenocorticolytic action of DMBA to differences between DMBA and BP with respect to metabolism and metabolites bound to protein were unsuccessful. Various possible mechanisms by which DMBA causes necrosis in the rat adrenal are discussed.