EXPRESSION AND ZONAL DISTRIBUTION OF CYP2D16 IN THE GUINEA-PIG ADRENAL-CORTEX - RELATIONSHIP TO XENOBIOTIC METABOLISM

We recently cloned a CYP2D subfamily member (CYP2D16) from a guinea pi g adrenal cDNA library and investigated the expression of CYP2D16 in t he guinea pig adrenal cortex and its relationship to adrenal xenobioti c metabolism. A modified sodium dodecyl sulfate-polyacrylamide gel ele ctrophoresis technique revealed three major bands in the molecular mas s range of cytochromes P450 in guinea pig adrenal microsomes, Two of t he bands were immunoreactive with anti-CYP17 (54 kDa) or anti-CYP21 (5 2 kDa) antibody. The third band (50 kDa) was immunoreactive with antib ody raised against CYP2D1 and with anti-CYP1A1/1A2 antibody. Microsequ encing of the 50-kDa band yielded an amino-terminal sequence of 38 ami no acids identical to that deduced from the CYP2D16 cDNA. In addition, Northern blot analyses indicated the CYP1A1 was not expressed in the adrenal gland, suggesting that only CYP2D16 composed the microsomal 50 -kDa band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis a nd Western blot analyses demonstrated greater expression of CYP2D16 in microsomes from the inner zone (zona reticularis) of the adrenal cort ex than from the outer zones, coinciding with the major site of adrena l xenobiotic metabolism. Bufuralol-1'-hydroxylase activity, a marker f or CYP2D isozymes, was also greater in inner- than in outer-zone micro somal preparations and was highly correlated with CYP2D16 concentratio ns. Northern blot analyses with a full-length CYP2D16 cDNA as the prob e gave strong bands with adrenal inner zone RNA preparations and relat ively weak bands with outer zone RNA. CYP2D16 mRNA was also detectable in liver and kidney RNA preparations, but at lower levels than in the adrenal inner zone, and it was not detectable in testes, lung, intest ines, or heart. Overall, the results demonstrate that CYP2D16 is expre ssed at highest levels in the inner zone of the guinea pig adrenal cor tex and suggest a major role for this isozyme in adrenal xenobiotic me tabolism.