CHARACTERIZATION OF THE ADRENAL CYTOCHROME P450C17 IN THE HAMSTER, A SMALL ANIMAL-MODEL FOR THE STUDY OF ADRENAL DEHYDROEPIANDROSTERONE BIOSYNTHESIS

The hamster, like the human produces cortisol as its major glucocortic oid, rather than corticosterone, typical of most enzyme rodents. It is not known, however, if the hamster cytochrome P450C17 (P450C17), a ke y enzyme for cortisol formation, also exhibits 17,20-lyase activity an d if it catalyzes the formation of dehydroepiandrosterone (DHEA) at th e adrenal level. To study this, we isolated the cDNA of P450C17 from a hamster adrenal library. This cDNA was sequenced and was found to hav e an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids. The hamster P450 C17 cDNA, in the coding region, is 76% homologous with the human P450C 17 cDNA. The cDNA was then cloned in the expression vector pSV-SPORT 1 , which was transiently transfected into COS 1 cells. The transfected cells were used for temporal studies on the transformation of radiolab eled C-21-Delta(5)- and C-21-Delta(4)-precursors. When transfected cel ls were incubated with [C-14]pregnenolone, rapid formation of [C-14]DH EA occurred. The intermediate 17 alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA. Likewise, when incubate d with C-21-Delta(4)-steroids, [C-14]progesterone and [H-3]17 alpha-hy droxyprogesterone, the 17,20-lyase product androstenedione was produce d efficiently. In these studies, with respect to the Delta(5) pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector. However, in contrast to t he bovine enzyme, which converted low amounts of progesterone to andro stenedione, the expressed hamster P450C17 enzyme showed an active meta bolism via the Delta(4) pathway. Northern blot analysis, using the com plete alpha-(32)Plabeled hamster P450C17 cDNA as the probe, demonstrat ed a strong presence of P450C17 mRNA in hamster adrenals, a weaker pre sence in testes and ovaries, and no detectable species in brain, mesen tery, and kidney. Immunoblotting analysis using an anti-rat P450C17 an tibody demonstrated the presence of P450C17 protein in hamster adrenal s, testes, and ovaries. Hamster adrenal cell suspensions and microsoma l preparations were used to demonstrate the biosynthesis of [C-14]17 a lpha-hydroxypregnenolone and [C-14]DHEA from [C-14]pregnenolone; both metabolites were formed during incubations. However, the ratio of [C-1 4]DHEA/[C-14]17 alpha-hydroxypregnenolone was much lower in adrenal ce lls than in transfected COS 1 cells, indicating the presence of putati ve factors in hamster adrenal cells, favoring the 17 alpha-hydroxylase activity rather than that of the 17,20-lyase. In conclusion, these st udies demonstrate that the hamster adrenal is both a DHEA and a cortis ol producer, and, therefore, this animal could be a suitable small ani mal model for the study of the role of DHEA in relation to human bioch emistry and physiology.