ADRENAL PROTEINS BOUND BY A REACTIVE INTERMEDIATE OF MITOTANE

Purpose: Mitotane (o,p'-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have s hown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride interm ediate. It was the objective of this study to compare the electrophore sis separation patterns of such adducts following activation of mitota ne by various adrenocortical sources. Methods: With the use of a I-125 -labeled analog of mitotane, 1-(2-chlorophenyl)- 1-(4-iodophenyl)-2,2- dichloroethane, gel electrophoresis patterns were obtained for homogen ates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-45 0(scc) and adrenodoxin, Results: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immuno-blotting analyses indic ated that the band at 49.5 kDa corresponded in molecular weight to tha t of adrenal cytochrome P-450(scc), but the band at 11.5 kDa did not c orrespond to adrenodoxin. Conclusions: The similarity of the results w ith canine and bovine adrenal preparations to that of human material o ffers useful systems for studying mitotane and its analogs. This shoul d aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.