Abstract 537 Endocrinology & Diabetes II Platform, Saturday, 5/1

Low doses of medroxyprogesterone acetate (MPA) can suppress steroidogenesis by suppressing the hypothalamic-pituitary-gonadal axis, but in high doses, MPA can also suppress steroidogenesis at the level of the gonad. Thus doses of 0.1 mg/kg/d will induce withdrawal bleeding in adult women, but doses of 2 to 5 mg/kg/d are needed to suppress steroidogenesis in testes of boys with familial gonadotropin-independent sexual precocity (testotoxicosis) or in the ovarian cysts of girls with the McCune-Albright syndrome, and doses up to 20 mg/kg/d are used in breast cancer. Nearly 20 years ago, studies using cultured rat Leydig cells indicated that MPA inhibited several enzymes, primarily 17 α-hydroxylase and 17,20-lyase (Acta Endocrinol 94:419, 1980); these two activities are now known to be catalyzed by a single enzyme, cytochrome P450c17. We examined the effects of MPA on the first three enzymes in the steroidogenic pathway, the human forms of P450scc (the cholesterol side-chain cleavage enzyme), P450c17, and 3 βHSDII. Concentrations of MPA up to 100 µM failed to inhibit P450scc activity in human placental JEG3 cells, as measured by a lack of effect on immunoassayable pregnenolone synthesis. To study the effect of MPA on P450c17 and 3 βHSDII, we developed a genetically engineered yeast system in which we can express human forms of steroidogenic enzymes and their cofactor proteins, prepare subcellular fractions containing the enzymes, and perform detailed, quantitative kinetic analyses. MPA failed to inhibit either the 17 α-hydroxylase or 17,20-lyase activities of human P450c17 in microsomes from these genetically engineered yeast as well as in microsomes from human adrenal glands. Thus MPA does not inhibit human P450c17, even if MPA does inhibit rat P450c17. However, in yeast expressing human 3 βHSDII, MPA was a competitive inhibitor of both the microsomal and cytoplasmic forms of 3 βHSDII with a Ki of 3.0 µM. This Ki is similar to the 5.2-5.5 µM Km that we determined for each of the three principal substrates of 3 βHSDII: pregnenolone, 17 α-hydroxypregnenolone, and DHEA. Recent studies show that doses of 5-20 mg MPA/kg/d achieve micromolar concentrations (Eur J Cancer 33:1407, 1997). Thus, the action of MPA to inhibit steroidogeneis is by substrate competition with 3 βHSDII. The genetically engineered yeast system permits us to assess the actions of drugs on each individual steroidogenic enzyme, providing an important in vitro tool for assessing pediatric endocrine therapies.