title: Screening for drug-induced alterations in the production and release of steroid hormones by porcine adrenocortical cells in vitro.
abstract: To screen drugs rapidly and at minimal expense for their potential to alter steroidogenesis, an in vitro model using porcine adrenocortical cells was developed. Pregnenolone, progesterone, deoxycorticosterone or corticosterone (all at 1 muM) were used as substrates. Drug-induced changes in the production/release of aldosterone were measured after 1-hr incubation. With pregnenolone, drug-induced effects on the release of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, HO-progesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. For assessment of cell viability and the amount of steroids produced/released, a cheap, simple modified Krebs solution was at least comparable to an elaborate cell culture medium. Within the conditions adopted, the cell suspension reacted to varying potassium concentrations as expected. ACTH stimulated steroid production/release only without added substrate. 11 agents known to interfere with steroid biogenesis were tested at 0.1-100 muM. Although all known points of action of the test compounds were located, several showed additional activity. Spironolactone shifted steroid biogenesis from aldosterone and cortisol towards androgenic steroids. Aminoglutethimide inhibited the release of aldosterone with corticosterone as substrate, but not with deoxycorticosterone or progesterone as substrate, revealing an alternative pathway in the biogenesis of aldosterone by-passing corticosterone. Trilostane (0.1-1 muM) completely blocked conversion of pregnenolone to progesterone and OH-progesterone; the release of androstenedione was at most only halved, whereas the release of dehydroepiandrosterone and testosterone was greatly enhanced. This implies isoenzymes of 3beta-hydroxysteroid dehydrogenase/isomerase with different sensitivities towards trilostane. Mitotane, metyrapone, ketoconazole and etomidate all inhibited the mitochondrial P450 (11beta 18 ) enzymes. In addition, mitotane and ketoconazole also inhibited (albeit to a lesser extent) endoplasmic enzymes involved in transformations at C21 and at C17, respectively. Cyproheptadine blocks all transformations with progesterone or HO-progesterone as starting point. Finasteride reduced the release of most steroids, except the androgens, presumably by inhibition of transformations at C3 and at C11. Carbadox and related quinoxalines inhibited not only C18 oxidation but also C21 hydroxylation. Steroidogenesis in these porcine adrenocortical cells in vitro could be described as similar to that in other mammals. A notable feature was that inhibition of the release/production of a steroid hormone was usually accompanied by an increased release of other steroid hormones. This screening model also yields information about the point of action of drugs interfering with steroidogenesis.
authors.affiliation: D.L.O.-Central Veterinary Institute (CDI-DLO), Department of Biochemistry and Toxicology, Postbus 65, 8200 AB, Lelystad, The Netherlands.
authors.forename: L P
authors.initials: LP
authors.lastname: Jager
authors.forename: G J
authors.initials: GJ
authors.lastname: De Graaf
authors.forename: H C
authors.initials: HC
authors.lastname: Widjaja-Greefkes