title: Steroid synthesis-dependent, oxygen-mediated damage of mitochondrial and microsomal cytochrome P-450 enzymes in rat Leydig cell cultures.
abstract: Treatment of primary cultures of rat Leydig cells with 1 mM 8-bromo-cAMP for 2 days at ambient oxygen tension (19%) caused a 59% decrease in mitochondrial cholesterol side-chain cleavage (P-450scc) activity. This decrease was completely prevented when the oxygen tension was reduced to 1% O2 or when steroid synthesis was inhibited by aminoglutethimide. When the endogenous concentration of pregnenolone was increased by inhibiting its further metabolism, P-450scc activity was reduced by 80% in unstimulated cultures and was completely eliminated in cAMP-treated cultures. These losses were prevented when cells were maintained at 1% O2. The amount of immunoreactive P-450scc was also decreased by treatments that reduced P-450scc activity. Stimulation with cAMP also lowered microsomal C17-20 lyase activity by an oxygen-mediated, steroid synthesis-dependent mechanism. Treatment of cultures with testosterone caused a similar oxygen tension-sensitive decrease in C17-20 lyase activity. These results suggest that the enhanced loss of mitochondrial and microsomal cytochrome P-450 activities in cAMP-treated cultures is caused by the increased production of pregnenolone and testosterone, respectively, which generate reactive damaging species derived from reduced dioxygen. The increased catalytic turnover of these P-450 enzymes may also contribute to their damage. Although P-450 activities were preserved at 1% O2, the ability of cAMP-treated cells to synthesize testosterone in response to subsequent cAMP stimulation was still reduced. If, however, 25-hydroxycholesterol was supplied to these cells the decrease in testosterone-producing capacity was prevented, which demonstrates that the reduced steroidogenic capacity of cAMP-treated Leydig cells is caused, primarily, by the reduced availability of endogenous cholesterol.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0278.
authors.forename: M
authors.initials: M
authors.lastname: Georgiou
authors.forename: L M
authors.initials: LM
authors.lastname: Perkins
authors.forename: A H
authors.initials: AH
authors.lastname: Payne
mesh_terms: D000446:Aldehyde-Lyases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006888:Hydroxycholesterols / Q000494:pharmacology; D007985:Leydig Cells / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D011284:Pregnenolone / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000502:physiology; D011959:Receptors, Estradiol / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase; D013256:Steroids / Q000096:biosynthesis*
chemical_list: D006888:Hydroxycholesterols; D011944:Receptors, Androgen; D011959:Receptors, Estradiol; D013256:Steroids; D011284:Pregnenolone; C007997:25-hydroxycholesterol; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases; D010100:Oxygen