Oxygen-mediated damage of microsomal cytochrome P-450 enzymes in cultured leydig cells. Role in steroidogenic desensitization.

P G Quinn,A H Payne

doi:10.1016/s0021-9258(17)43019-5

Abstract

Cultured Leydig cells exhibited time-dependent decreases in the microsomal cytochrome P-450 enzyme activities, 17 alpha-hydroxylase and C17-20 lyase when maintained under standard culture conditions (95% air, 5% CO2). Inclusion of the hydroxyl radical scavenger dimethyl sulfoxide in the culture medium, or the reduction of oxygen tension from 19 to 1% O2 was effective in preserving these enzyme activities and the combined effects of low O2 and dimethyl sulfoxide were synergistic. Leydig cells in culture were treated with 1 mM 8-Br-cAMP to induce steroidogenic desensitization which resulted in greater decreases in 17 alpha-hydroxylase and C17-20 lyase activities, as well as a diminished capacity to produce testosterone in response to subsequent acute stimulation with 8-Br-cAMP. Reduction of the oxygen tension from 19 to 1% O2 prevented this enhanced loss of microsomal P-450 activities in desensitized Leydig cells. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase, a microsomal enzyme which is not a P-450 enzyme, was stable in cultures of both control and desensitized Leydig cells under all culture conditions. These data are consistent with the hypothesis that oxygen-mediated damage is responsible for the time-dependent decrease in 17 alpha-hydroxylase and C17-20 lyase activities of control Leydig cells, and is the mechanism by which these microsomal P-450 activities are further decreased in desensitized Leydig cells. Desensitized Leydig cells exhibited a 50 and 70% decrease at 24 and 48 h, respectively, in their ability to produce testosterone in response to subsequent acute stimulation with 8-Br-cAMP, regardless of the culture conditions. Since desensitized Leydig cells cultured at 1% O2 showed no greater loss of enzyme activity than did controls, loss of microsomal P-450 activities is not the cause of the diminished testosterone biosynthetic capacity of desensitized Leydig cells.

Highlights

Cultured Leydig cells exhibited time-dependent de- zation of the steroidogenic pathway which, by definition, creases in the microsomal cytochrome P-450 enzyme results in a diminished capacity to produce testosterone in activities, 17a-hydroxylaseand c17-20 lyase when response to subsequent acute stimulationand is accompanied maintained under standard culture conditions (95% by decreases of the microsomal P-4501activities, 17a-hydroxair, 5%CO2)
A recent report from this laboratory responsible for the time-dependent decrease in 17a- demonstrated that decreases in 17a-hydroxylase and C17-20 hydroxylase and c17-20lyase activities of control Ley- lyase activities following administration of a single high dose dig cells, and is themechanism bywhich these micro- of luteinizing hormone were not necessarily associated with a somal P-450 activitiesare further decreased in desen- decrease in testosterone biosynthetic capacity (13)
Since desensitized Leydig cells cultured at 1% O2 showed no greater loss of enzyme activity than did controls, loss of microsomal P-450 activities isnot tion, reports from this laboratory and others have indicated that depletion of the precursor cholesterol, due to prolonged stimulation of steroidogenesis is an important factor in the limited steroidogenic response of desensitized Leydig cells (14-16)

Summary

Introduction

Cultured Leydig cells exhibited time-dependent de- zation of the steroidogenic pathway which, by definition, creases in the microsomal cytochrome P-450 enzyme results in a diminished capacity to produce testosterone in activities, 17a-hydroxylaseand c17-20 lyase when response to subsequent acute stimulationand is accompanied maintained under standard culture conditions (95% by decreases of the microsomal P-4501activities, 17a-hydroxair, 5%CO2). Decreased metabolism of substrate at1%O2 cannot account for the differences discussed below in the P450 activities of desensitized Leydig cell cultures maintained at high and low oxygen tension.

Results

Conclusion