Quinn P G, Payne A H
J Biol Chem. 1984 Apr 10;259(7):4130-5.
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.
在标准培养条件（95%空气，5%二氧化碳）下培养时，睾丸间质细胞的微粒体细胞色素P-450酶活性、17α-羟化酶和C17-20裂解酶活性呈现出时间依赖性下降。在培养基中加入羟自由基清除剂二甲基亚砜，或将氧张力从19%降至1% O₂，可有效维持这些酶的活性，低氧和二甲基亚砜的联合作用具有协同性。用1 mM 8-溴环磷腺苷（8-Br-cAMP）处理培养的睾丸间质细胞以诱导类固醇生成脱敏，这导致17α-羟化酶和C17-20裂解酶活性进一步下降，以及对随后用8-Br-cAMP进行急性刺激产生睾酮的能力减弱。将氧张力从19%降至1% O₂可防止脱敏的睾丸间质细胞中微粒体P-450活性的这种增强的损失。δ5-3β-羟类固醇脱氢酶-异构酶是一种微粒体酶，不是P-450酶，在所有培养条件下，其活性在对照和脱敏的睾丸间质细胞培养物中均稳定。这些数据与以下假设一致：氧介导的损伤是对照睾丸间质细胞中17α-羟化酶和C17-20裂解酶活性随时间下降的原因，并且是脱敏的睾丸间质细胞中这些微粒体P-450活性进一步下降的机制。无论培养条件如何，脱敏的睾丸间质细胞在24小时和48小时时，对随后用8-Br-cAMP进行急性刺激产生睾酮的能力分别下降了50%和70%。由于在1% O₂下培养的脱敏睾丸间质细胞与对照相比没有显示出更大的酶活性损失，因此微粒体P-450活性的丧失不是脱敏的睾丸间质细胞睾酮生物合成能力减弱的原因。
