Li Haikun, Xu Liping, Dunbar Joseph C, Dhabuwala C B
Department of Urology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Urology. 2003 Mar;61(3):646-50. doi: 10.1016/s0090-4295(02)02263-x.
We have previously demonstrated that cocaine exposure leads to apoptosis in rat testes. To understand further the mechanism of cocaine-induced testicular damage, we studied the effect of cocaine on cytochrome c release from the mitochondria. We also determined the caspase 3, caspase 8, and caspase 9 activities in rat testes after chronic cocaine exposure.
Thirty-day-old male Sprague-Dawley rats received cocaine hydrochloride or equal volumes of normal saline subcutaneously daily for 90 days. The testes were removed at 15, 30, and 90 days of cocaine or saline administration. Mitochondria and cytosolic fractions from testes were isolated. Western blotting was performed in both fractions using anti-cytochrome c antibody. Caspase 3, caspase 8, and caspase 9 activities were determined by fluorometric assay.
The expression of cytochrome c protein in the cytosolic fraction was increased on day 15 and persisted for up to 90 days after cocaine injection compared with controls. However, the expression of cytochrome c in testes was decreased in the mitochondria fraction on days 15, 30, and 90 after cocaine injections compared with the corresponding controls. The caspase activity study showed caspase 3 and caspase 9 activities increased in cocaine-treated testes at each point of the study compared with the corresponding controls. However, the caspase 8 activity in cocaine-treated testes did not change significantly at each point of the study compared with the corresponding controls.
Our results suggest that the release of cytochrome c from mitochondria and its subsequent activation of caspase 9 and caspase 3 in testes play a key role in cocaine-induced germ cell apoptosis. Our findings also indicate that cocaine-induced testicular germ cell apoptosis in rats is at least initiated through a mitochondria-associated pathway.
我们之前已证明，接触可卡因会导致大鼠睾丸细胞凋亡。为进一步了解可卡因诱导睾丸损伤的机制，我们研究了可卡因对线粒体细胞色素c释放的影响。我们还测定了慢性接触可卡因后大鼠睾丸中半胱天冬酶3、半胱天冬酶8和半胱天冬酶9的活性。
30日龄雄性斯普拉格-道利大鼠每天皮下注射盐酸可卡因或等体积生理盐水，持续90天。在给予可卡因或生理盐水后的第15、30和90天取出睾丸。分离睾丸的线粒体和胞质部分。使用抗细胞色素c抗体对两部分进行蛋白质免疫印迹分析。通过荧光测定法测定半胱天冬酶3、半胱天冬酶8和半胱天冬酶9的活性。
与对照组相比，注射可卡因后第15天，胞质部分细胞色素c蛋白的表达增加，并持续至90天。然而，与相应对照组相比，注射可卡因后第15、30和90天，睾丸线粒体部分细胞色素c的表达降低。半胱天冬酶活性研究表明，与相应对照组相比，在研究的每个时间点，可卡因处理的睾丸中半胱天冬酶3和半胱天冬酶9的活性均增加。然而，与相应对照组相比，在研究的每个时间点，可卡因处理的睾丸中半胱天冬酶8的活性没有显著变化。
我们的结果表明，线粒体中细胞色素c的释放及其随后在睾丸中对半胱天冬酶9和半胱天冬酶3的激活在可卡因诱导的生殖细胞凋亡中起关键作用。我们的研究结果还表明，可卡因诱导的大鼠睾丸生殖细胞凋亡至少是通过线粒体相关途径启动的。
