Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis
Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis
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DOI：
10.1016/j.freeradbiomed.2004.02.067
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发表时间：
2004-06-01
影响因子：
7.4
通讯作者：
Luderer, U
中科院分区：
文献类型：
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作者：
Lopez, SG;Luderer, U
抗癌药物环磷酰胺 (CPA) 治疗会破坏卵泡。 CPA 的活性代谢物通过与谷胱甘肽 (GSH) 结合而解毒。我们测试了以下假设：CPA 导致卵巢卵泡细胞凋亡，以及在 CPA 给药前抑制卵巢 GSH 合成可增强 CPA 诱导的细胞凋亡。发情前期大鼠注射两次，间隔2小时，（1）生理盐水，然后生理盐水； (2)生理盐水，然后50mg/kg CPA； (3)生理盐水，然后300mg/kg CPA； (4) 5 mmol/kg 丁硫氨酸亚砜亚胺 (BSO) 抑制谷氨酸半胱氨酸连接酶 (GCL)（GSH 合成的限速酶），然后使用 50 mg/kg CPA。第二次注射后 24 小时，经 CPA 处理的卵巢通过琼脂糖凝胶电泳观察到 DNA 断裂明显增加，通过 TUNEL 观察到颗粒细胞凋亡明显增加，但 BSO 并没有增强 50 mg/kg CPA 的效果。接下来，我们测试了 CPA 抑制卵巢 GSH 浓度和 GSH 合成限速酶 GCL 表达的假设。给发情前期的大鼠注射300或50mg/kg CPA或媒介物，并在8或24小时后处死。 CPA治疗后，卵巢和肝脏GSH水平显着降低，卵巢GCL亚基mRNA水平显着升高。 GCL亚基蛋白水平没有显着变化。最后，我们检验了 GSH 消耗导致卵泡细胞凋亡的假设。发情前期大鼠在0700和1900小时注射5mmol/kg BSO或生理盐水。 BSO 治疗开始 24 小时后，组织学上闭锁卵泡的百分比显着增加，而凋亡、TUNEL 阳性卵泡的百分比则无显着增加。我们的结果表明，CPA 通过诱导颗粒细胞凋亡来破坏卵泡，并且 CPA 治疗会导致卵巢 GSH 水平下降。 BSO 治疗后更明显的 GSH 抑制并未导致卵泡细胞凋亡出现统计学上显着的增加。因此，GSH 消耗似乎并不是 CPA 引起滤泡细胞凋亡的机制。 (C) 2004 Elsevier Inc. 保留所有权利。
Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis. (C) 2004 Elsevier Inc. All rights reserved.