Department of Medicine, University of California Irvine, Irvine, CA 92617, United States.
Department of Medicine, University of California Irvine, Irvine, CA 92617, United States; Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92617, United States; Program in Public Health, University of California Irvine, Irvine, CA 92617, United States.
Toxicol Appl Pharmacol. 2018 Aug 1;352:38-45. doi: 10.1016/j.taap.2018.05.024. Epub 2018 May 22.
Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in glutathione (GSH) synthesis, have decreased tissue GSH. We previously showed that Gclm-/- embryos have increased sensitivity to the prenatal in vivo ovarian toxicity of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) compared with Gclm+/+ littermates. We also showed that BaP-induced germ cell death in cultured wild type embryonic ovaries is caspase-dependent. Here, we hypothesized that GSH deficiency increases sensitivity of cultured embryonic ovaries to BaP-induced germ cell death. 13.5 days post coitum (dpc) embryonic ovaries of all Gclm genotypes were fixed immediately or cultured for 24 h in media supplemented with DMSO vehicle or 500 ng/ml BaP. The percentage of activated caspase-3 positive germ cells varied significantly among groups. Within each genotype, DMSO and BaP-treated groups had increased germ cell caspase-3 activation compared to uncultured. Gclm+/- ovaries had significantly increased caspase-3 activation with BaP treatment compared to DMSO, and caspase-3 activation increased non-significantly in Gclm-/- ovaries treated with BaP compared to DMSO. There was no statistically significant effect of BaP treatment on germ cell numbers at 24 h, consistent with our prior observations in wild type ovaries, but Gclm-/- ovaries in both cultured groups had lower germ cell numbers than Gclm+/+ ovaries. There were no statistically significant BaP-treatment or genotype-related differences among groups in lipid peroxidation and germ cell proliferation. These data indicate that Gclm heterozygous or homozygous deletion sensitizes embryonic ovaries to BaP- and tissue culture-induced germ cell apoptosis.
缺乏谷氨酸半胱氨酸连接酶（GCL）修饰亚基的小鼠（GSH 合成的限速酶）组织 GSH 减少。我们之前表明，与 Gclm+/+ 同窝仔鼠相比，Gclm-/-胚胎对多环芳烃苯并[a]芘（BaP）体内产前卵巢毒性更敏感。我们还表明，BaP 诱导培养的野生型胚胎卵巢中的生殖细胞凋亡依赖于半胱天冬酶。在这里，我们假设 GSH 缺乏会增加培养的胚胎卵巢对 BaP 诱导的生殖细胞死亡的敏感性。所有 Gclm 基因型的 13.5 天孕龄（dpc）胚胎卵巢立即固定或在补充有 DMSO 载体或 500ng/ml BaP 的培养基中培养 24 小时。活化半胱天冬酶-3 阳性生殖细胞的百分比在各组之间差异显著。在每个基因型中，与未培养相比，DMSO 和 BaP 处理组的生殖细胞半胱天冬酶-3 激活增加。与 DMSO 相比，Gclm+/-卵巢中 BaP 处理的半胱天冬酶-3 激活显著增加，而 Gclm-/-卵巢中 BaP 处理的半胱天冬酶-3 激活与 DMSO 相比略有增加。24 小时时，BaP 处理对生殖细胞数量没有统计学上的显著影响，与我们之前在野生型卵巢中的观察结果一致，但在培养的两组 Gclm-/-卵巢中，生殖细胞数量均低于 Gclm+/+卵巢。各组间在脂质过氧化和生殖细胞增殖方面，BaP 处理或基因型无显著差异。这些数据表明，Gclm 杂合或纯合缺失使胚胎卵巢对 BaP 和组织培养诱导的生殖细胞凋亡敏感。
