Joint Graduate Program in Toxicology, Graduate School of Biomedical Sciences at Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Neurotoxicology. 2011 Oct;32(5):535-44. doi: 10.1016/j.neuro.2011.06.003. Epub 2011 Jun 29.
The developing brain is particularly sensitive to environmental teratogens, such as methylmercury (MeHg), which may induce cell death. Although several mechanisms of MeHg-induced apoptosis have been defined in culture models, pathways mediating caspase-3 activation in vivo remain unclear, especially in the developing hippocampus. To explore apoptotic mechanisms, Sprague-Dawley rats were exposed to 5 μg/g MeHg or PBS vehicle on postnatal day 7 (P7) and the hippocampus was assessed at various times for levels of apoptotic proteins. MeHg induced a 38% increase in Bax protein and an increase in cytosolic cytochrome c at 4h, followed by later increases in caspase-9 (40% at 12h; 33% at 24h) and caspase-8 (33% at 24h), compared to controls. MeHg also induced an increase in executioner caspase-3, a protease activated by both mitochondrial-dependent caspase-9 and mitochondrial-independent caspase-8. To further define pathways, we used a forebrain culture model and found that the MeHg-induced increases in caspase-3 and caspase-8 were completely blocked by a caspase-9-specific inhibitor, while caspase-9 induction was unperturbed by the caspase-8 inhibitor. These observations suggest that MeHg acts primarily through the mitochondrial-dependent cascade to activate caspase-3 in forebrain precursors, a pathway that may contribute to previously documented neurotoxicity in developing hippocampus. In turn, using the endpoint protein, caspase-3, as a sensitive marker for neural injury, we were able to detect hippocampal cell death in vivo at ten-fold lower levels of MeHg exposure (0.6 μg/g) than previously reported. Thus mitochondrial-dependent cell death in the hippocampus may serve as a sensitive index for teratogenic insults to the developing brain.
发育中的大脑对环境致畸物特别敏感，例如甲基汞（MeHg），它可能诱导细胞死亡。尽管在培养模型中已经定义了几种 MeHg 诱导细胞凋亡的机制，但体内介导半胱天冬酶-3 激活的途径仍不清楚，尤其是在发育中的海马体中。为了探索凋亡机制，Sprague-Dawley 大鼠在出生后第 7 天（P7）接受 5μg/g 的 MeHg 或 PBS 载体处理，并在不同时间评估海马体中凋亡蛋白的水平。MeHg 在 4 小时时诱导 Bax 蛋白增加 38%，细胞质细胞色素 c 增加，并随后在 12 小时时（增加 40%）和 24 小时时（增加 33%）分别增加 caspase-9 和 caspase-8，与对照组相比。MeHg 还诱导了执行器半胱天冬酶-3（一种由线粒体依赖性 caspase-9 和线粒体非依赖性 caspase-8 激活的蛋白酶）的增加。为了进一步定义途径，我们使用前脑培养模型发现，MeHg 诱导的 caspase-3 和 caspase-8 的增加完全被 caspase-9 特异性抑制剂阻断，而 caspase-9 的诱导不受 caspase-8 抑制剂的影响。这些观察结果表明，MeHg 主要通过线粒体依赖性级联反应激活前脑前体细胞中的 caspase-3，这一途径可能导致发育中的海马体中先前记录的神经毒性。反过来，我们使用终点蛋白 caspase-3 作为神经损伤的敏感标志物，能够在 MeHg 暴露水平低十倍（0.6μg/g）时在体内检测到海马体细胞死亡，这比以前报道的要低。因此，海马体中线粒体依赖性细胞死亡可能作为发育中大脑对致畸物损伤的敏感指标。
