Kim Sang Hyun, Sharma Raghubir P
Interdisciplinary Program in Toxicology, Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7389, USA.
Toxicol Appl Pharmacol. 2004 Apr 1;196(1):47-57. doi: 10.1016/j.taap.2003.11.020.
The current study characterizes the mechanism by which mercury, a toxic metal, induces death in murine macrophages. The cytotoxic EC(50) of mercury ranged from 62.7 to 81.1 microM by various assays in J774A.1 cultures; accordingly, we employed 70 microM of mercuric chloride in most experiments. Mercury-induced intracellular calcium modulated reactive oxygen species (ROS) production, which resulted in both cell apoptosis and necrosis indicated by annexin V binding and caspase-3 activity, and propidium-iodide binding. Calcium antagonists abolished ROS production. Mercury stimulated p38 mitogen-activated protein kinase (MAPK) and additively stimulated lipopolysaccharide-activated p38. Mercury-activated p38 was decreased by pretreatment of cells with antioxidants, N-acetylcysteine (NAC) and silymarin, indicating that mercury-induced ROS were involved in p38 activation. Mercury increased the expression of tumor necrosis factor alpha (TNFalpha); antioxidants and a specific p38 inhibitor decreased this effect. Pretreatment with antioxidants, p38 inhibitor, and anti-TNFalpha antibody decreased mercury-induced necrosis; however, anti-TNFalpha antibody did not decrease mercury-induced apoptosis. Results suggest that mercury-induced macrophage death is a mix of apoptosis and necrosis employing different pathways. P38-mediated caspase activation regulates mercury-induced apoptosis and p38-mediated TNFalpha regulates necrosis in these cells. Calcium regulates ROS production and mercury-induced ROS modulate downstream p38 that regulates both apoptosis and necrosis.
本研究描述了有毒金属汞诱导小鼠巨噬细胞死亡的机制。在J774A.1培养物中，通过各种测定方法，汞的细胞毒性半数有效浓度（EC50）范围为62.7至81.1微摩尔；因此，在大多数实验中我们使用了70微摩尔的氯化汞。汞诱导的细胞内钙调节活性氧（ROS）的产生，这导致细胞凋亡和坏死，通过膜联蛋白V结合、半胱天冬酶-3活性以及碘化丙啶结合来指示。钙拮抗剂消除了ROS的产生。汞刺激p38丝裂原活化蛋白激酶（MAPK），并对脂多糖激活的p38产生累加刺激作用。用抗氧化剂N-乙酰半胱氨酸（NAC）和水飞蓟宾预处理细胞后，汞激活的p38降低，这表明汞诱导的ROS参与了p38的激活。汞增加了肿瘤坏死因子α（TNFα）的表达；抗氧化剂和一种特异性p38抑制剂降低了这种作用。用抗氧化剂、p38抑制剂和抗TNFα抗体预处理可减少汞诱导的坏死；然而，抗TNFα抗体并未减少汞诱导的凋亡。结果表明，汞诱导的巨噬细胞死亡是凋亡和坏死的混合，采用了不同的途径。在这些细胞中，p38介导的半胱天冬酶激活调节汞诱导的凋亡，p38介导的TNFα调节坏死。钙调节ROS的产生，汞诱导的ROS调节下游的p38，而p38调节凋亡和坏死。
