Datta Soma, Mazumder Shibnath, Ghosh Debabrata, Dey Saibal, Bhattacharya Shelley
Immunobiology Laboratory, School of Life Sciences, Visva-Bharati University, Santiniketan 731 235, India.
Toxicol Appl Pharmacol. 2009 Dec 15;241(3):329-38. doi: 10.1016/j.taap.2009.09.007. Epub 2009 Sep 24.
We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 microM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V-propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47(phox). Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47(phox) translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH(2)-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38-JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.
我们之前已经证明，长期暴露（30天）于微摩尔浓度（0.50微摩尔）的砷会导致胡子鲶的头肾巨噬细胞（HKM）死亡。本研究的目的是确定砷诱导的HKM死亡的性质，并阐明该过程中涉及的信号转导途径。从DNA凝胶、膜联蛋白V-碘化丙啶、Hoechst 33342染色及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）分析可以明显看出，砷诱导的HKM死亡本质上是凋亡性的。抑制剂研究和免疫印迹分析进一步表明，砷诱导的HKM凋亡涉及半胱天冬酶-3的激活以及聚（ADP-核糖）聚合酶的裂解，聚（ADP-核糖）聚合酶是一种已被充分表征的半胱天冬酶-3底物。用抗氧化剂N-乙酰半胱氨酸或二甲基亚砜预孵育可显著降低砷处理的HKM中的活性氧（ROS）水平，并防止半胱天冬酶激活、丙二醛形成及HKM凋亡。砷诱导烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶亚基p47（phox）的膜易位。用NADPH氧化酶的两种选择性抑制剂夹竹桃麻素和二亚苯基碘鎓氯化物预孵育可防止p47（phox）易位、ROS产生及HKM死亡。将HKM暴露于砷会诱导丝裂原活化蛋白激酶家族（MAPK）蛋白的激活，包括c-Jun氨基末端蛋白激酶（JNK）和p38丝裂原活化蛋白激酶（p38）。用p38抑制剂SB203580和JNK抑制剂SP600125对HKM进行预孵育可保护HKM免受砷诱导的凋亡。我们得出结论，暴露于微摩尔浓度的砷会通过激活NADPH氧化酶产生活性氧，进而导致半胱天冬酶-3介导的HKM凋亡。此外，该研究还表明p38-JNK途径在砷诱导的胡子鲶HKM凋亡中起作用。
