Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.
Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.
复制标题
DOI：
10.1021/tx500323h
复制
发表时间：
2015-04-20
影响因子：
4.1
通讯作者：
Ross MK
中科院分区：
文献类型：
--
作者：
Mangum LC;Borazjani A;Stokes JV;Matthews AT;Lee JH;Chambers JE;Ross MK
生物累积性有机卤素化学品，如有机氯（OC）杀虫剂，已越来越多地与疾病的病因学;然而，化学品暴露和疾病，如动脉粥样硬化，癌症和糖尿病之间的机械联系，是复杂的和不明确的。OC暴露引起的全身氧化应激可能在这些病理学的发展中起着至关重要的作用。单核细胞是先天性免疫系统的重要监视细胞，其通过合成氧自由基（例如超氧化物）来响应具有β-相关分子模式的细胞外信号，以对抗感染性病原体。我们假设OC化学物质可能对单核细胞有毒，因为超氧化物衍生的活性氧（ROS）能够引起细胞氧化损伤。反应性氧自由基在单核细胞中大部分由NADPH氧化酶（Nox）产生。本研究旨在研究两种氯代环二烯化合物trans-nonachlor和dieldrin以及DDT的氯代脂环族代谢物p，p′-DDE对人单核细胞系中Nox活性的刺激作用，并阐明这种激活作用的机制。用反式九氯草胺或狄氏剂（培养基中0.1-10 μM）处理的人THP-1单核细胞显示出细胞内ROS水平升高，这一点通过补充方法得到证实，包括使用探针DCFH-DA和基于氢乙啶的荧光和UPLC-MS分析的流式细胞术分析。此外，在另外两种细胞系，小鼠J774巨噬细胞和人HL-60细胞中也观察到由反式-九甲草胺引起的诱导活性氧通量。Nox在OC介导的氧化应激中的中心作用通过用Nox抑制剂diphenyleneiodonium和VAS-2870处理的OC暴露的单核细胞中的减弱的超氧化物产生来证明。此外，与载体处理的细胞相比，用OC挑战的单核细胞表现出增加的磷酸化p47 phox水平和增强的p47 phox膜定位。p47 phox是Nox的胞质调节亚基，其磷酸化和转运到膜中的NOX 2催化亚基是Nox组装和活化的必要步骤。狄氏剂和反式九甲草胺治疗的单核细胞也导致花生四烯酸（AA）和类花生酸的生产显着增加，这可以被废除的磷脂酶A2（PLA 2）抑制剂花生四烯酸三氟甲基酮（ATK），但不是由钙非依赖性PLA 2抑制剂溴烯醇内酯。这表明，胞浆磷脂酶A2通过增加激活蛋白激酶C（磷酸化p47 phox）的AA的细胞内池，在诱导Nox活性中起着至关重要的作用。此外，ATK还阻断OC诱导的p47 phox丝氨酸磷酸化并减弱ROS水平，这进一步支持了由胞质PLA 2释放的AA池负责Nox激活的观点。总之，结果表明，反式-九甲草胺和狄氏剂能够通过依赖于细胞内AA水平升高的Nox依赖性机制增加细胞内超氧化物水平。这些发现是重要的，因为环境毒物对单核细胞的慢性激活可能导致致病性氧化应激和炎症。
Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p′-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1–10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47phox levels and enhanced p47phox membrane localization compared to that in vehicle-treated cells. p47phox is a cytosolic regulatory subunit of Nox, and its phosphorylation and translocation to the NOX2 catalytic subunit in membranes is a requisite step for Nox assembly and activation. Dieldrin and trans-nonachlor treatments of monocytes also resulted in marked increases in arachidonic acid (AA) and eicosanoid production, which could be abrogated by the phospholipase A2 (PLA2) inhibitor arachidonoyltrifluoromethyl ketone (ATK) but not by calcium-independent PLA2 inhibitor bromoenol lactone. This suggested that cytosolic PLA2 plays a crucial role in the induction of Nox activity by increasing the intracellular pool of AA that activates protein kinase C, which phosphorylates p47phox. In addition, ATK also blocked OC-induced p47phox serine phosphorylation and attenuated ROS levels, which further supports the notion that the AA pool liberated by cytosolic PLA2 is responsible for Nox activation. Together, the results suggest that trans-nonachlor and dieldrin are capable of increasing intracellular superoxide levels via a Nox-dependent mechanism that relies on elevated intracellular AA levels. These findings are significant because chronic activation of monocytes by environmental toxicants might contribute to pathogenic oxidative stress and inflammation.