New mechanism of organophosphorus pesticide-induced immunotoxocity : organophosphorus pesticide-induced apoptosis in immune cells

我们先前发现有机磷农药（OPs）显著抑制自然杀伤（NK）活性，这是通过以下机制介导的：1）OPs通过抑制颗粒酶的活性损害NK、LAK和CTL细胞的颗粒胞吐途径（Li et al.，2002）和通过降低穿孔素、颗粒酶A和颗粒溶素的细胞内水平（这是通过诱导NK细胞的脱粒和通过抑制穿孔素、颗粒酶A和颗粒溶素的mRNA的转录来介导的）;（2）OPs对NK细胞、LAK细胞和CTL细胞的FasL/Fas通路有损伤作用，其中NK细胞的颗粒胞吐途径不起作用，只有FasL/Fas途径保持功能。但上述两种机制不能完全解释OP抑制NK活性的全部机制，还可能有其他机制参与。这促使我们推测OPs可能诱导NK细胞和CTL细胞凋亡， ...更多信息 为了探讨OP农药诱导免疫毒性的机制，我们研究了OP农药是否能诱导人自然杀伤（NK）细胞凋亡。NK-92 CI和NK-92 MI细胞是不依赖于白细胞介素-2的人NK细胞系，表达CD 56、穿孔素、颗粒酶A、B、3/K和颗粒溶素，并且在铬释放试验中对K562细胞具有高度细胞毒性。结果表明，敌敌畏和CP可诱导NK-92细胞凋亡，并呈剂量和时间依赖性。DDVP还以剂量和时间依赖性方式诱导NK-92 CI细胞内活性caspase-3的增加，并且caspase-3抑制剂Z-DEVD-FMK显著抑制DDVP诱导的凋亡，表明这种凋亡部分由细胞内caspase-3的激活介导。CP诱导的细胞凋亡模式与DDVP不同。CP在高剂量下的反应快于DDVP，而DDVP在低剂量下的诱导能力慢于CP，但比CP强。NK-92 CI和NK-92 MI细胞对OP农药的反应不同，NK-92 CI细胞对OP农药的敏感性高于NK-92 MI细胞。这与DDVP对NK细胞活性的抑制作用相似，即NK-92 CI细胞比NK-92 MI细胞更容易被DDVP抑制，这表明DDVP诱导的NK细胞凋亡与NK细胞杀伤活性的抑制有关。综上所述，这些发现表明，OP农药诱导的NK细胞活性的抑制可能至少部分介导的OP农药诱导的NK细胞的凋亡。为了探讨OP农药诱导的细胞毒性T淋巴细胞（CTL）活性的抑制机制，它也被调查是否OP农药可以诱导T细胞在Jurkat人T细胞在体外细胞死亡/凋亡。发现CP以剂量和时间依赖性方式诱导Jurkat人T细胞的细胞死亡。CP还以剂量和时间依赖性方式诱导Jurkat T细胞凋亡，表明CP诱导的细胞死亡由凋亡组成。CP还以剂量和时间依赖性方式诱导Jurkat T细胞中细胞内活性caspase-3的增加，Z-DEVD-FMK显著抑制CP诱导的凋亡。为探讨OP对人Jurkat T细胞凋亡的影响及其机制，以Wistar大鼠为实验动物，经口给予杀螟松（FNT）及其主要代谢产物3-甲基-4-硝基苯酚（MNP）0、5和10 mg/kg，4-5天/周，共9周。从对照和暴露大鼠收获脾细胞，并通过流式细胞术定量以下细胞表型：（1）B细胞，（2）T细胞，（3）T细胞亚群（CD 4/CD 8），（4）自然杀伤（NK）细胞，和（5）巨噬细胞。还检查了体重、脾脏重量和脾脏的组织病理学改变。FNT 10 mg/kg组脾脏CD 8 + T细胞百分率、CD 8/CD 4比值及MNP 10 mg/kg组脾脏CD 3+、CD 8 + T细胞百分率均显著低于对照组。FNT暴露还显著降低脾脏重量和体重。透射电镜下观察到FNT染毒大鼠脾脏淋巴细胞凋亡。然而，FNT和MNP暴露不影响脾NK细胞、B细胞和巨噬细胞。提示FNT和MNP可选择性地影响大鼠脾T细胞。少

We previously found that organophosphorus pesticides (OPs) significantly inhibited natural killer (NK) activity, which is mediated by the following mechanisms : 1) OPs impair the granule exocytosis pathway of NK, LAK and CTL cells by inhibiting the activity of granzymes (Li et al., 2002) and by decreasing the intracellular level of perforin, granzyme A, and granulysin, which was mediated by inducing degranulation of NK cells, and by inhibiting the transcription of mRNAs of perforin, granzyme A, and granulysin ; 2) OPs impair the FasL/Fas pathway of NK, LAK and CTL cells, as investigated by using perforin-knockout mice, in which the granule exocytosis pathway of NK cells does not function and only the FasL/Fas pathway remains functional. However, the above two mechanisms cannot completely explain the all mechanism of OP-induced inhibition of NK activity and there must be other mechanisms involve. This encourages us to speculate that OPs may induce apoptosis in NK and CTL and ultimately … More inhibited cytolytic activity of effectors.In order to explore the mechanism of OP pesticide-induced immunotoxicity, we investigated whether OP pesticides can induce apoptosis in human natural killer (NK) cells. NK-92CI and NK-92MI cells, which are interleukin-2 independent human NK cell lines, express CD56, perforin, granzymes A, B, 3/K, and granulysin and are highly cytotoxic to K562 cells in the chromium release assay were treated with DDVP or CP in vitro. It was found that DDVP and CP significantly induced apoptosis in NK-92 cells in a dose- and time-dependent manner. DDVP also induced an increase in intracellular active caspase-3 in NK-92CI cells in a dose- and time-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited DDVP-induced apoptosis, suggesting that this apoptosis is partially mediated by the activation of intracellular caspase-3. The pattern of apoptosis induced by CP differed from that induced by DDVP. CP showed a faster response than DDVP at higher doses ; whereas, DDVP showed a slower but stronger apoptosis-inducing ability than CP at lower doses. Moreover, the response to OP pesticides differed between NK-92CI and NK-92MI cells, and NK-92CI cells were more sensitive to OP pesticides than NK-92MI cells. This is similar to the inhibition of NK activity induced by DDVP, in which NK-92CI cells were more easily inhibited by DDVP than NK-92MI cells and strongly suggested a relationship between DDVP-induced apoptosis and the inhibition of cytolytic activity in NK cells. Taken together, these findings suggest that OP pesticide-induced inhibition of NK activity may be at least partially mediated by OP pesticide-induced apoptosis in NK cells.To explore the mechanism of OP pesticide-induced inhibition of cytotoxic T lymphocyte (CTL) activity, it was also investigated whether OP pesticides can induce cell death/apoptosis in T cells using Jurkat human T cells in vitro. It was found that CP induced the cell death of Jurkat human T cells in a dose- and time-dependent manner. CP also induced apoptosis in Jurkat T cells in a dose- and time-dependent manner suggesting that CP-induced cell death consisted of apoptosis. CP also induced an increase in intracellular active caspase-3 in Jurkat T cells in a dose- and time-dependent manner, and Z-DEVD-FMK significantly inhibited CP-induced apoptosis. These findings indicate that CP can induce apoptosis in human Jurkat T cell cells, and this effect is partially mediated by the activation of intracellular caspase-3.To investigate the effect of OP on the splenocytes and the underlying mechanism in vivo, Fenitrothion (FNT) and its main metabolite, 3-methyl-4-nitrophenol (MNP) were administered orally to Wistar rats in daily doses of 0, 5 and 10mg/kg, 4-5 days/week for 9 weeks. Splenocytes were harvested from control and exposed rats, and the following cell phenotypes were quantified by flow cytometry : (1) B cells, (2) T cells, (3) T cell subsets (CD4/CD8), (4) natural killer (NK) cells, and (5) macrophages. Body weight, weight of the spleen and histopathological alterations of spleens were also examined. The percentage of splenic CD8+ T cells and the ratio of CD8/CD4 in the group receiving 10mg/kg FNT, and the percentages of splenic CD3+ and CD8+ T cells in the group receiving 10mg/kg MNP were significantly decreased compared with those in the controls. FNT exposure also significantly decreased the weight of the spleen and body weight. In addition, apoptotic lymphocytes in spleen were observed in FNT-exposed rats under transmission electron microscope. However, FNT and MNP exposures did not affect splenic NK cells, B cells, and macrophages. The above findings indicate that FNT and MNP may selectively affect splenic T cells in rats. Less