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)显著抑制自然杀伤细胞(NK)活性，其机制如下：1)OPs通过抑制NK、LAK和CTL细胞的颗粒酶活性，降低细胞内穿孔素、颗粒酶A和颗粒溶素的水平，抑制穿孔素、颗粒酶A和颗粒溶素的转录，抑制穿孔素、颗粒酶A和颗粒溶素的转录，从而损害NK、LAK和CTL细胞的颗粒吐出途径；2)OPs可损伤NK、LAK和CTL细胞的FasL/Fas途径，穿孔素基因敲除小鼠的研究表明，在这种情况下，NK细胞的颗粒吐出途径不起作用，仅有FasL/Fas途径起作用。但以上两种机制并不能完全解释OP抑制NK活性的全部机制，一定还有其他机制参与其中。这促使我们推测，OPs可能诱导NK和CTL细胞凋亡，最终诱导…细胞凋亡为了探讨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细胞(NK细胞)的凋亡。体外铬释放实验显示，NK-92CI和NK-92MI细胞表达CD56、穿孔素、颗粒酶A、B、3/K和颗粒溶素，对K562细胞具有较强的细胞毒作用，是IL-2非依赖的人NK细胞株。结果表明，敌敌畏和CP均能显著诱导NK-92细胞发生凋亡，并呈剂量和时间依赖关系。DDVP还以剂量和时间依赖的方式诱导NK-92CI细胞内caspase-3活性增加，caspase-3抑制剂Z-DEVD-FMK可显著抑制DDVP诱导的细胞凋亡，提示这种凋亡部分是通过激活细胞内caspase-3介导的。CP诱导的细胞凋亡模式与敌敌畏诱导的不同。在高剂量时，CP的反应比敌敌畏快，而在低剂量时，敌敌畏诱导细胞凋亡的能力比CP慢，但比CP强。此外，NK-92CI和NK-92MI细胞对OP农药的反应不同，NK-92CI细胞对OP农药的敏感性高于NK-92MI细胞。这类似于敌敌畏对NK细胞活性的抑制，其中NK-92CI细胞比NK-92MI细胞更容易被DDVP抑制，并强烈地提示了DDVP诱导的NK细胞凋亡与抑制NK细胞的杀伤活性之间的关系。为探讨OP农药抑制细胞毒性T淋巴细胞(CTL)活性的机制，本研究还通过体外培养Jurkat人T细胞，研究了OP农药是否能诱导T细胞死亡/凋亡。结果表明，CP以剂量和时间依赖的方式诱导Jurkat人T细胞死亡。CP还以剂量和时间依赖的方式诱导Jurkat T细胞凋亡，提示CP诱导的细胞死亡由细胞凋亡组成。CP还以剂量和时间依赖的方式诱导Jurkat T细胞内caspase-3活性增加，Z-DEVD-FMK显著抑制CP诱导的细胞凋亡。这些结果表明，CP可诱导人Jurkat T细胞发生凋亡，这种作用部分是通过激活细胞内caspase-3来实现的。为探讨OP对脾细胞的作用及其机制，采用Wistar大鼠灌胃给予杀菌硫磷(FNT)及其主要代谢产物3-甲基-4-硝基苯酚(MNP)，剂量分别为0、5和10 mg/kg，每周4~5天，共9周。采集正常大鼠和染毒大鼠的脾细胞，用流式细胞仪检测B细胞、T细胞、T细胞亚群(CD_4/CD_8)、自然杀伤细胞(NK)和巨噬细胞的数量。同时检测体重、脾重量和脾组织病理学改变。10 mg/kg FNT组小鼠脾CD8+T细胞百分率、CD8/CD4比值和10 mg/kg MNP组小鼠脾CD3+、CD8+T细胞百分率均显著低于对照组。FNT暴露还显著降低了小鼠的脾重量和体重。此外，在透射电子显微镜下还观察到FNT染毒大鼠脾内淋巴细胞的凋亡。然而，FNT和MNP暴露对脾NK细胞、B细胞和巨噬细胞没有影响。以上结果提示，FNT和MNP可能对大鼠脾T细胞有选择性作用。较少

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