| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2009: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2008: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Research Abstract |
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
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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
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