ABSTRACT
OBJECTIVES: This study was performed to evaluate the critical role of glutathione(GSH) in methyl mercury chloride(MeHgCl)induced cell apoptosis. METHODS: The effect of GSH in MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium(DMEM). RESULTS: MeHgCl exerted a dose dependent cytotoxicity,as demonstrated by the MTT assay, which is an assay dependent partially on the mitochondrial function. Moreover, in the presence of NAC, a GSH precursor, the MeHgCl induced cytotoxicity was significantly decreased whereas BSO, a specific GSH synthesis inhibitor,increased the MeHgCl induced cytotoxicity.The MeHgCl induced DNA fragmentation and chromatin condensation was consistent with the morphological alterations. The MeHgCl treated cells exhibited increasing annexin V-FITC binding to the phos-phatidylserine(PS)translocated from the inner to the outer leaflet of the plasma membrane and those cells with NAC pretreatment significantly exhibited decreasing annexin V-FITC binding compared to the cells treated with MeHgCl only. However BSO pretreatment markedly exhibited the increasing annexin V-FITC binding. The MeHgCl treated cells generated ROS, which was evidenced by the oxidation of dihydroethidine and the generation of the fluorescent product, ethidium. In addition, BSO pretreatment further enhanced the extent of ROS generation caused by MeHgCl whereas NAC pretreatment decreased the amount of ROS generation. MeHgCl led to a dose dependent decrease in the GSH content. Although MeHgCl exposure significantly reduced the GSH level, those cells that had a NAC pretreatment contained a higher level of GSH compared to the cells treated with MeHgCl only. In contrast, BSO pretreatment futher enhanced the extent of GSH depletion caused by MeHgCl. CONCLUSIONS: These results indicate that MeHgCl reduced the GSH content and impaired the defense against oxidative damage caused by ROS formation in RAW 264.7 cells. It is possible that these factors leads to the activation of the apoptosis signaling pathway. Ultimately these results suggest that GSH plays a crucial role in protecting the activity against MeHgCl induced apoptosis.
Subject(s)
Animals , Mice , Antioxidants , Apoptosis , Cell Membrane , Chromatin , DNA Fragmentation , Ethidium , GlutathioneABSTRACT
Thl cloned cell line 28-4 which is an I-A + KLH - specific Th1 type clone of (C57BU6xC 3H) F1 origin was kindly provided by professor Tomio Tada. In these studies, employing these cloned cells, the author investigated both proliferation responses of Thl cells in the presence of various concentrations of cytokines, such as IL-2, IL-4 or IL-6 and proliferation of Thl cells to various concentration of mitogens such as PHA, ConA or PWM. In addition, the author also investigated the proliferation response of Th1 cells to the optimal dose of PHA, ConA or PWM in the presence or absence of above mentioned cytokines. It was found that IL-2, IL-4 or IL-6 alone their growth stimulation degree was dependent on cytokine concentration and that PHA, ConA or PWM stimulated Thl cell proliferation and optimal dose of PHA ConA and PWM was 3 g, 4 g and 2 g per ml, respectively. In addition, proliferation response of Th1 cells to ConA or PWM in the presence of IL-2 was significantly enhanced, but the proliferation response to PHA was not increased significantly. However, IL-4 did not significantly modulate mitogen-activated Thl cell proliferation response. Interestingly, IL-6 decreased PHA- or ConA-activated proliferation of Thl cells, but did not change PWM-activated proliferation. Taken together, these studies strongly suggested that IL-2, IL-4 or IL-6 itself clone stimulated the Thl cell proliferation and that PHA, ConA or PWM also stimulated Thl cell proliferation. In addition, these studies also indicated that IL-2 increased ConA- or PWM-activated Thl cell proliferation, but IL6 inhibited PHA- or ConA-activated Th1 cell proliferation and that IL-4 did not significantly change the mitogen-activated Th1 cell proliferation.