Masaaki Okabe

Homocysteine and copper induce cellular apoptosis via caspase activation and nuclear translocation of apoptosis-inducing factor in neuronal cell line SH-SY5Y.

Hyperhomocysteinemia has been implicated in dementia and neurodegenerative disease. Physiological homocysteine concentrations did not result in apoptosis in SH-SY5Y cells in the present study. The apoptosis was recognized in millimolar level of homocysteine. However, SH-SY5Y cell death was observed following exposure to micromolar level of homocysteine in combination with copper. Exposure to 250microM homocysteine and 10microM CuCl(2) for one day decreased cell viability by 40%. Homocysteine and copper caused apoptosis, because hallmarks of apoptosis were recognized, such as loss of mitochondrial membrane potential, TUNEL-positive cells, release of cytochrome c from mitochondria, and caspase-3 activation, but not nucleosomal DNA fragmentation. Homocysteine and copper generated the intracellular reactive oxygen species, and homocysteine and copper-induced apoptosis was due to an accumulation of intracellular reactive oxygen species, which was inhibited by catalase. Pan-caspase inhibitor, z-VAD-fmk, could not completely inhibited homocysteine and copper-induced cell death. Homocysteine and copper also caused the nuclear translocation of apoptosis-inducing factor. These results suggested that homocysteine and copper induced not only caspase-dependent apoptosis but also caspase-independent apoptosis-inducing factor related apoptosis.

The Differentiation of C2C12 Cells to Myotube by Paraquat

Paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride), producing reactive oxygen species (ROS) to show its toxicity is one of herbicides once widely used. Although the toxicity of paraquat on human and animals is too strong, paraquat is still in use in many developing countries. Therefore it is important to know the risk of paraquat on mammals. Effect of paraquat on the muscle of mammals is not well investigated, but it has been observed that interstitial cells differentiated into muscle cells in the lung of paraquat-injected monkey (Fukuda et al., Am J Pathol, 118(3): 452–475). Here we studied the effect of paraquat on the differentiation of mouse myoblast cell line C2C12 cells to myotube. C2C12 cells were maintained in Dulbecco’s modified eagle medium containing 10% fetal bovine serum at 37°C, 5% CO2. Paraquat (0.1 ˜ 1000 ng/mL) was added to the medium and C2C12 cells were cultured in each medium for 12 days. Then the cells were harvested and applied to SDS-PAGE and western-blotting with anti-myosin heavy chain (MCH) antibody. Light microscope observation revealed that C2C12 cells morphologically changed to myotube depending on the concentration of paraquat even in undifferentiating condition. The amount of MCH, the molecular marker of muscle differentiation, increased depending on the concentration of paraquat. Paraquat produces ROS to show its toxicity. We also studied the effect of ascorbic acid on paraquat-dependent differentiation of C2C12 cells. C2C12 cells were cultured with paraquat and ascorbic acids in the same conditions stated above. As a result it was observed that ascorbic acid (10˜100 μM) did not inhibit the differentiation of C2C12 cells, and the overproduction of MCH by paraquat was not decreased by the ascorbic acid. Furthermore, the existence of H2O2 (0.1˜100 μM) did not induce the differentiation of C2C12 cells. These results suggest that the differentiation of C2C12 cells to myotube is modulated by paraquat in ROS-independent manner.

The Effects of Pesticides on Immune Cells

The environmental pollution induced by the chemical substances is regarded as a serious problem, and has various influences on the ecosystem. Recently, many people are interested in safety of foods. We focused on the pesticide chemicals presently used on farm in many countries, and chose methoxychlor and endosulfan. To investigate the influence of methoxychlor and endosulfan on immunoglobulin production, human hybridoma HB4C5 cells and mouse splenic lymphocytes were cultured in the medium containing these compounds at various concentrations. As a result, human IgM production of HB4C5 cells was slightly decreased in a dose-dependent manner. On the other hand, mouse splenic lymphocytes treated with each pesticide were markedly decreased both IgA and IgG production level except for IgM. Moreover, to assess the influence of methoxychlor and endosulfan in the body, we examined the effects of these compounds on mice by oral administration. The spleen was remarkably atrophied whereas the body weights were same as a control group. In addition, the weight loss was observed not only in liver and kidney. These results suggest that these pesticides affect immune system as well as detoxification tissues. It is supposed from these results that the exposure to these compounds via foods leads us to the decline in immune activity of our body, resulting in immune diseases.