Nobuyoshi Chiba

Determination of 5-hydroperoxyeicosatetraenoic acid produced in rat basophilic leukemia cell line RBL-2H3 by high-performance liquid chromatography with chemiluminescence detection

A simple and sensitive method, applicable to quantification of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) produced in cells has been developed using high-performance liquid chromatography on a silica gel column with chemiluminescence detection. 5-HPETE was clearly separated from other positional isomers of HPETEs and hydroxyeicosatetraenoic acids with hexane-isopropanol-acetic acid (97:3:0.01, v/v) as the mobile phase. The lower limit of detection was about 100 pg. 5-HPETE produced in 10(7) cells of RBL-2H3 cells stimulated with A23187 was determined as 480+/-30 pg. In the present study, 5-HPETE, which occurs naturally, was detected and quantitated for the first time in intact cells.

Alteration of gene expressions by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx).

To determine the effect on gene expression of trace levels of reactive oxygen species from mitochondria, we used the mRNA differential display technique to compare gene expression in two cell lines: M15, which overexpresses mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx), in rat basophilic leukemia RBL-2H3 cells, and a control cell line, S1. We isolated 27 differentially expressed genes, including 10 previously unreported sequences. These genes included cytoskeletal proteins (beta-tubulin, nonmuscle myosin alkali light chain, and vimentin), growth or proliferation regulators [growth differentiation factor 1 (Gdf-1), Rap1a, and inhibitor of growth 3 (Ing3)], and others. Although the expression of most of the isolated genes did not respond to ROS (hydrogen peroxide) or antioxidant (pyrolidine dithiocarbamate) treatment, the expression of Gdf-1 was downregulated by hydrogen peroxide treatment. Thus, low levels of ROS produced in mitochondria during normal cellular metabolism can modulate gene expression.