Hydrogen peroxide-induced cellular injury is associated with increase in endogenous fluorescence from rat gastric mucosal epithelial cell culture: A new method for detecting oxidative cellular injury by fluorescence measurement

Abstract

To develop a new method of detecting cellular injury caused by oxygen radicals, we studied endogenous fluorescence from the cultured cells of a rat gastric mucosal epithelial cell line. Measurement with an ultra-high sensitivity camera-image processor system under an inverted epifluorescence microscope showed that the fluorescence intensity of the cells increased time- and dose-dependently after the addition of hydrogen peroxide (H2O2), an oxygen radical precursor, to the medium. This increase was inhibited by the presence of catalase. Phase-contrast and fluorescence microscopy revealed that the fluorescence was emitted from granular substances in the cytoplasm of the injured cells. The spectral pattern of excitation and emission indicated that the fluorescent substances were flavins. In cell-free experiments, glutathione reductase which has flavin adenine dinucleotide (FAD) at the active site, increased in fluorescence after incubation with H2O2 in the presence of reduced glutathione and glutathione peroxidase. These findings indicate that FAD in the cytoplasm of cells injured by H2O2 increased in endogenous fluorescence according to the extent of injury, and suggest that fluorescence measurement may be a simple method in cellular toxicology to detect oxygen radical-induced injuries.

Gastric acid induces mitochondrial superoxide production and lipid peroxidation in gastric epithelial cells

“…A gastric epithelial cell line previously established at our laboratory, RGM1 [12], was reobtained from RIKEN BioResource Center (Tsukuba, Japan). Cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM/F12; Cosmo Bio, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; Gibco, Grand Island, NY, USA) and 2 mM glutamine at 37°C in a humidified incubator with 5% CO 2 .…”

Cellular membrane fluidity measurement by fluorescence polarization in indomethacin-induced gastric cellular injury in vitro

“…18,19 Cells were grown in a 1 : 1 mixture of Dulbecco's modifi ed Eagle medium and Ham's F-12 medium (DMEM/F12) supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C in a humidifi ed incubator with 5% CO 2 . RGM-1 cells were incubated in 96-well, black, fl at-bottom assay plates (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) at a concentration of 10 5 cells/ml for fl uorescence imaging and in 96-well assay plates at the same concentration for the cytotoxicity assays.…”

Rebamipide Significantly Inhibits Indomethacin-Induced Mitochondrial Damage, Lipid Peroxidation, and Apoptosis in Gastric Epithelial RGM-1 Cells

“…Rat gastric epithelial cell line RGM-1, a diploid, and nontransformed rat epithelial cell line derived from normal Wister rat gastric mucosa (15,16) were used. Cells were grown in a 1:1 mixture of both Dulbecco's modified Eagle medium and Ham's F-12 medium (DMEM/F12) supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine at 37 • C in a humidified incubator with 5% CO 2 .…”