title: Oxidative stress and mitochondrial damage precedes gastric mucosal cell death induced by ethanol administration.
abstract: Although it has been speculated that active oxidants and mitochondrial membrane damages play roles in ethanol-induced gastric mucosal damages, its detail remains unknown. The present study was designed to investigate whether ethanol induces oxidative stress and mitochondrial permeability transition (MPT) before cell death of gastric mucosal cells. Rat gastric mucosal cells (RGM-1) were kept in serum-free Dulbecco's modified Eagle's medium before addition of various concentrations of ethanol. Nuclear morphological aftemations and membrane barrier dysfunction of RGM-1 cells were assessed by staining with Hoechst 33342 and propidium iodide, respectively. To assess the contribution of oxygen-derived free radicals and intracellular glutathione, scavenger of hydrogen peroxide and the hydroxyl radical, N,N-dimethylthiourea, glutathione precursor, N-acetyl-L-cysteine, and an inhibitor of alcohol dehydrogenase, 4-methylpyrazole were added before treatment with ethanol. To investigate MPT, calcein and tetramethylrhodamine methyl ester were loaded before addition of ethanol, and the changes of fluorescence intensity were monitored using a laser scanning confocal microscope. Ethanol (>5% v/v) dose-dependently increased the number of propidium iodide-positive cells, suggesting a diminished barrier function of cell membrane. After addition of ethanol, mitochondria were filled quickly with calcein indicating MPT, which was accompanied by mitochondrial depolarization, as shown by loss of tetramethylrodamine methyl ester before cell death. Ethanol-induced cell death was significantly attenuated by simultaneous incubation with either N,N-dimethylthiourea or N-acetyl-L-cysteine, suggesting the importance of intracellular redox states in inducing cellular damage, whereas such change was not attenuated by 4-methylpyrazole. Present results suggest that ethanol treatment induces intracellular oxidative stress and produces MPT and mitochondrial depolarization, which are preceding cell death in gastric mucosal cells. Intracellular antioxidants, such as glutathione, may have a significant protective action against ethanol in gastric mucosal cells.
authors.affiliation: Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Hirokawa
authors.forename: S
authors.initials: S
authors.lastname: Miura
authors.forename: H
authors.initials: H
authors.lastname: Yoshida
authors.forename: I
authors.initials: I
authors.lastname: Kurose
authors.forename: T
authors.initials: T
authors.lastname: Shigematsu
authors.forename: R
authors.initials: R
authors.lastname: Hokari
authors.forename: H
authors.initials: H
authors.lastname: Higuchi
authors.forename: N
authors.initials: N
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Yokoyama
authors.forename: H
authors.initials: H
authors.lastname: Kimura
authors.forename: S
authors.initials: S
authors.lastname: Kato
authors.forename: H
authors.initials: H
authors.lastname: Ishii
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000633:toxicity*; D005753:Gastric Mucosa / Q000187:drug effects*; D007425:Intracellular Membranes / Q000187:drug effects*; D015227:Lipid Peroxidation / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects*; D018384:Oxidative Stress; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D000431:Ethanol