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Role of oxidative stress in hypoxia‐reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells
Author(s) -
Samarasinghe Dev A.,
Tapner Michael,
Farrell Geoffrey C.
Publication year - 2000
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.510310124
Subject(s) - oxidative stress , glutathione , superoxide dismutase , viability assay , hypoxia (environmental) , trypan blue , endothelial stem cell , reactive oxygen species , superoxide , pharmacology , chemistry , reperfusion injury , biochemistry , andrology , biology , endocrinology , medicine , apoptosis , ischemia , oxygen , in vitro , organic chemistry , enzyme
To characterize the role of oxidative stress in cultured rat sinusoidal endothelial cells, we studied the production of superoxide after reoxygenation, the relationship of reduced glutathione (GSH) levels to cell injury, and the protective efficacy of antioxidants. Hypoxia (pO 2 1‐2 mm Hg) was achieved by culturing cells under 95% N 2 5% CO 2 for 4 hours. Reoxygenation was then reestablished, and viability was determined at 24 hours by trypan blue exclusion; putative protective agents were added at the time of reoxygenation (4 hours). As previously reported, reoxygenation after 4 hours hypoxia accentuated sinusoidal cell death fourfold compared with hypoxic or normoxic controls ( P < .0001). Superoxide was not produced on reoxygenation, and superoxide dismutase provided no protection against reoxygenation injury. Cellular levels of GSH fell to 37 ± 4% of normoxic controls ( P < .0001) following reoxygenation. These changes were essentially abrogated by Trolox (Aldrich Chemical Co., Milwaukee, WI) and dimethyl sulfoxide, both of which also completely protected against reoxygenation injury. When cellular GSH levels were lowered by addition of diethylmaleate (which conjugates GSH), this reduced the viability of endothelial cells cultured under normoxic conditions and greatly augmented reoxygenation injury. Conversely, addition of exogenous GSH partially protected endothelial cells against hypoxia‐reoxygenation injury. Desferrioxamine also protected against reoxygenation injury, but catalase was only partly protective. It is concluded that sinusoidal endothelial cells undergo significant intracellular oxidative stress following reoxygenation, and their viability is critically dependent on GSH levels. Reactive oxygen species are likely mediators of oxidative stress in hepatic sinusoidal endothelial cells.