Role of oxidative stress in hypoxia-reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells

To characterize the role of oxidative stress in cultured rat sinusoidal end othelial 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 achi eved by culturing cells under 95% N(2)5% CO2 for 4 hours. Reoxygenation was then reestablished, and viability was determined at 24 hours by trypan blu e exclusion; putative protective agents were added at the time of reoxygena tion (4 hours). As previously reported, reoxygenation after 4 hours hypoxia accentuated sinusoidal cell death fourfold compared with hypoxic or normox ic controls (P <.0001). Superoxide was not produced on reoxygenation, and s uperoxide dismutase provided no protection against reoxygenation Injury. Ce llular levels of GSH fell to 37 +/- 4% of normoxic controls (P <.0001) foll owing reoxygenation. These changes were essentially abrogated by Trolox (Al drich Chemical Co., Milwaukee, WI) and dimethyl sulfoxide, both of which al so completely protected against reoxygenation injury. When cellular GSH lev els were lowered by addition of diethylmaleate (which conjugates GSH), this reduced the viability of endothelial cells cultured under normoxic conditi ons and greatly augmented reoxygenation injury. Conversely, addition of exo genous GSH partially protected endothelial cells against hypoxia-reoxygenat ion injury. Desferrioxamine also protected against reoxygenation injury, bu t catalase was only partly protective. It is concluded that sinusoidal endo thelial cells undergo significant intracellular oxidative stress following reoxygenation, and their viability is critically dependent on GSH levels. R eactive oxygen species are likely mediators of oxidative stress in hepatic sinusoidal endothelial cells.