Hydrogen peroxide damage to scavenging function and ultrastructure of liver sinusoidal endothelial cells is prevented by n-acetyl-cysteine but not GSH

Abstract Reactive oxygen species (ROS) are prevalent in the liver during intoxication, infection, inflammation, and ageing. Changes in liver sinusoidal endothelial cells (LSECs) are associated with various liver diseases. We investigated how oxidative stress induced by H 2 O 2 affects isolated rat LSECs at different concentrations (0.5-1000µM) and exposure times (10-120 min). Our findings show that H 2 O 2 exposure affects several LSEC functions in a dose- and time-dependent manner: (1) cell viability, reducing potential, and scavenging function decreased as H 2 O 2 concentration and exposure time increased; (2) intracellular ROS levels rose with higher H 2 O 2 concentrations; (3) fenestrations exhibited a dynamic response, initially closing but partially reopening at H 2 O 2 concentrations above 100µM after about 1 h; (4) scavenging function was affected after just 10 min of exposure, with the impact being irreversible and primarily affecting degradation rather than receptor-mediated uptake; (5) the tubulin network was disrupted in high H 2 O 2 concentration while the actin cytoskeleton appears to remain largely intact. Finally, we found that reducing agents and thiol donors such as N-Acetyl Cysteine (NAC) and Glutathione (GSH) could protect cells from ROS-induced damage but could not reverse existing damage. Pretreatment with NAC, but not GSH, reduced the negative effects of ROS exposure suggesting that LSEC does not store an excess amount of GSH but rather can readily produce it in the occurrence of oxidative stress conditions. The observed thresholds in dose and time-dependent changes as well as the treatments with NAC/GSH confirm the existence of ROS depleting system in LSEC. Abstract Figure Highlights ROS by H 2 O 2 irreversibly depletes LSEC endocytic/scavenging function in vitro H 2 O 2 exposure causes dynamic, dose-dependent defenestration of LSEC within 0.5 h Partial refenestration can occur after about 1h of exposure to H 2 O 2 NAC/GSH mitigate H 2 O 2 -induced ROS effects in LSEC LSEC do not store excess GSH but produce GSH when exposed to oxidative stress