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 H2O2 affects isolated rat LSECs at different concentrations (0.5-1000{micro}M) and exposure times (10-120 min). Our findings show that H2O2 exposure affects several LSEC functions in a dose- and time-dependent manner: (1) cell viability, reducing potential, and scavenging function decreased as H2O2 concentration and exposure time increased; (2) intracellular ROS levels rose with higher H2O2 concentrations; (3) fenestrations exhibited a dynamic response, initially closing but partially reopening at H2O2 concentrations above 100{micro}M 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 H2O2 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/609175v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1f25779org.highwire.dtl.DTLVardef@14cda58org.highwire.dtl.DTLVardef@92dea4org.highwire.dtl.DTLVardef@56e97c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIROS by H2O2 irreversibly depletes LSEC endocytic/scavenging function in vitro C_LIO_LIH2O2 exposure causes dynamic, dose-dependent defenestration of LSEC within 0.5 h C_LIO_LIPartial refenestration can occur after about 1h of exposure to H2O2 C_LIO_LINAC/GSH mitigate H2O2-induced ROS effects in LSEC C_LIO_LILSEC do not store excess GSH but produce GSH when exposed to oxidative stress C_LI