title: Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells.
abstract: 1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown. The aim of the present study was to study the role of CPR in the generation of ROS and cellular injury under basal conditions, and after simulated in vitro ischaemia-reperfusion (IR). 2. Plasmid DNA or siRNA approaches were used to transiently overexpress or knockdown the human CPR gene in rat liver epithelial (WB-F344) or human hepatoblastoma (HepG2) cells, respectively. The generation of ROS and/or cellular injury was then studied under the basal conditions and after simulated IR (4 h of ischaemia plus 30 min of reoxygenation). 3. Under the basal conditions, transient overexpression of CPR protein in WB-F344 cells caused a 90% increase in the CPR activity, which was associated with a 100% increase in the ROS production. In contrast, after simulated IR, a 2.5-fold higher CPR activity did not significantly affect the magnitude of ROS generation or cell death. Similarly, although the knockdown of CPR protein resulted in a significant reduction (∼30%) in the CPR activity, the ROS production was not substantially altered after simulated IR in HepG2 cells. 4. Our data suggest that CPR plays a major role in the ROS generation by liver cells under the basal conditions. However, the role of CPR in the ROS generation during simulated in vitro IR injury in these cells is minimal, if any.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA.
authors.forename: Venkateswaran C
authors.initials: VC
authors.lastname: Pillai
authors.forename: Russell O
authors.initials: RO
authors.lastname: Snyder
authors.forename: Upendra
authors.initials: U
authors.lastname: Gumaste
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Thekkumkara
authors.forename: Reza
authors.initials: R
authors.lastname: Mehvar
mesh_terms: D000818:Animals; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D009251:NADPH-Ferrihemoprotein Reductase