CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells

Abstract

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca 2+ -ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cells lines: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca 2+ -dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca 2+ -dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes vs 4.7 nmol Pi/min/mg prot in E47 microsomes). Saturation curves with Ca 2+ or ATP showed that CYP2E1 overexpression produced a decrease in V MAX but did not affect the KM for either Ca 2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATPdependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2 mM N-acetylcysteine prevented the decrease in microsomal Ca 2+ ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca 2+ ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

“…The contrasted results observed for these two compounds (Table I) suggest that this difference lies in the cell's metabolic capability. While CPA's genotoxicity for HepG2 cells has previously been reported [Westerink et al, 2011;Tsamou et al, 2012], the genotoxic potential of DMNA for these cells without the addition of a bioactivation system has not been demonstrated [Westerink et al, 2007b;Tsamou et al, 2012], probably due to poor CYP2E1 expression in this cell line [Caro et al, 2009]. Our results for the five other compounds (BaP, DMBA, AFB1, 2-AAF, and DAT), which share a genotoxic MOA related to a specific biotransformation process, confirmed that HepG2 cells express functional CYP1A1, 1A2, 1B1, and 3A4 activities.…”

Selective Modulation of Hepatic Cytochrome P450 and Flavin Monooxygenase 3 Expression duringCitrobacter rodentiumInfection in Severe Combined Immune-Deficient Mice

“…The intensity of chemiluminescent protein bands was visualized by fluorography on X-ray film and was scanned, and the protein bands were quantified by densitometry. All assays were performed within a linear range, and bands' intensities were normalized to G␤, a loading control for microsomal samples (Caro et al, 2009).…”

Bimodal targeting of microsomal cytochrome P450s to mitochondria: implications in drug metabolism and toxicity

“…CYP2E1 is believed to contribute to alcoholic liver disease and nonalcoholic steatohepatitis through effects on lipid hydroperoxides [127–129]. There is a great deal of in vitro evidence to suggest that induction of CYP2E1 in cultured cell systems results in oxidative damage to the cells [130–132]. There is also accumulating in vivo evidence to suggest that CYP2E1 plays a role in alcohol-mediated liver injury [114].…”