Elsevier

Toxicology

Volume 255, Issue 3, 31 January 2009, Pages 171-176
Toxicology

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

https://doi.org/10.1016/j.tox.2008.10.020Get rights and content

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 Ca2+-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 cell line: 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 Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent 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 Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

Introduction

Calcium homeostasis is essential for cell survival, and changes in intracellular calcium are pivotal to signal transduction (Grover et al., 2003). In mammalian cells, a 10,000× concentration gradient across the plasma membrane is achieved by permanent active cytoplasmic Ca2+ extrusion, through the Na+/Ca2+ exchanger, by using the Na+ gradient generated by Na+/K+ ATPase, and is fine-tuned by the plasma membrane Ca2+-ATPase (PMCA) activity. In addition, most of the intracellular calcium is sequestered in the luminal side of the endoplasmic reticulum, by active pumping from the cytoplasm promoted by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), creating another similar transmembrane gradient (Souza dos Santos et al., 2007).

Several reports have described that reactive oxygen species (ROS) and reactive nitrogen species (RNS) can inhibit SERCA, leading to alterations in calcium homeostasis. Oxidative damage to SERCA was reported in sarcoplasmic reticulum vesicles treated in vitro with Fe2+/H2O2/ascorbate (Castilho et al., 1996), hypoxanthine/xanthine oxidase (Barnes et al., 2000), hydrogen peroxide (Grover et al., 1997), and peroxynitrite (Gutierrez-Martin et al., 2004). Partial inactivation of SERCA caused by oxidative stress has been associated with several pathological conditions such as biological aging (Viner et al., 1999), obesity (Li et al., 2006), heart failure (Lokuta et al., 2005), and high intensity exercise (Matsunaga et al., 2003).

An active source of cellular ROS is cytochrome P450. The cytochrome P450 enzymes are a superfamily of hemeproteins that serve as terminal oxidases in the mixed function oxidase system for metabolizing various endogenous substrates such as steroids and fatty acids, and xenobiotics including drugs, toxins and carcinogens (Caro and Cederbaum, 2004). Oxygen activation by P450, necessary for the enzyme catalytic function, can also result in the production of ROS. Small amounts of the superoxide anion radical (O2radical dot) can be produced from decay of the oxygenated P450 complex, while hydrogen peroxide (H2O2) can form from either dismutation of O2radical dot or decay of the peroxy P450 complex (Puntarulo and Cederbaum, 1998). Among the different P450 isoforms, CYP2E1 has a high capacity to reduce dioxygen to reactive oxy radicals (Caro and Cederbaum, 2004).

In this work, we evaluated the possibility that CYP2E1-generated reactive oxygen species may inhibit microsomal Ca2+-ATPase activity in intact liver cells.

Section snippets

Chemicals

Phosphate-buffered saline (PBS) was from Roche (Newark, NJ). G418 was from Invitrogen (Carlsbad, CA). Protein concentration was measured using the Bio-Rad DC protein assay (Hercules, CA). Most of the other chemicals used were from Sigma Chemical Co. (St. Louis, MO). Antibodies for immunoblots were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture

Two human hepatoma HepG2 cell sublines described in Chen and Cederbaum (1998) were used as models in this study: (1) E47 cells, which constitutively

Microsomal Ca2+-ATPase activity in CYP2E1 overexpressing cells

Previous work has shown that E47 cells overexpress active microsomal CYP2E1 and generate higher levels of reactive oxygen species with respect to control C34 cells, which do not express significantly any cytochrome P450 (Caro and Cederbaum, 2004). In this work, we measured the microsomal Ca2+-ATPase activity in E47 versus C34 cells. The selected assay for microsomal Ca2+-ATPase activity measures inorganic phosphate (Pi) released by the ATPase activity in one-step, without removing proteins from

Discussion

Microsomal ATPase activity in HepG2 cells showed two components: (1) a Ca2+-independent activity (around 10% of the total), which did not show significant differences between C34 and E47 cells; and (2) a Ca2+-dependent activity (around 90% of the total), which showed a significant 55% decrease in E47 with respect to C34 cells. This suggests that CYP2E1 overexpression inhibits microsomal Ca2+-dependent ATPase activity in intact HepG2 cells. The ATPase activity of rat liver microsomes also showed

Conflict of interest

The authors declare that there are no conflicts of interest.

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