Superoxide anion mediated mitochondrial dysfunction leads to hepatocyte apoptosis preferentially in the periportal region during copper toxicity in rats

doi:10.1016/j.cbi.2009.08.014

Chemico-Biological Interactions

Volume 182, Issues 2–3, 10 December 2009, Pages 136-147

https://doi.org/10.1016/j.cbi.2009.08.014 Get rights and content

Abstract

Chronic exposure to copper induces hepatocellular apoptosis with greater injury in the periportal region compared to the perivenous region. Here we have identified the factors responsible for the development of regional damage in the liver under in vivo conditions. Enhanced production of reactive oxygen species (ROS) with predominance of superoxide radical (O₂ radical dot ⁻) indicates the contribution of redox imbalance in the process. This may be linked with copper catalyzed oxidation of GSH to GSSG resulting in the generation of O₂⁻. Downregulation of Cu-Zn SOD in consequence of the degradation of this enzyme, causes decreased dismutation of O₂⁻, that further contributes to the enhanced level of O₂ radical dot ⁻ in the periportal region. Decreased functioning of Mn SOD activity, reduction in mitochondrial thiol/disulphide ratio and generation of O₂⁻ were much higher in the mitochondria from periportal region, which point to the involvement of this organelle in the regional hepatotoxicity observed during copper exposure. This was supported by copper-mediated enhanced mitochondrial dysfunction as evident from ATP depletion, collapse of mitochondrial membrane potential (MMP) and induction of mitochondrial permeability transition (MPT). Results suggest the active participation of O₂ radical dot ⁻ in inducing mitochondrial dysfunction preferentially in the periportal region that eventually leads to the development of hepatotoxicity due to copper exposure under in vivo condition.

Introduction

Diseases like Wilson Disease, Indian Childhood Cirrhosis, Idiopathic Copper Toxicosis and Chronic Cholestasis are all associated with hepatic copper accumulation which eventually leads to cirrhosis of the liver [1], [2], [3]. Copper is absorbed from the gastrointestinal tract and transported to the hepatocytes via portal transporters. Intracellular copper then binds to metalochaperones and becomes destined for storage within hepatocytes, secretion in plasma or excretion in bile [4]. Although liver is considered to be the main site for copper homeostasis in the body, excess of this transition metal induces apoptotic changes in the hepatocytes followed by cirrhosis which eventually culminates in liver failure [5].

Hepatocytes display distinct heterogeneity towards structure and function within different zones of the liver [6], [7]. Even though wide variance has been observed in metabolic and transport processes specifically between the periportal and perivenous regions, it is not yet clear whether this zonal dissimilarity has any reflection on the cellular death process associated with hepatotoxicity. Keeping this in mind, the first aim of our study was to find out the response of hepatocytes from periportal and perivenous regions in copper toxicity under in vivo conditions.

Recent reports implicate mitochondria as the major target for liver toxicity resulting from copper overload, either through high dietary intake or due to a genetic defect in transport mechanism [8]. However, the sequence of events in the development of pathologies related to mitochondrial dysfunction with an emphasis on its applicability under in vivo conditions remained largely unknown.

Literature available so far suggests that dissipation of mitochondrial transmembrane potential (Δψ_m), followed by induction of mitochondrial permeability transition (MPT) eventually lead to the release of proapoptotic factors and activate cellular apoptosis [9]. Whilst the redox property of copper is essential for the activity of cuproenzymes, excess of this element in the cells can also be a source of reactive oxygen species (ROS), that can damage lipids, proteins and nucleic acids [10]. Since enhanced generation of ROS is known to play an important role in MPT induction, our second aim was to explore whether copper toxicity can induce ROS mediated MPT opening followed by mitochondrial dysfunction, and to identify the ROS involved in the process.

In the presence of glutathione (GSH), Cu²⁺ is reduced to Cu⁺ which then catalyzes the generation of ROS via the Fenton reaction. Considering the importance of GSH in the mobilization of copper in hepatocytes [11], our third objective was to determine whether GSH is involved in the generation of ROS and induction of mitochondrial dysfunction that may ultimately converge to hepatocyte apoptosis during copper toxicity.

Section snippets

Chemicals

Unless otherwise noted, all chemicals were obtained from Sigma (St. Louis, MO). RPMI Medium 1640 (GIBCO™) and FBS (GIBCO™) were obtained from Invitrogen Corporation (Grand Island, NY). Serum Alanine Transaminase (ALT) measurement kit was obtained from TECO Diagnostics (Anaheim, CA). ATP Bioluminescence Assay Kit was purchased from Biovision Inc. (Mountain View, CA). TUNEL assay kit (APO-DIRECT™ kit) was purchased from Calbiochem (San Diego, CA). The primary antibody [SOD-1 (FL-154); Santa Cruz

Copper toxicity induces morphological and functional alterations in liver

In order to elucidate the mechanism of copper induced liver toxicity under in vivo condition, we first determined the endurance of rats towards exposure to increasing doses of copper for different durations. As evident from Fig. 1A, exposure to CuSO₄ beyond 45 days affected the mean survival of all the animals. Rats receiving lower doses (10 mg/kg and 15 mg/kg) showed 100% survival up to 45 days, whereas the animals receiving CuSO₄ at the dose of 20 mg/kg could not tolerate the treatment showing

Discussion

Copper is an essential transition metal which is required for normal growth and development of living organisms. It functions as a cofactor for several mammalian enzymes like cytochrome C oxidase, Cu-Zn SOD, and ceruloplasmin [37]. Hepatocellular copper homeostasis involves copper uptake into hepatocytes followed by its incorporation into major copper containing proteins and excretion through bile [4]. When copper accumulation surpasses the normal storage capacity of the liver, it causes

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

Authors are grateful to Dr. Anil Ghosh of Drug Development Diagnostics & Biotechnology Division and Dr. S.N. Kabir of Cell Biology & Physiology Division at the Indian Institute of Chemical Biology, Kolkata, for providing facilities for histological & microphotographic studies. Authors thank Mrs. Banasri Das for confocal microscopic studies. This work was supported by Council of Scientific and Industrial Research (CSIR), India, Project NWP 0009, and CSIR fellowships (to D.N.R. and S.M.).

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Superoxide anion mediated mitochondrial dysfunction leads to hepatocyte apoptosis preferentially in the periportal region during copper toxicity in rats

Abstract

Introduction

Section snippets

Chemicals

Copper toxicity induces morphological and functional alterations in liver

Discussion

Conflict of interest statement

Acknowledgments

Lancet

Gastroenterology

Am. J. Clin. Nutr.

Am. J. Clin. Nutr.

Gastroenterology

Method Enzymol.

Methods Cell Biol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Blood Cells Mol. Dis.

Anal. Biochem.

Anal. Biochem.

Biochem. Biophys. Acta

Hepatology

Mol. Aspects Med.

J. Comp. Pathol.

Toxicology

Biochim. Biophys. Acta Gen. Subjects

Chem. Biol. Interact.

Biochim. Biophys. Acta Protein Struct. Mol. Enzymol.

Free Radic. Biol. Med.

Wilson's Disease

Functional specialization of different hepatocyte populations

Phys. Rev.

Cu2+ toxicity inhibition of mitochondrial dehydrogenases in vitro and in vivo

Ann. Neurol.

Mitochondrial control of cell death

Nat. Med.

Basic and clinical aspects of copper

Crit. Rev. Clin. Lab. Sci.

An overview of the metabolism of copper

Eur. J. Med. Res.

Copper chelation therapy in intrahepatic cholestasis of childhood

Gut

Cu²⁺ toxicity inhibition of mitochondrial dehydrogenases in vitro and in vivo