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Title: Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes

Abstract

Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca{sup 2+} {sub c}) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 {mu}M). At 8 h, diclofenac caused increases in [Ca{sup 2+}]{sub c} (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca{sup 2+} chelator) inhibited cell injury, indicating that Ca{sup 2+} plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, {delta}{psi}{sub m} (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca{sup 2+}]{sub c}. The CYP2C9 inhibitor sulphaphenazole (10 {mu}M) protected from diclofenac-induced cell injury and prevented increases in [Ca{sup 2+}]{sub c}, whilemore » it had no effect on the dissipation of the {delta}{psi}{sub m}. Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca{sup 2+}]{sub c}. In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca{sup 2+}]{sub c}, triggering the mPT and precipitating cell death.« less

Authors:
 [1];  [1];  [1];  [2]
  1. Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore)
  2. Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore) and Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117597 (Singapore). E-mail: phcbua@nus.edu.sg
Publication Date:
OSTI Identifier:
20850506
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 217; Journal Issue: 3; Other Information: DOI: 10.1016/j.taap.2006.09.012; PII: S0041-008X(06)00345-0; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; CALCIUM IONS; DEPOLARIZATION; DRUGS; FLUORESCENCE; INFLAMMATION; INJURIES; IODIDES; LEAD; LIVER CELLS; MEMBRANES; METABOLITES; MITOCHONDRIA; PERMEABILITY; PHOSPHORYLATION

Citation Formats

Lim, M.S., Lim, Priscilla L.K., Gupta, Rashi, and Boelsterli, Urs A.. Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes. United States: N. p., 2006. Web. doi:10.1016/j.taap.2006.09.012.
Lim, M.S., Lim, Priscilla L.K., Gupta, Rashi, & Boelsterli, Urs A.. Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes. United States. doi:10.1016/j.taap.2006.09.012.
Lim, M.S., Lim, Priscilla L.K., Gupta, Rashi, and Boelsterli, Urs A.. Fri . "Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes". United States. doi:10.1016/j.taap.2006.09.012.
@article{osti_20850506,
title = {Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes},
author = {Lim, M.S. and Lim, Priscilla L.K. and Gupta, Rashi and Boelsterli, Urs A.},
abstractNote = {Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca{sup 2+} {sub c}) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 {mu}M). At 8 h, diclofenac caused increases in [Ca{sup 2+}]{sub c} (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca{sup 2+} chelator) inhibited cell injury, indicating that Ca{sup 2+} plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, {delta}{psi}{sub m} (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca{sup 2+}]{sub c}. The CYP2C9 inhibitor sulphaphenazole (10 {mu}M) protected from diclofenac-induced cell injury and prevented increases in [Ca{sup 2+}]{sub c}, while it had no effect on the dissipation of the {delta}{psi}{sub m}. Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca{sup 2+}]{sub c}. In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca{sup 2+}]{sub c}, triggering the mPT and precipitating cell death.},
doi = {10.1016/j.taap.2006.09.012},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 217,
place = {United States},
year = {Fri Dec 15 00:00:00 EST 2006},
month = {Fri Dec 15 00:00:00 EST 2006}
}