1887

Abstract

The widely used drug diclofenac can cause serious heart, liver and kidney injury, which may be related to its ability to cause mitochondrial dysfunction. Using as a model system, we studied the mechanisms of diclofenac toxicity and the role of mitochondria therein. We found that diclofenac reduced cell growth and viability and increased levels of reactive oxygen species (ROS). Strains increasingly relying on respiration for their energy production showed enhanced sensitivity to diclofenac. Furthermore, oxygen consumption was inhibited by diclofenac, suggesting that the drug inhibits respiration. To identify the site of respiratory inhibition, we investigated the effects of deletion of respiratory chain subunits on diclofenac toxicity. Whereas deletion of most subunits had no effect, loss of either Rip1p of complex III or Cox9p of complex IV resulted in enhanced resistance to diclofenac. In these deletion strains, diclofenac did not increase ROS formation as severely as in the wild-type. Our data are consistent with a mechanism of toxicity in which diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death.

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2011-03-01
2024-12-06
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References

  1. Alem M. A. S., Douglas L. J. 2004; Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans . Antimicrob Agents Chemother 48:41–47
    [Google Scholar]
  2. Armstrong J. S., Yang H. Y., Duan W., Whiteman M. 2004; Cytochrome bc 1 regulates the mitochondrial permeability transition by two distinct pathways. J Biol Chem 279:50420–50428
    [Google Scholar]
  3. Beckmann J. D., Ljungdahl P. O., Trumpower B. L. 1989; Mutational analysis of the mitochondrial Rieske Iron–Sulfur protein of Saccharomyces cerevisiae . 1. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants. J Biol Chem 264:3713–3722
    [Google Scholar]
  4. Bharucha N., Kumar A. 2007; Yeast genomics and drug target identification. Comb Chem High Throughput Screen 10:618–634
    [Google Scholar]
  5. Boelsterli U. A., Lim P. L. 2007; Mitochondrial abnormalities – a link to idiosyncratic drug hepatotoxicity?. Toxicol Appl Pharmacol 220:92–107
    [Google Scholar]
  6. Bort R., Ponsoda X., Jover R., Gómez-Lechón M. J., Castell J. V. 1999; Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity. J Pharmacol Exp Ther 288:65–72
    [Google Scholar]
  7. Calder K. M., McEwen J. E. 1991; Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII in assembly of remaining subunits. Mol Microbiol 5:1769–1777
    [Google Scholar]
  8. Cecere F., Iuliano A., Albano F., Zappelli C., Castellano I., Grimaldi P., Masullo M., De Vendittis E., Ruocco M. R. 2010; Diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y: possible involvement of the mitochondrial superoxide dismutase. J Biomed Biotechnol 2010801726
    [Google Scholar]
  9. Crivellone M. D., Wu M. A., Tzagoloff A. 1988; Assembly of the mitochondrial membrane system. Analysis of structural mutants of the yeast coenzyme QH2-cytochrome c reductase complex. J Biol Chem 263:14323–14333
    [Google Scholar]
  10. Dowhan W., Bibus C. R., Schatz G. 1985; The cytoplasmically-made subunit-IV is necessary for assembly of cytochrome c oxidase in yeast. EMBO J 4:179–184
    [Google Scholar]
  11. Dykens J. A., Will Y. 2007; The significance of mitochondrial toxicity testing in drug development. Drug Discov Today 12:777–785
    [Google Scholar]
  12. Fendt S. M., Sauer U. 2010; Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates. BMC Syst Biol 4:12
    [Google Scholar]
  13. Fosbøl E. L., Gislason G. H., Jacobsen S., Folke F., Hansen M. L., Schramm T. K., Sorensen R., Rasmussen J. N., Andersen S. S. other authors 2009; Risk of myocardial infarction and death associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) among healthy individuals: a nationwide cohort study. Clin Pharmacol Ther 85:190–197
    [Google Scholar]
  14. Gietz R. D., Sugino A. 1988; New yeast- Escherichia-coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking 6-base pair restriction sites. Gene 74:527–534
    [Google Scholar]
  15. Goldring E. S., Grossman L. I., Krupnick D., Cryer D. R., Marmur J. 1970; The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol 52:323–335
    [Google Scholar]
  16. Gómez-Lechón M. J., Ponsoda X., O'Connor E., Donato T., Castell J. V., Jover R. 2003; Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS. Biochem Pharmacol 66:2155–2167
    [Google Scholar]
  17. Guzy R. D., Hoyos B., Robin E., Chen H., Liu L. P., Mansfield K. D., Simon M. C., Hammerling U., Schumacker P. T. 2005; Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing. Cell Metab 1:401–408
    [Google Scholar]
  18. Guzy R. D., Mack M. M., Schumacker P. T. 2007; Mitochondrial complex III is required for hypoxia-induced ROS production and gene transcription in yeast. Antioxid Redox Signal 9:1317–1328
    [Google Scholar]
  19. Hallstrom T. C., Moye-Rowley W. S. 2000; Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae . J Biol Chem 275:37347–37356
    [Google Scholar]
  20. Inoue A., Muranaka S., Fujita H., Kanno T., Tamai H., Utsumi K. 2004; Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway. Free Radic Biol Med 37:1290–1299
    [Google Scholar]
  21. Katzmann D. J., Burnett P. E., Golin J., Mahe Y., Moye-Rowley W. S. 1994; Transcriptional control of the yeast PDR5 gene by the PDR3 gene product. Mol Cell Biol 14:4653–4661
    [Google Scholar]
  22. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. 1983; A general method for polyethylene-glycol-induced genetic-transformation of bacteria and yeast. Gene 25:333–341
    [Google Scholar]
  23. Kowaltowski A. J., de Souza-Pinto N. C., Castilho R. F., Vercesi A. E. 2009; Mitochondria and reactive oxygen species. Free Radic Biol Med 47:333–343
    [Google Scholar]
  24. Krause M. M., Brand M. D., Krauss S., Meisel C., Vergin H., Burmester G. R., Buttgereit F. 2003; Nonsteroidal antiinflammatory drugs and a selective cyclooxygenase 2 inhibitor uncouple mitochondria in intact cells. Arthritis Rheum 48:1438–1444
    [Google Scholar]
  25. Labbe G., Pessayre D., Fromenty B. 2008; Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies. Fundam Clin Pharmacol 22:335–353
    [Google Scholar]
  26. Laine L., Goldkind L., Curtis S. P., Connors L. G., Yanqiong Z., Cannon C. P. 2009; How common is diclofenac-associated liver injury? Analysis of 17,289 arthritis patients in a long-term prospective clinical trial. Am J Gastroenterol 104:356–362
    [Google Scholar]
  27. Lee J. Y., Hwang G. W., Naganuma A. 2009; Rip1 enhances methylmercury toxicity through production of reactive oxygen species (ROS) in budding yeast. J Toxicol Sci 34:715–717
    [Google Scholar]
  28. Lemire B. D., Oyedotun K. S. 2002; The Saccharomyces cerevisiae mitochondrial succinate : ubiquinone oxidoreductase. Biochim Biophys Acta 1553102–116
    [Google Scholar]
  29. Lenaz G., Genova M. L. 2010; Structure and organization of mitochondrial respiratory complexes: a new understanding of an old subject. Antioxid Redox Signal 12:961–1008
    [Google Scholar]
  30. Lim M. S., Lim P. L., Gupta R., Boelsterli U. A. 2006; 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. Toxicol Appl Pharmacol 217:322–331
    [Google Scholar]
  31. Masubuchi Y., Yamada S., Horie T. 2000; Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs. J Pharmacol Exp Ther 292:982–987
    [Google Scholar]
  32. Masubuchi Y., Nakayama S., Horie T. 2002; Role of mitochondrial permeability transition in diclofenac-induced hepatocyte injury in rats. Hepatology 35:544–551
    [Google Scholar]
  33. McEwen J. E., Ko C., Kloeckner-Gruissem B., Poyton R. O. 1986; Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae . Characterization of mutants in 34 complementation groups. J Biol Chem 261:11872–11879
    [Google Scholar]
  34. Mima S., Ushijima H., Hwang H. J., Tsutsumi S., Makise M., Yamaguchi Y., Tomofusa T., Mizushima H., Mizushima T. 2007; Identification of the TPO1 gene in yeast, and its human orthologue TETRAN, which cause resistance to NSAIDs. FEBS Lett 581:1457–1463
    [Google Scholar]
  35. Muller F. L., Roberts A. G., Bowman M. K., Kramer D. M. 2003; Architecture of the Qo site of the cytochrome bc 1 complex probed by superoxide production. Biochemistry 42:6493–6499
    [Google Scholar]
  36. Nieminen A. L., Byrne A. M., Herman B., Lemasters J. J. 1997; Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species. Am J Physiol 272:C1286–C1294
    [Google Scholar]
  37. Niklas J., Noor F., Heinzle E. 2009; Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2. Toxicol Appl Pharmacol 240:327–336
    [Google Scholar]
  38. Noverr M. C., Phare S. M., Toews G. B., Coffey M. J., Huffnagle G. B. 2001; Pathogenic yeasts Cryptococcus neoformans and Candida albicans produce immunomodulatory prostaglandins. Infect Immun 69:2957–2963
    [Google Scholar]
  39. Orij R., Postmus J., Ter Beek A., Brul S., Smits G. J. 2009; In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. Microbiology 155:268–278
    [Google Scholar]
  40. Poyton R. O., Ball K. A., Castello P. R. 2009; Mitochondrial generation of free radicals and hypoxic signaling. Trends Endocrinol Metab 20:332–340
    [Google Scholar]
  41. Rosenfeld E., Beauvoit B. 2003; Role of the non-respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae . Yeast 20:1115–1144
    [Google Scholar]
  42. Sapienza K., Bannister W., Balzan R. 2008; Mitochondrial involvement in aspirin-induced apoptosis in yeast. Microbiology 154:2740–2747
    [Google Scholar]
  43. Seo B. B., Kitajima-Ihara T., Chan E. K., Scheffler I. E., Matsuno-Yagi A., Yagi T. 1998; Molecular remedy of complex I defects: rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells. Proc Natl Acad Sci U S A 95:9167–9171
    [Google Scholar]
  44. Srikanth C. V., Chakraborti A. K., Bachhawat A. K. 2005; Acetaminophen toxicity and resistance in the yeast Saccharomyces cerevisiae . Microbiology 151:99–111
    [Google Scholar]
  45. Taanman J. W., Capaldi R. A. 1992; Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme. J Biol Chem 267:22481–22485
    [Google Scholar]
  46. Turrens J. F. 2003; Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344
    [Google Scholar]
  47. Wohnsland F., Faller B. 2001; High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J Med Chem 44:923–930
    [Google Scholar]
  48. Wright R. M., Dircks L. K., Poyton R. O. 1986; Characterization of COX9 , the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa. Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme. J Biol Chem 261:17183–17191
    [Google Scholar]
  49. Yang S. Q., Ma H. W., Yu L., Yu C. A. 2008; On the mechanism of quinol oxidation at the QP site in the cytochrome bc 1 complex. Studied using mutants lacking cytochrome b L or b H . J Biol Chem 283:28767–28776
    [Google Scholar]
  50. Zara V., Conte L., Trumpower B. L. 2009; Biogenesis of the yeast cytochrome bc1 complex. Biochim Biophys Acta 179389–96
    [Google Scholar]
  51. Zhang X., Moye-Rowley W. S. 2001; Saccharomyces cerevisiae multidrug resistance gene expression inversely correlates with the status of the F0 component of the mitochondrial ATPase. J Biol Chem 276:47844–47852
    [Google Scholar]
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