In Vitro Assessment of Mitochondrial Toxicity to Predict Drug-Induced Liver Injury

  • Mathieu Porceddu
  • Nelly Buron
  • Pierre Rustin
  • Bernard Fromenty
  • Annie Borgne-SanchezEmail author
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Mitochondrial liability of drugs and other xenobiotics is a major issue for patients because such toxicity can damage different tissues and organs such as liver, heart, and muscle. Drug-induced mitochondrial toxicity is also a major concern for pharmaceutical industries. Indeed, it is now acknowledged that such mechanism of toxicity can induce severe, and sometimes fatal, liver injury which can lead to the interruption of clinical trials, or drug withdrawal after marketing, such as in the case of troglitazone. Therefore, drug-induced mitochondrial dysfunction is increasingly sought after by pharmaceutical companies by using reliable in vitro assays in order to discard potential mitochondrion-toxic drugs during drug discovery stage. This chapter presents the in vitro methods used to identify potential mitochondrion-toxic drugs. To this end, different types of biological materials are used such as isolated mouse liver mitochondria and the human hepatic HepaRG® cell line, which expresses the main enzymes and transcription factors involved in drug metabolism. The in vitro method we discussed allows to investigate several key mitochondrial parameters such as oxygen consumption, transmembrane potential, respiratory chain complex activities, and mtDNA levels. These investigations are able to detect not only direct and acute mitochondrial alterations due to parent drugs but also indirect and chronic mitochondrial liability that can be induced by secondary metabolites. Hence, it could be used to detect potential drug-induced mitochondrial liability and to understand the involved mechanisms.

Key words

DILI Drug-induced liver injury Hepatocytes Hepatotoxicity Liver Mitochondria Mitochondrial toxicity Oxidative stress Respiratory chain Transmembrane potential 



This review was supported by a grant from the Agence Nationale de la Recherche (ANR-16-CE18-0010-03 MITOXDRUGS). We are very grateful to Biopredic International (Rennes, France) and especially Dr. Christophe Chesné for providing HepaRG cells for the characterization experiments and Sandrine Camus for recommendations on HepaRG cell culture.


  1. 1.
    Begriche K, Massart J, Robin MA, Borgne-Sanchez A, Fromenty B (2011) Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. J Hepatol 54:773–794CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Gougeon ML, Penicaud L, Fromenty B, Leclercq P, Viard JP, Capeau J (2004) Adipocytes targets and actors in the pathogenesis of HIV-associated lipodystrophy and metabolic alterations. Antivir Ther 9:161–177PubMedPubMedCentralGoogle Scholar
  3. 3.
    Varga ZV, Ferdinandy P, Liaudet L, Pacher P (2015) Drug-induced mitochondrial dysfunction and cardiotoxicity. Am J Physiol Heart Circ Physiol 309:H1453–H1467CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Fromenty B, Pessayre D (1995) Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity. Pharmacol Ther 67:101–154CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Knockaert L, Descatoire V, Vadrot N, Fromenty B, Robin MA (2011) Mitochondrial CYP2E1 is sufficient to mediate oxidative stress and cytotoxicity induced by ethanol and acetaminophen. Toxicol In Vitro 25:475–484CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lieber CS, DeCarli L, Rubin E (1975) Sequential production of fatty liver, hepatitis, and cirrhosis in sub-human primates fed ethanol with adequate diets. Proc Natl Acad Sci U S A 72:437–441CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hardonniere K, Saunier E, Lemarie A, Fernier M, Gallais I, Helies-Toussaint C, Mograbi B, Antonio S, Benit P, Rustin P, Janin M, Habarou F, Ottolenghi C, Lavault MT, Benelli C, Sergent O, Huc L, Bortoli S, Lagadic-Gossmann D (2016) The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival. Sci Rep 6:30776CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jiang Y, Xia W, Yang J, Zhu Y, Chang H, Liu J, Huo W, Xu B, Chen X, Li Y, Xu S (2015) BPA-induced DNA hypermethylation of the master mitochondrial gene PGC-1alpha contributes to cardiomyopathy in male rats. Toxicology 329:21–31CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Labbe G, Pessayre D, Fromenty B (2008) Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies. Fundam Clin Pharmacol 22:335–353CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Will Y, Dykens J (2014) Mitochondrial toxicity assessment in industry—a decade of technology development and insight. Expert Opin Drug Metab Toxicol 10:1061–1067CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Nadanaciva S, Will Y (2011) Investigating mitochondrial dysfunction to increase drug safety in the pharmaceutical industry. Curr Drug Targets 12:774–782CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wallace DC, Fan W, Procaccio V (2010) Mitochondrial energetics and therapeutics. Annu Rev Pathol 5:297–348CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Naven RT, Swiss R, Klug-McLeod J, Will Y, Greene N (2013) The development of structure-activity relationships for mitochondrial dysfunction: uncoupling of oxidative phosphorylation. Toxicol Sci 131:271–278CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Schon E, Fromenty B (2015) Alteration of mitochondrial DNA in liver diseases, vol 150. Taylor & Francis, New-YorkGoogle Scholar
  15. 15.
    Govindarajan R, Leung GP, Zhou M, Tse CM, Wang J, Unadkat JD (2009) Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3. Am J Physiol Gastrointest Liver Physiol 296:G910–G922CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lee EW, Lai Y, Zhang H, Unadkat JD (2006) Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine. J Biol Chem 281:16700–16706CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Berson A, Renault S, Letteron P, Robin MA, Fromenty B, Fau D, Le Bot MA, Riche C, Durand-Schneider AM, Feldmann G, Pessayre D (1996) Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction. Gastroenterology 110:1878–1890CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Al Maruf A, O’Brien PJ, Naserzadeh P, Fathian R, Salimi A, Pourahmad J (2017) Methotrexate induced mitochondrial injury and cytochrome c release in rat liver hepatocytes. Drug Chem Toxicol:1–11Google Scholar
  19. 19.
    Kowaltowski AJ, Castilho RF, Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress. FEBS Lett 495:12–15CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Pessayre D, Mansouri A, Berson A, Fromenty B (2010) Mitochondrial involvement in drug-induced liver injury. Handb Exp Pharmacol:311–365Google Scholar
  21. 21.
    Gardner K, Hall PA, Chinnery PF, Payne BA (2014) HIV treatment and associated mitochondrial pathology: review of 25 years of in vitro, animal, and human studies. Toxicol Pathol 42:811–822CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Mansouri A, Haouzi D, Descatoire V, Demeilliers C, Sutton A, Vadrot N, Fromenty B, Feldmann G, Pessayre D, Berson A (2003) Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver. Hepatology 38:715–725CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Antherieu S, Rogue A, Fromenty B, Guillouzo A, Robin MA (2011) Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells. Hepatology 53:1895–1905CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Aubert J, Begriche K, Knockaert L, Robin MA, Fromenty B (2011) Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role. Clin Res Hepatol Gastroenterol 35:630–637CrossRefGoogle Scholar
  25. 25.
    Lee WM (2003) Drug-induced hepatotoxicity. N Engl J Med 349:474–485CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Russmann S, Kullak-Ublick GA, Grattagliano I (2009) Current concepts of mechanisms in drug-induced hepatotoxicity. Curr Med Chem 16:3041–3053CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Porceddu M, Buron N, Roussel C, Labbe G, Fromenty B, Borgne-Sanchez A (2012) Prediction of liver injury induced by chemicals in human with a multiparametric assay on isolated mouse liver mitochondria. Toxicol Sci 129:332–345CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Buron N, Porceddu M, Roussel C, Begriche K, Trak-Smayra V, Gicquel T, Fromenty B, Borgne-Sanchez A (2017) Chronic and low exposure to a pharmaceutical cocktail induces mitochondrial dysfunction in liver and hyperglycemia: differential responses between lean and obese mice. Environ Toxicol 32:1375–1389CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    El-Khoury R, Dufour E, Rak M, Ramanantsoa N, Grandchamp N, Csaba Z, Duvillie B, Benit P, Gallego J, Gressens P, Sarkis C, Jacobs HT, Rustin P (2013) Alternative oxidase expression in the mouse enables bypassing cytochrome c oxidase blockade and limits mitochondrial ROS overproduction. PLoS Genet 9:e1003182CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Bénit P, Chrétien D, Porceddu M, Rustin P, Rak M (2017) A performing, versatile and inexpensive device for oxygen uptake measurement. J Clin Med 6(6), pii: E58. doi:10.3390/jcm6060058
  31. 31.
    Will Y, Hynes J, Ogurtsov VI, Papkovsky DB (2006) Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat Protoc 1:2563–2572CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Dykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y (2008) In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone. Toxicol Sci 103:335–345CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Buron N, Porceddu M, Brabant M, Desgue D, Racoeur C, Lassalle M, Pechoux C, Rustin P, Jacotot E, Borgne-Sanchez A (2010) Use of human cancer cell lines mitochondria to explore the mechanisms of BH3 peptides and ABT-737-induced mitochondrial membrane permeabilization. PLoS One 5:e9924CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C (2007) The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact 168:66–73CrossRefGoogle Scholar
  35. 35.
    Peyta L, Jarnouen K, Pinault M, Guimaraes C, Pais de Barros JP, Chevalier S, Dumas JF, Maillot F, Hatch GM, Loyer P, Servais S (2016) Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells. Biochim Biophys Acta 1857:443–453CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Chretien D, Rustin P, Bourgeron T, Rotig A, Saudubray JM, Munnich A (1994) Reference charts for respiratory chain activities in human tissues. Clin Chim Acta 228:53–70CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Aninat C, Piton A, Glaise D, Le Charpentier T, Langouet S, Morel F, Guguen-Guillouzo C, Guillouzo A (2006) Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 34:75–83CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Benit P, Goncalves S, Philippe Dassa E, Briere JJ, Martin G, Rustin P (2006) Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples. Clin Chim Acta 374:81–86CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Bradbury DA, Simmons TD, Slater KJ, Crouch SP (2000) Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J Immunol Methods 240:79–92CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Pertuiset C, Porceddu M, Buron N, Camus S, Chesné C, Borgne-Sanchez A (2015) Identification of drug-induced mitochondrial alterations using HepaRG cell line. Toxicol Lett 238:S316–S317CrossRefGoogle Scholar
  41. 41.
    Porceddu M, Pertuiset C, Camus S, Chesné C, Buron N, Borgne-Sanchez A (2016) In vitro prediction of antiretroviral drug-induced hepatotoxicicty by using mitochondrial MiToxView screening platform. Toxicol Sci 150:S598Google Scholar
  42. 42.
    Igoudjil A, Massart J, Begriche K, Descatoire V, Robin MA, Fromenty B (2008) High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes. Toxicol In Vitro 22:887–898CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Rossignol R, Gilkerson R, Aggeler R, Yamagata K, Remington SJ, Capaldi RA (2004) Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Res 64:985–993CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y (2007) Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol Sci 97:539–547CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Hewitt NJ, Hewitt P (2004) Phase I and II enzyme characterization of two sources of HepG2 cell lines. Xenobiotica 34:243–256CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Guo L, Dial S, Shi L, Branham W, Liu J, Fang JL, Green B, Deng H, Kaput J, Ning B (2011) Similarities and differences in the expression of drug-metabolizing enzymes between human hepatic cell lines and primary human hepatocytes. Drug Metab Dispos 39:528–538CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Hynes J, Nadanaciva S, Swiss R, Carey C, Kirwan S, Will Y (2013) A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays. Toxicol In Vitro 27:560–569CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Michaut A, Le Guillou D, Moreau C, Bucher S, McGill MR, Martinais S, Gicquel T, Morel I, Robin MA, Jaeschke H, Fromenty B (2016) A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: application to acetaminophen. Toxicol Appl Pharmacol 292:40–55CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Pessayre D, Fromenty B, Berson A, Robin MA, Letteron P, Moreau R, Mansouri A (2012) Central role of mitochondria in drug-induced liver injury. Drug Metab Rev 44:34–87CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Nadanaciva S, Rana P, Beeson GC, Chen D, Ferrick DA, Beeson CC, Will Y (2012) Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform. J Bioenerg Biomembr 44:421–437CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Fromenty B, Freneaux E, Labbe G, Deschamps D, Larrey D, Letteron P, Pessayre D (1989) Tianeptine, a new tricyclic antidepressant metabolized by beta-oxidation of its heptanoic side chain, inhibits the mitochondrial oxidation of medium and short chain fatty acids in mice. Biochem Pharmacol 38:3743–3751CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Freneaux E, Fromenty B, Berson A, Labbe G, Degott C, Letteron P, Larrey D, Pessayre D (1990) Stereoselective and nonstereoselective effects of ibuprofen enantiomers on mitochondrial beta-oxidation of fatty acids. J Pharmacol Exp Ther 255:529–535PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Mathieu Porceddu
    • 1
    • 2
  • Nelly Buron
    • 1
    • 2
  • Pierre Rustin
    • 3
  • Bernard Fromenty
    • 4
  • Annie Borgne-Sanchez
    • 1
    • 2
    Email author
  1. 1.MITOLOGICS Research LabHôpital Robert DebréParisFrance
  2. 2.MITOLOGICS SASParc BiocitechRomainvilleFrance
  3. 3.INSERM, UMR1141-PROTECTHôpital Robert DebréParisFrance
  4. 4.INSERM, INRA, Univ Rennes 1, Univ Bretagne Loire, Nutrition, Metabolism and Cancer (NuMeCan)RennesFrance

Personalised recommendations