Archives of Toxicology

, Volume 92, Issue 6, pp 2109–2118 | Cite as

Dysregulation of autophagy in rat liver with mitochondrial DNA depletion induced by the nucleoside analogue zidovudine

  • Ana Santos-Llamas
  • Maria J. Monte
  • Jose J. G. Marin
  • Maria J. PerezEmail author
Organ Toxicity and Mechanisms


The nucleoside reverse transcriptase inhibitor zidovudine (AZT), used in HIV infection treatment, induces mitochondrial DNA (mtDNA) depletion. A cause–effect relationship between mtDNA status alterations and autophagy has been reported. Both events are common in several liver diseases, including hepatocellular carcinoma. Here, we have studied autophagy activation in rat liver with mtDNA depletion induced by AZT administration in drinking water for 35 days. AZT at a concentration of 1 mg/ml, but not 0.5 mg/ml in the drinking water, decreased mtDNA levels in rat liver and extrahepatic tissues. In liver, mtDNA-encoded cytochrome c oxidase 1 protein levels were decreased. Although serum biomarkers of liver and kidney toxicity remained unaltered, β-hydroxybutyrate levels were increased in liver of AZT-treated rats. Moreover, autophagy was dysregulated at two levels: (i) decreased induction signalling of this process as indicated by increases in autophagy inhibitors activity (AKT/mTOR), and absence of changes (Beclin-1, Atg5, Atg7) or decreases (AMPK/ULK1) in the expression/activity of pro-autophagy proteins; and (ii) reduced autophagosome degradation as indicated by decreases in the lysosome abundance (LAMP2 marker) and the transcription factor TFEB controlling lysosome biogenesis. This resulted in increased autophagosome abundance (LC3-II marker) and accumulation of the protein selectively degraded by autophagy p62, and the transcription factor Nrf2 in liver of AZT-treated rats. Nrf2 was activated as indicated by the up-regulation of antioxidant target genes Nqo1 and Hmox-1. In conclusion, rat liver with AZT-induced mtDNA depletion presented dysregulations in autophagosome formation and degradation balance, which results in accumulation of these structures in parenchymal liver cells, favouring hepatocarcinogenesis.


Autophagy Hepatocarcinogenesis Mitochondria Retrograde regulation 



This study was supported by the Spanish “Instituto de Salud Carlos III” (Grant FIS PI15/00179 and PI16/00598) co-financed by European Regional Development Fund (ERDF), the Ministry of Science and Innovation, Spain (SAF2013-40620-R and SAF2016-75197-R), the “Junta de Castilla y León”, Spain (SA015U13 and SA063P17), the “Fundacion Mutua Madrileña”, Spain (Call 2015) and the “Fundación Memoria de D. Samuel Solórzano Barruso”, Spain (FS/8-2017). The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT) and CIBERehd. The authors thank Emma Keck for editing the English.

Compliance with ethical standards

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

204_2018_2200_MOESM1_ESM.tif (27.1 mb)
Supplementary material 1 (TIF 27726 KB)
204_2018_2200_MOESM2_ESM.tif (24.3 mb)
Supplementary material 2 (TIF 24855 KB)
204_2018_2200_MOESM3_ESM.tif (22.5 mb)
Supplementary material 3 (TIF 22991 KB)
204_2018_2200_MOESM4_ESM.tif (24.9 mb)
Supplementary material 4 (TIF 25480 KB)
204_2018_2200_MOESM5_ESM.tif (22.3 mb)
Supplementary material 5 (TIF 22806 KB)
204_2018_2200_MOESM6_ESM.tif (27.5 mb)
Supplementary material 6 (TIF 28109 KB)
204_2018_2200_MOESM7_ESM.docx (13 kb)
Supplementary material 7 (DOCX 13 KB)
204_2018_2200_MOESM8_ESM.docx (16 kb)
Supplementary material 8 (DOCX 15 KB)


  1. Adebiyi OO, Adebiyi OA, Owira PM (2015) Naringin reverses hepatocyte apoptosis and oxidative stress associated with HIV-1 nucleotide reverse transcriptase inhibitors-induced metabolic complications. Nutrients 7(12):10352–10368CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alers S, Löffler AS, Wesselborg S et al (2012) Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: crosstalk, shortcuts, and feedbacks. Mol Cell Biol 32(1):2–11CrossRefPubMedPubMedCentralGoogle Scholar
  3. Arnaudo E, Dalakas M, Shanske S et al (1991) Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 337(8740):508–510CrossRefPubMedGoogle Scholar
  4. Baixauli F, Acín-Pérez R, Villarroya-Beltrí C et al (2015) Mitochondrial respiration controls lysosomal function during inflammatory T cell responses. Cell Metab 22(3):485–498CrossRefPubMedPubMedCentralGoogle Scholar
  5. Barile M, Valenti D, Quagliariello E et al (1998) Mitochondria as cell targets of AZT (zidovudine). Gen Pharmacol 31(4):531–538CrossRefPubMedGoogle Scholar
  6. Blazquez AG, Briz O, Gonzalez-Sanchez E et al (2014) The effect of acetaminophen on the expression of BCRP in trophoblast cells impairs the placental barrier to bile acids during maternal cholestasis. Toxicol Appl Pharmacol 277(1):77–85CrossRefPubMedGoogle Scholar
  7. Brinkman K, Smeitink JA, Romijn JA et al (1999) Mitochondrial toxicity induced by nucleoside analogue reverse transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral therapy related lipodystrophy. Lancet 354(9184):1112–1115CrossRefPubMedGoogle Scholar
  8. Chariot P, Drogou I, de Lacroix-Szmania I et al (1999) Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion. J Hepatol 30(1):156–160CrossRefPubMedGoogle Scholar
  9. Corcuera T, Alonso MJ, Picazo A et al (1996) Hepatic Morphological alterations induced by zidovudine (ZDV) in an experimental model. Pathol Res Pract 192(2):182–187CrossRefPubMedGoogle Scholar
  10. Dash S, Chava S, Chandra PK et al (2016) Autophagy in hepatocellular carcinomas: from pathophysiology to therapeutic response. Hepat Med 8:9–20CrossRefPubMedPubMedCentralGoogle Scholar
  11. Eskelinen EL, Tanaka Y, Saftig P (2003) At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol 13(3):137–145CrossRefPubMedGoogle Scholar
  12. Fernandez-Mosquera L, Diogo CV, Yambire KF et al (2017) Acute and chronic mitochondrial respiratory chain deficiency differentially regulate lysosomal biogenesis. Sci Rep 7:45076CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gaou I, Malliti M, Guimont MC et al (2001) Effect of stavudine on mitochondrial genome and fatty acid oxidation in lean and obese mice. J Pharmacol Exp Ther 297(2):516–523PubMedGoogle Scholar
  14. Gonzalez-Sanchez E, Marin JJ, Perez MJ (2014) The expression of genes involved in hepatocellular carcinoma chemoresistance is affected by mitochondrial genome depletion. Mol Pharm 11(6):1856–1868CrossRefPubMedGoogle Scholar
  15. Hsu CC, Lee HC, Wei YH (2013) Mitochondrial DNA alterations and mitochondrial dysfunction in the progression of hepatocellular carcinoma. World J Gastroenterol 19(47):8880–8886CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hu Y, Suarez J, Fricovsky E et al (2009) Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose. J Biol Chem 284(1):547–555CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hurley JH, Schulman BA (2014) Atomistic autophagy: the structures of cellular self-digestion. Cell 157(2):300–311CrossRefPubMedPubMedCentralGoogle Scholar
  18. Inami Y, Waguri S, Sakamoto A et al (2011) Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J Cell Biol 193(2):275–284CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lewis W, Gonzalez B, Chomyn A et al (1992) Zidovudine induces molecular, biochemical, and ultrastructural changes in rat skeletal muscle mitochondria. J Clin Invest 89(4):1354–1360CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lewis W, Simpson JF, Meyer RR (1994) Cardiac mitochondrial DNA polymerase-gamma is inhibited competitively and noncompetitively by phosphorylated zidovudine. Circ Res 74(2):344–348CrossRefPubMedGoogle Scholar
  21. Li-Harms X, Milasta S, Lynch J et al (2015) Mito-protective autophagy is impaired in erythroid cells of aged mtDNA-mutator mice. Blood 125(1):162–174CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lindqvist LM, Heinlein M, Huang DC et al (2014) Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc Natl Acad Sci USA 111(23):8512–8517CrossRefPubMedPubMedCentralGoogle Scholar
  23. Liu L, Liao JZ, He XX et al (2017) The role of autophagy in hepatocellular carcinoma: friend or foe. Oncotarget 8(34):57707–57722PubMedPubMedCentralGoogle Scholar
  24. Macias RI, Hierro C, de Juan SC et al (2011) Hepatic expression of sodium-dependent vitamin C transporters: ontogeny, subtissular distribution and effect of chronic liver diseases. Br J Nutr 106(12):1814–1825CrossRefPubMedGoogle Scholar
  25. Marin JJ, Hernandez A, Revuelta IE et al (2013) Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: role of the Akt/mTOR survival pathway and Bcl-2 family proteins. Free Radic Biol Med 61:218–228CrossRefPubMedGoogle Scholar
  26. Marin JJG, Lozano E, Perez MJ (2016) Lack of mitochondrial DNA impairs chemical hypoxia-induced autophagy in liver tumour cells through ROS-AMPK-ULK1 signaling dysregulation independently of HIF-1α. Free Radic Biol Med 101:71–84CrossRefPubMedGoogle Scholar
  27. Marquez RT, Xu L (2012) Bcl-2: beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. Am J Cancer Res 2(2):214–221PubMedPubMedCentralGoogle Scholar
  28. Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3(6):542–545CrossRefPubMedGoogle Scholar
  29. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326CrossRefPubMedPubMedCentralGoogle Scholar
  30. Mutter FE, Park BK, Copple IM (2015) Value of monitoring Nrf2 activity for the detection of chemical and oxidative stress. Biochem Soc Trans 43(4):657–662CrossRefPubMedPubMedCentralGoogle Scholar
  31. Pelicano H, Xu RH, Du M et al (2006) Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. J Cell Biol 175(6):913–923CrossRefPubMedPubMedCentralGoogle Scholar
  32. Perez MJ, Macías RI, Duran C et al (2005) Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid. J Hepatol 43(2):324–332CrossRefPubMedGoogle Scholar
  33. Powles T, Robinson D, Stebbing J et al (2009) Highly active antiretroviral therapy and the incidence of non-AIDS-defining cancers in people with HIV infection. J Clin Oncol 27(6):884–890CrossRefPubMedGoogle Scholar
  34. Raghu R, Karthikeyan S (2016) Zidovudine and isoniazid induced liver toxicity and oxidative stress: evaluation of mitigating properties of silibinin. Environ Toxicol Pharmacol 46:217–226CrossRefPubMedGoogle Scholar
  35. Ramanathan R, Sivanesan K (2017) Evaluation of ameliorative ability of Silibinin against zidovudine and isoniazid-induced hepatotoxicity and hyperlipidaemia in rats: role of silibinin in phase I and II drug metabolism. Chem Biol Interact 273:142–153CrossRefPubMedGoogle Scholar
  36. Rambold AS, Lippincott-Schwartz J (2011) Mechanisms of mitochondria and autophagy crosstalk. Cell Cycle 10(23):4032–4038CrossRefPubMedPubMedCentralGoogle Scholar
  37. Ruderman NB, Xu XJ, Nelson L et al (2010) AMPK and SIRT1: a long-standing partnership? Am J Physiol Endocrinol Metab 298(4):E751-E760CrossRefPubMedCentralGoogle Scholar
  38. Rusten TE, Stenmark H (2010) p62, an autophagy hero or culprit? Nat Cell Biol 12(3):207–209CrossRefPubMedGoogle Scholar
  39. Scruggs ER, Dirks AJ (2008) Mechanisms of zidovudine-induced mitochondrial toxicity and mytophagy. Pharmacology 82(2):83–88CrossRefPubMedGoogle Scholar
  40. Sridharan S, Jain K, Basu A (2011) Regulation of autophagy by kinases. Cancers 3(2):2630–2654CrossRefPubMedPubMedCentralGoogle Scholar
  41. Stankov MV, Panayotova-Dimitrova D, Leverkus M et al (2012) Autophagy inhibition due to thymidine analogues as novel mechanism leading to hepatocyte dysfunction and lipid accumulation. AIDS 26(16):1995–2006CrossRefPubMedGoogle Scholar
  42. Takamura A, Komatsu M, Hara T et al (2011) Autophagy-deficient mice develop multiple liver tumours. Genes Dev 25(8):795–800CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tanaka J, Ozawa K, Tobe T (1979) Significance of blood ketone body ratio as an indicator of hepatic cellular energy status in jaundiced rabbits. Gastroenterology 76(4):691–696PubMedGoogle Scholar
  44. Tanaka Y, Guhde G, Suter A et al (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 406(6798):902–906CrossRefPubMedGoogle Scholar
  45. Tian Y, Kuo CF, Sir D et al (2015) Autophagy inhibits oxidative stress and tumour suppressors to exert its dual effect on hepatocarcinogenesis. Cell Death Differ 22(6):1025–1034CrossRefPubMedGoogle Scholar
  46. Umemura A, He F, Taniguchi K et al (2016) p62, upregulated during preneoplasia, induces hepatocellular carcinogenesis by maintaining survival of stressed HCC-initiating cells. Cancer Cell 29(6):935–948CrossRefPubMedPubMedCentralGoogle Scholar
  47. Venhoff N, Lebrecht D, Pfeifer D et al (2012) Muscle-fiber transdifferentiation in an experimental model of respiratory chain myopathy. Arthritis Res Ther 14(5):R233CrossRefPubMedPubMedCentralGoogle Scholar
  48. Voss M, Künzel U, Higel F et al (2014) Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation. EMBO J 33(24):2890–2905CrossRefPubMedPubMedCentralGoogle Scholar
  49. Williamson DH, Lund P, Krebs HA (1967) The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103(2):514–527CrossRefPubMedPubMedCentralGoogle Scholar
  50. Zhang Y, Song F, Gao Z et al (2014) Long-term exposure of mice to nucleoside analogues disrupts mitochondrial DNA maintenance in cortical neurons. PLoS One 9(1):e85637CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Experimental Hepatology and Drug Targeting, Institute of Biomedical Research of Salamanca (IBSAL), CIBERehdUniversity of SalamancaSalamancaSpain
  2. 2.Research UnitUniversity Hospital of SalamancaSalamancaSpain

Personalised recommendations