Valproic Acid-Induced Hepatotoxicity and the Protective Role of Thiol Reductants

Document Type : Research(Original) Article


Shiraz University of Medical Sciences, Pharmaceutical Sciences Research Center


Valproic acid (VPA) is a widely administered drug against epilepsy and several other neurological disorders. On the other hand, liver injury is a deleterious side effect associated with VPA. Oxidative stress seems to play a critical role in VPA-induced hepatotoxicity. The current investigation was designed to evaluate if N-acetylcysteine (NAC) and dithiothreitol (DTT) as thiol reducing agents have any protective effects against VPA-induced liver injury. Isolated rat hepatocytes (in vitro) were exposed to increasing concentrations of VPA (25, 50, 100, 150, and 250 µM) and markers of cytotoxicity were evaluated. Furthermore, animals received VPA (250 and 500 mg/kg, i.p for 15 consecutive days) (in vivo) and markers of liver injury were monitored. It was found that 250 µM of VPA caused marked cytotoxicity toward isolated hepatocytes as judged by trypan blue exclusion test. Moreover, markers of oxidative stress including glutathione depletion and lipid peroxidation were detected in VPA-treated hepatocytes. On the other hand, VPA caused a significant increase in plasma markers of hepatotoxicity in drug-treated group. Liver histopathological changes and markers of oxidative stress were also detected in VPA-treated animals. It was found that administration of NAC (1 mM), and DTT (1 mM) significantly alleviated VPA-induced cytotoxicity (In vitro). NAC (250 and 500 mg/kg) and DTT (15 and 30 mg/kg) also significantly mitigated VPA hepatotoxicity (In vivo). The data obtained from the current investigation indicate potential therapeutic properties of thiol reductants against VPA-induced liver injury.


  1. Davies JA, Valproic Acid, in xPharm: The Comprehensive Pharmacology Reference2007, Elsevier: New York. p. 1-5.
  2. Bryant AE, Dreifuss FE. Valproic acid hepatic fatalities. III. U.S. experience since 1986. Neurology. 1996;46;465-469.
  3. Chateauvieux S, Morceau F, Diederich M, Valproic Acid, in Encyclopedia of Toxicology (Third Edition), P. Wexler, Editor 2014, Academic Press: Oxford. p. 905-908.
  4. Nanau RM, Neuman MG. Adverse drug reactions induced by valproic acid. Clin Biochem. 2013;46;1323-1338.
  5. Heidari R, Jafari F, Khodaei F, Shirazi Yeganeh B, Niknahad H. The Mechanism of Valproic Acid-Induced Fanconi Syndrome Involves Mitochondrial Dysfunction and Oxidative Stress in Rat Kidney. Nephrology (Carlton, Vic). 2017;In-Press.
  6. Li S, Guo J, Ying Z, Chen S, Yang L, Chen K, Long Q, Qin D, Pei D, Liu X. Valproic acid‐induced hepatotoxicity in alpers syndrome is associated with mitochondrial permeability transition pore opening‐dependent apoptotic sensitivity in an induced pluripotent stem cell model. Hepatology. 2015;61;1730-1739.
  7. Vitins AP, Kienhuis AS, Speksnijder EN, Roodbergen M, Luijten M, Ven LTMvd. Mechanisms of amiodarone and valproic acid induced liver steatosis in mouse in vivo act as a template for other hepatotoxicity models. Arch Toxicol. 2014;88;1573-1588.
  8. Gezginci-Oktayoglu S, Turkyilmaz IB, Ercin M, Yanardag R, Bolkent S. Vitamin U has a protective effect on valproic acid-induced renal damage due to its anti-oxidant, anti-inflammatory, and anti-fibrotic properties. Protoplasma. 2015.
  9. Ahangar N, Naderi M, Noroozi A, Ghasemi M, Zamani E, Shaki F. Zinc Deficiency and Oxidative Stress Involved in Valproic Acid Induced Hepatotoxicity: Protection by Zinc and Selenium Supplementation. Biol Trace Element Res. 2017;1-8.
  10. Chang TKH, Abbott FS. Oxidative stress as a mechanism of valproic acid-associated hepatotoxicity. Drug Metab Rev. 2006;38;627-639.
  11. Michoulas A, Tong V, Teng XW, Chang TKH, Abbott FS, Farrell K. Oxidative stress in children receiving valproic acid. J Pediatric. 2006;149;692-696.
  12. Fisher R, Nau H, Gandolfi AJ, Putnam CW, Brendel K. Valproic Acid Hepatotoxicity in Human Liver Slices. Drug Chem Toxicol. 1991;14;375-394.
  13. Heidari R, Babaei H, Eghbal MA. Amodiaquine-induced toxicity in isolated rat hepatocytes and the cytoprotective effects of taurine and/or N-acetyl cysteine. Res Pharm Sci. 2014;9;97-105.
  14. Khan S, O'Brien PJ. 1-bromoalkanes as new potent nontoxic glutathione depletors in isolated rat hepatocytes. Biochem Biophy Res Commun. 1991;179;436-41.
  15. Heidari R, Babaei H, Eghbal MA. Cytoprotective Effects of Taurine Against Toxicity Induced by Isoniazid and Hydrazine in Isolated Rat Hepatocytes. Arch Indust Hyg Toxicol. 2013;64;201-210.
  16. Jamshidzadeh A, Niknahad H, Kashafi H. Cytotoxicity of chloroquine in isolated rat hepatocytes. J Appl Toxicol. 2007;27;322-6.
  17. Abdoli N, Heidari R, Azarmi Y, Eghbal MA. Mechanisms of the Statins Cytotoxicity in Freshly Isolated Rat Hepatocytes. J Biochem Mol Toxicol. 2013;n/a-n/a.
  18. Heidari R, Babaei H, Eghbal M. Mechanisms of methimazole cytotoxicity in isolated rat hepatocytes. Drug Chem Toxicol. 2013;36;403-411.
  19. Heidari R, Babaei H, Eghbal MA. Ameliorative effects of taurine against methimazole-induced cytotoxicity in isolated rat hepatocytes. Scientia pharmaceutica. 2012;80;987.
  20. Sokmen BB, Tunali S, Yanardag R. Effects of vitamin U (S-methyl methionine sulphonium chloride) on valproic acid induced liver injury in rats. Food Chem Toxicol. 2012;50;3562-3566.
  21. Gezginci-Oktayoglu S, Turkyilmaz IB, Ercin M, Yanardag R, Bolkent S. Vitamin U has a protective effect on valproic acid-induced renal damage due to its anti-oxidant, anti-inflammatory, and anti-fibrotic properties. Protoplasma. 2015;253;127-135.
  22. Jamshidzadeh A, Heidari R, Mohammadi-Samani S, Azarpira N, Najbi A, Jahani P, Abdoli N. A Comparison between the Nephrotoxic Profile of Gentamicin and Gentamicin Nanoparticles in Mice. J Biochem Mol Toxicol. 2015;29;57-62.
  23. Heidari R, Taheri V, Rahimi HR, Yeganeh BS, Niknahad H, Najibi A. Sulfasalazine-induced renal injury in rats and the protective role of thiol-reductants. Renal Failure. 2016;38;137-141.
  24. Jamshidzadeh A, Heidari R, Golzar T, Derakhshanfar A. Effect of Eisenia foetida Extract against Cisplatin-Induced Kidney Injury in Rats. J Diet Suppl. 2016;13;551-559.
  25. Jamshidzadeh A, Heidari R, Abazari F, Ramezani M, Khodaei F, Ommati MM, Ayarzadeh M, Firuzi R, Saeedi A, Azarpira N, others. Antimalarial Drugs-Induced Hepatic Injury in Rats and the Protective Role of Carnosine. Pharm Sci. 2016;22.
  26. Niknahad H, Heidari R, Firuzi R, Abazari F, Ramezani M, Azarpira N, Hosseinzadeh M, Najibi A, Saeedi A. Concurrent Inflammation Augments Antimalarial Drugs-Induced Liver Injury in Rats. Adv Pharm Bull. 2016;6.
  27. Moezi L, Heidari R, Amirghofran Z, Nekooeian AA, Monabati A, Dehpour AR. Enhanced anti-ulcer effect of pioglitazone on gastric ulcers in cirrhotic rats: The role of nitric oxide and IL-1b. Pharmacol Report. 2013;65;134-143.
  28. Heidari R, Jamshidzadeh A, Niknahad H, Mardani E, Ommati MM, Azarpira N, Khodaei F, Zarei A, Ayarzadeh M, Mousavi S, Abdoli N, Yeganeh BS, Saeedi A, Najibi A. Effect of taurine on chronic and acute liver injury: Focus on blood and brain ammonia. Toxicol Report. 2016;3;870-879.
  29. Heidari R, Niknahad H, Jamshidzadeh A, Azarpira N, Bazyari M, Najibi A. Carbonyl Traps as Potential Protective Agents against Methimazole-Induced Liver Injury. J Biochem Mol Toxicol. 2014;29;173-181.
  30. Niknahad H, Heidari R, Mokhtebaz T, Mansouri S, Dehshahri S, Abdoli N, Najibi A. Evaluating the effects of different fractions obtained from Gundelia tournefortii extract against carbon tetrachloride-induced liver injury in rats. Trend Pharm Sci. 2016;2;25-34.
  31. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analyt Bioch. 1968;25;192-205.
  32. Heidari R, Jamshidzadeh A, Niknahad H, Safari F, Azizi H, Abdoli N, Ommati MM, Khodaei F, Saeedi A, Najibi A. The Hepatoprotection Provided by Taurine and Glycine against Antineoplastic Drugs Induced Liver Injury in an Ex Vivo Model of Normothermic Recirculating Isolated Perfused Rat Liver. Trend Pharm Sci. 2016;2;59-76.
  33. Heidari R, Babaei H, Roshangar L, Eghbal MA. Effects of Enzyme Induction and/or Glutathione Depletion on Methimazole-Induced Hepatotoxicity in Mice and the Protective Role of N-Acetylcysteine. Adv Pharm Bull. 2014;4;21-28.
  34. Tong V, Teng XW, Chang TKH, Abbott FS. Valproic acid II: effects on oxidative stress, mitochondrial membrane potential, and cytotoxicity in glutathione-depleted rat hepatocytes. Toxicol Sci. 2005;86;436-443.
  35. Labbe G, Pessayre D, Fromenty B. Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies. Fundament Clin Pharmacol. 2008;22;335-353.
  36. Heidari R, Niknahad H, Jamshidzadeh A, Eghbal MA, Abdoli N. An Overview on the Proposed Mechanisms of Antithyroid Drugs-Induced Liver Injury. Adv Pharm Bull. 2015;5;1-11.
  37. Silva MFB, Aires CCP, Luis PBM, Ruiter JPN, Ijlst L, Duran M, Wanders RJA, Almeida ITd. Valproic acid metabolism and its effects on mitochondrial fatty acid oxidation: A review. J Inherit Metab Dis. 2008;31;205-216.
  38. Banaclocha MMn, Hernandez AI, Martı́nez N, Ferrandiz MaL. N-Acetylcysteine protects against age-related increase in oxidized proteins in mouse synaptic mitochondria. Brain Res. 1997;762;256-258.
  39. Kamboj SS, Sandhir R. Protective effect of N-acetylcysteine supplementation on mitochondrial oxidative stress and mitochondrial enzymes in cerebral cortex of streptozotocin-treated diabetic rats. Mitochondrion. 2011;11;214-222.