Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2018 Volume: 12 Drug Design, Development and Therapy Dovepress Running head verso: Abdulkhaleq et al Running head recto: Antioxidative stress effects of vitamins C, E, and B and their combination open access to scientific and medical research 12 DOI: 172487 Open Access Full Text Article ORIGINAL RESEARCH Antioxidative stress effects of vitamins C, E, and B12, and their combination can protect the liver against acetaminophen-induced hepatotoxicity in rats 1 Farah M Abdulkhaleq Background: Several vitamins, including C, E, and B12, have been recognized as antioxidants Tawfiq M Alhussainy1 and have shown hepatoprotective effects against the hepatotoxicity caused by acetaminophen Mujtaba M Badr2 (APAP) overdose. The current investigation aims to study the effect of these vitamins and their Asad A Abu Khalil2 combination in protecting the liver from APAP hepatotoxicity in rats. Omar Gammoh3 Materials and methods: An in vitro model of freshly isolated rat hepatocytes was utilized Bayan Y Ghanim2 for assessing hepatocyte mitochondrial activity conducted by cell proliferation assay (MTT). The isolated hepatocytes were treated with vitamin C, vitamin E, vitamin B and their combina- Nidal A Qinna1,2 12 tion, with and without further addition of toxic concentrations of APAP. In addition, an in vivo 1 Department of Pharmacology and experiment was carried out on Sprague Dawley rats treated intraperitoneally for 8 days with Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, emulsions of the vitamins or their combination prior to injecting them with APAP. University of Petra, Amman, Jordan; Results: In vitro results showed that vitamins C and B and the combination preparation sig- 2 University of Petra Pharmaceutical nificantly increased the percentage of hepatocyte mitochondrial activity, both with and without Center (UPPC), University of Petra, Amman, Jordan; 3Department of the addition of APAP (P,0.01). The mitochondrial activity in the isolated cultured hepatocytes Pharmacy, Faculty of Health Sciences, was further enhanced with APAP addition. In vivo, the vitamins and their combination effec- American University of Madaba, tively reduced APAP-induced serum liver enzymes levels, namely ALT, AST, and ALP, and Madaba, Jordan also attenuated oxidative stress and lipids peroxidation confirmed by the results of glutathione, superoxide dismutase, and maloondialdehyde. Conclusion: Pretreatment with vitamins C, E, B12, or their combination was found to be ben- eficial in preventing in vivo hepatic oxidative stress induced by APAP overdose. Vitamin C on its own showed superior protection against APAP-induced liver injury in rats compared to the other vitamins. The proliferation of APAP-intoxicated liver cells in vitro was highest when protected with the vitamins’ combination. Keywords: glutathione, hepatocytes, proliferation, hepatoprotective, superoxide dismutase, primary cell culture Introduction Drug-induced hepatotoxicity is encountered in clinical practice and rises within easily accessible compounds like over-the-counter drugs. Acetaminophen, N-acetyl-para- Correspondence: Nidal A Qinna aminophenol (APAP) or paracetamol, was originally introduced to the market as an Department of Pharmacology and analgesic drug by Von Mering in 1893,1 mostly used as an analgesic and antipyretic Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, for relieving mild and moderate pain and fever.2,3 APAP-induced liver injury is trans- PO Box 961343, 11196 Amman, Jordan lated to acute liver failure, with admission of more than 30,000 patients every year.4 Tel +962 7 9556 5027 Email [email protected] The spectrum of injury caused by APAP triggers the liver all the way down to its submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 3525–3533 3525 Dovepress © 2018 Abdulkhaleq et al. This work is published and licensed by Dove Medical Press Limited. 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Abdulkhaleq et al Dovepress hepatocytes and nuclear molecules.5 Interestingly, infants and injury is mainly attributed to acute induction of oxidative young children are less susceptible to acute APAP toxicity stress in hepatocytes, the aim of the present study was to 6 due to age-associated differences in drug metabolism. evaluate the hepatoprotective effects of VC, VE, and VB12 At therapeutic levels, most of the administered dose is on APAP-induced hepatotoxicity. Moreover, we also wanted normally metabolized by glucuronidation and sulfonation to investigate whether a preparation comprising a combina- (Phase II) to produce inactive nontoxic metabolites that are tion of all three vitamins could augment the expected hepatic easily excreted by kidney.7 On the other hand, a small por- protection. tion is metabolized by oxidation (Phase I) through CYP2E1, a member of the CYP family,8 to a highly toxic metabolite, Materials and methods namely, N-acetyl-p-benzoquinoneimine (NAPQI), a reactive Materials and chemicals metabolite that depletes glutathione (GSH) and covalently All the materials used for the experiment were of analytical binds to mitochondrial proteins. NAPQI is efficiently detoxi- grade. VC, VE, and VB12, EDTA, MgCl2, FBS, streptomy- fied by conjugation with GSH. However, at toxic doses, GSH cin, penicillin, RPMI-1640 medium, olive oil, and GSH is depleted by the conjugation reaction, and NAPQI cova- ELISA assay kit were all purchased from Sigma Aldrich lently binds to proteins to produce reactive oxygen species (St Louis, MO, USA). Hank’s balanced salt solution (ROS). ROS induce oxidative stress leading to lipid peroxida- (HBSS) was obtained from Invitrogen (Carlsbad, CA, USA). tion, mitochondrial dysfunction, disruption of calcium, and Collagenase II and L-glutamine were purchased from Gibco nitric oxide homeostasis, and finally, cell death by apoptosis BRL (Gaithersburg, MD, USA). Tween 80 and DMSO were and necrosis.9 obtained from Scharlan Chemie S.A. (Barcelona, Spain), Antioxidants may exert their effects on biological sys- whereas Tris base and TACS® MTT kit were purchased tems by several mechanisms including electron donation, from Promega (Madison, WI, USA) and Trevigen (Gaith- chelation of metal ions, regulation of gene expression, or by ersburg, MD, USA), respectively. Acetaminophen and iso- acting as co-antioxidants.10,11 Among biologically important florane were kind gifts from the Jordanian Pharmaceutical antioxidants, vitamins C, E, and B12 function mainly by two Manufacturing Company JPM (Amman, Jordan) and Hikma principle mechanisms of action; initially by donating elec- Pharmaceuticals (Amman, Jordan), respectively. ELISA trons to free radicals: the chain-breaking mechanism. The kits for superoxide dismutase (SOD) and malondialdehyde second mechanism involves removal of initiators of reactive (MDA) were purchased from MyBioSource, (San Diego, oxygen or nitrogen species, acting as secondary antioxidants CA, USA). by quenching a chain-initiating catalyst.12–14 Vitamin C (VC), also known as ascorbic acid, is a potent Experimental animals naturally occurring water-soluble antioxidant.15 Vitamin E Adult male Sprague Dawley rats with average weights of (VE), a potent lipid-soluble antioxidant,16 is found in the 240±20 g were housed at the Animal House Unit of the plasma and tissues as an isomer, namely, α-tocopherol, University of Petra (Amman, Jordan). Rats were kept in the most important antioxidant in cell membranes.17 It air-conditioned environment under controlled tempera- also regulates cell proliferation, platelet aggregation, and tures, humidity, and photoperiod cycles and acclimatized NADPH-oxidase activation, as well as the expression of for 10 days before experimenting. Rats were fasted over- genes responsible for its own metabolism, cell adhesion, night (for 18–22 hours) with free access to water, unless 18 inflammation, and fibrosis. Vitamin B12 (VB12) (cyano otherwise stated. All experiments were approved by the cobalamin) is a water-soluble vitamin essential for maintain- Research Council at the Faculty of Pharmacy and Medi- ing the normal functions of the peripheral nervous system19 cal Sciences (approval number: 15/2/2016) and conducted and the brain, as it plays a considerably critical role in the in accordance with the University of Petra Institutional myelination of the white matter and nerves.20 The potential Guidelines on Animal Use, which adopts the guidelines antioxidative properties and the ability of cobalamins in of the Federation of European Laboratory Animal Science regulating inflammatory cytokines and have been previously Association. reported.13 Furthermore, the synthetic cyanocobalamin is one of cobalamin forms that is available commercially as a Preparation of buffers dietary supplement.21 Liver perfusion buffers were freshly prepared under sterile Since APAP-induced liver injury represents the most