39J Contemp Med Sci | Vol. 4, No. 1, Winter 2018: 39–44 Research Antioxidants as recipes for efavirenz-induced liver damage: A study in albino rats Elias Adikwu,a Bonsome Bokolob aDepartment of Pharmacology, Faculty of Basic Medical Sciences, University of Port Harcourt, Rivers State, Nigeria bDepartment of Pharmacology, Faculty of basic medical Sciences, Niger Delta University Wilberforce Island, Bayelsa State, Nigeria Correspondence to Elias Adikwu (email: adikwuelias@gmail.com). (Submitted: 02 September 2017 – Revised version received: 24 September 2017 – Accepted: 11 October 2017 – Published online: 26 March 2018) Introduction The liver plays vital role in the biotransformation of drugs and toxins.1 In view of the function of the liver, in the biotransfor- mation of drugs and toxins, liver cells which include Kupffer, hepatic stellate, endothelial and parenchymal cells are major target of oxidative radicals produced by drugs and toxins.2,3 Oxidative stress has been shown to be an essential originating factor in the pathogenesis of drug-induced hepatic damage.4,5 The over-production of oxidative radicals is toxic to hepato- cytes and initiates reactive oxygen species (ROS)-mediated cascade causing hepatocyte death, and leading to acute or chronic hepatic damage.6,7 The use of Efavirenz (EFV) a member of the non- nucleoside reverse transcriptase inhibitors (NNRTIs) has dramatically reduced human immunodeficiency virus (HIV)-related morbidity and mortality in the world.8 However, studies have associated the use of EFV with hepatic damage which is a fre- quent class problem of the non-NNRTIs.9,10 The molecular pathogenesis of this effect is poorly understood, but recent reports have highlighted features of mitochondrial dysfunction in hepatic cells exposed to clinically relevant concentrations of EFV.11 Also, recent studies have demonstrated endoplasmic reticulum stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cell lines.12 N-acetylcysteine (NAC) is the acetylated derivative of the amino acid L-cysteine. It is frequently employed as a source of sulfhydryl groups to cells as an acetylated precursor of reduced GSH. It is an antioxidant that interacts directly with reactive oxygen and nitrogen species and up-regulate endogenous GSH status, thereby preventing oxidative stress-induced alter- ations in biomolecules.13 Furthermore, in experimental animal model and clinical studies, NAC showed ameliorative effect on drug-induced acute liver injury and has been clinically adopted for the treatment of acetaminophen associated hepa- totoxicity.14,15 Moreover, animal and human studies of NAC suggest that it is a very safe as an effective tool for the treat- ment of many diseases considered to be mediated by oxidative radical and it has been used therapeutically in several disor- ders related to oxidative stress.16 Vitamin E is the major lipid-soluble and potent chain- breaking antioxidant that inhibits the production of oxidative radicals when fat undergoes oxidation and during the propa- gation of free radical reactions.17 It is primarily located in cell and organelle membranes where it can exert its protective effect. It acts as the first line of defence against lipid peroxida- tion, by protecting polyunsaturated fatty acids present in membrane phospholipids and in plasma lipoproteins from free radical attack.18,19 It is the only major lipid-soluble, chain breaking antioxidant found in plasma, red cells and tissues, allowing it to protect the integrity of lipid structures, mainly membranes.20 In addition, previous studies have demonstrated the potential of vitamin E in the amelioration of drug-induced hepatotoxicity in animal model . 21,22 Vitamin C is a six-carbon compound structurally related to glucose. It consists of two inter-convertible compounds: L-ascorbic acid, which is a strong reducing agent, and its oxi- dized derivative, L-dehydroascorbic acid.23 It is a water-soluble antioxidant with diverse biological functions. Its actions include acting as a cofactor for the enzymes involved in collagen hydrox- ylation, biosynthesis of carnitine and norepinephrine, tyrosine metabolism and peptide hormone amidation.24 Its antioxidant property includes inhibition of lipid peroxidation, oxidative cell damage and stimulatory effects on other antioxidants especially ISSN 2413-0516 Objective Hepatotoxicity is a clinical challenge associated with the use of efavirenz (EFV). This study investigated the effects of n-acetylcysteine (NAC), vitamins C and E on EFV-induced hepatotoxicity in albino rats. Methods Rats were divided into groups and administered with NAC (20 mg/kg), vitamin C (50 mg/kg), vitamin E (50 mg/kg), vitamins C + E and 60 mg/kg of EFV, respectively. Rats were also divided into groups and pretreated with NAC, vitamins C, E, and combined doses of vitamins C + E prior to treatment with EFV for 15 days, respectively. After drug administration rats were sacrificed and serum was col- lected and evaluated for liver function parameters. Rats were dissected, liver was collected weighed and, homogenized and evaluated for alkaline phosphatase (ALP), alanine aminotransferase (AST), aspartate aminotransferase (ALT), γ-glutamyl transferase (GGT), lactate dehy- drogenase (LDH), malondialdehyde (MDA), super oxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPX) levels and pathological damage. Results Effects were not significant (p > 0.05) on body and liver weights, however, the levels of AST, ALT, AST, GGT, LDH, CB, TB and MDA were increased significantly (P < 0.05) whereas SOD, CAT, SOD, GSH and GPX were decreased significantly (P < 0.05) in EFV-treated rats in comparison to control. The liver of EFV-treated rats showed necrosis of hepatocytes hepatocytes necroses. Nevertheless, EFV-induced alter- ations in the above parameters were significantly (P < 0.05) ameliorated in antioxidants pretreated rats. The combined doses of vitamins C and E producedbest and significant (P < 0.05) in comparison to their individual doses. Conclusion This study shows the showed prospects of antioxidants as candidates for the treatments of efavirenz-induced hepatotoxicity. Keywords antioxidants, efavirenz, liver, toxicity, mitigation, rat 40 J Contemp Med Sci | Vol. 4, No. 1, Winter 2018: 39–44 Antioxidants ameliorate efavirenz-induced liver damage Research vitamin E.25,26 Furthermore, vitamin C has shown ameliorative effect on drug-induced hepatotoxity as shown in in-vivo animal studies.27,28 The present study was therefore designed to evaluate the effects of NAC, vitamins C and E on efavirenz-induced hepatotoxicity in albino rats. Materials and Methods Drugs and Chemicals The vitamin E used for this study was manufactured by strides pharmaceuticals India while vitamin C was manufac- tured by Emzor pharmaceuticals industries Lagos, Nigeria. Efavirenz was manufactured by Miland laboratory, India. The present study used 20 mg/kg/day of NAC, 50 mg/kg/day of vitamin C and 50 mg/kg/day of vitamin E29,30 and 60 mg/kg/ day of EFV. NAC was dissolved normal saline, vitamin C was dissolved in water while arachis oil was used as the vehicle for vitamin E and EFV. Grouping of Rats and Drug Administration Fifty five albino rats were randomized into six groups A-E. Groups A, B, D contained five rats each while group C and E contained twenty rats each which were further divided into four sub-groups of five rats each. The rats in group A served as placebo control whereas group B served as the solvent control and were administered with normal saline and arachis oil respectively. The rats in group C were orally administered with NAC (20 mg/kg/day), vitamin C (50 mg/kg/day), vitamin E (50 mg/kg/day) and a combination of vitamins C+ E respec- tively. The rats in group D were orally administered with 60 mg/kg/day of EFV. The rats in groups E were orally pretreated with NAC, vitamin C, vitamin E, and combined doses of vitamins C+E prior to the administration of EFV for 15 days respectively. Collection of Samples and Biochemical Analysis After the termination of drug administration on day 15, the rats were fasted overnight and sacrificed under diethyl ether. The blood was collected via cardiac puncture; serum was extracted and evaluated for alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehy- drogenase, gamma glutamyl transferase conjugated and total bilirubin using Randox Diagnostic kits (Randox Laboratories Ltd., Crumlin, U.K.). Liver was excised, weighed and washed in cold 1.15% KCL solution, then homogenized and centri- fuged. The supernatant was decanted and used for the evaluation of oxidative stress indices. Glutathione peroxidase (GPX) activity was evaluated as reported by Rotruck et al., 197331 SOD activity was determined by the method of Sun and Zigma (1978)32 while CAT activity was measured according to Sinha et al., 1972.33 Reduced Glutathione was analyzed according as described by Sedlak and Lindsay (1968)34 while malondialdehyde (MDA) was measured using the method of Buege and Aust (1978).35 Histological Examination of the Liver The histological examination of the liver was performed in Anatomical Pathology Department of University of Port Harcourt Teaching Hospital, Choba, Rivers State, Nigeria. The liver tissue was excised and rinsed in normal saline. Liver sec- tions were taken and processed and embedded in paraffin. Liver tissues were then processed into sections 4-5 μm thick, stained with Hematoxylin and Eosin and observed under a light microscope for any morphological changes. Statistical Analysis Results are expressed as mean ± SD. Differences among the experimental groups were identified by one-way analysis of variance and Tukey’s multiple comparison test and a p < 0.05 was considered significant. Results Effects on Body, Liver Weights and Liver Function Parameters The administration of NAC, vitamins C and E for 15 days did not produce significant (p > 0.05) effects on serum levels of AST, ALT, ALP, GGT, LDH, TB and CB when compared to control (Table 2 and 3).On the other hand, the above param- eters were significantly (p < 0.05) increased in rats adminis- tered with 60mg/kg/day of EFV for 15 days with no significant (p > 0.05) effects on the body and liver weights in comparison to control (Table 1-3). However, rats pretreated with indi- vidual doses of NAC, vitamins C and E showed significant (p < 0.05) reductions in the serum levels of AST, ALT, ALP, GGT, LDH, CB and TB when compared to EFV-treated rats (Table 2 and 3). Furthermore, a combination of vitamins C and E produced most and significant (p < 0.05) reductions in the serum levels of AST, ALT, ALP, GGT, LDH, CB and TB when compared to their individual doses, but effects did not differ significantly (p < 0.05) from pretreatment with NAC (Table 1and 2). Furthermore, treatment with NAC, Vit C and E did not produce significant (p < 0.05) effects on liver levels of AST, ALT, ALP, GGT, and LDH in comparison to control. On the contrary, these parameters were significantly (p < 0.05) increased in EFV-treated rats in comparison to control (Table 1 and 2). However, the liver levels of AST, ALT, ALP, GGT, and LDH were decreased significantly (p < 0.05) in rats pretreated with NAC, vitamins C and E prior to treat- ment with EFV. The observed decreases in these parameters were most and significant (p < 0.05) in rats pretreated with combined doses of vitamins C and E when compared to their individual doses. However, decreases observed in these parameters in rats pretreated with combined doses of vita- mins C and E did not differ (p > 0.05) when compared to NAC pretreated rats (Table 2 and 3). Effects on Oxidative Stress Indices and Liver Histology The administration of NAC, vitamins C and E did not produce significant (p > 0.05) effects on the liver levels of SOD, CAT, GSH, GPX and MDA when compared to control. In contrast, liver levels of SOD, CAT, GSH and GPX were decreased significantly (p < 0.05) whereas MDA levels were increased significantly (p < 0.05) in rats treated with EFV when com- pared to control (Table 4). However, rats pretreated with NAC, vitamin C, and E prior to treatment with EFV showed significant (p < 0.05) increases in SOD, CAT, GSH and GPX levels whereas MDA levels were decreased significantly (p < 0.05) when compared to EFV-treated rats. Interestingly, pretreatment with a combination of vitamins C and E pro- duced the most and significant (p < 0.05) effects on SOD, CAT, Elias Adikwu and Bonsome Bokolo 41J Contemp Med Sci | Vol. 4, No. 1, Winter 2018: 39–44 Research Antioxidants ameliorate efavirenz-induced liver damage Table 3. Effects of n-acetylcysteine, vitamins C and E on liver levels of biomarkers of liver function in efavirenz-treated albino rats Treatment ALT (U/L) AST (U/L) ALP (U/L) GGT (U/L) LDH (U/L) Control 70.6 ± 8.07a 65.6 ± 6.33a 63.0 ± 4.12a 80.0 ± 7.15a 1.81 ± 4.12a NAC 64.3 ± 5.25a 60.2 ± 4.00a 59.4 ± 3.01a 72.9 ± 6.72a 1.72 ± 3.01a Vitamin C 69.4 ± 6.21a 63.5 ± 4.20a 61.8 ± 5.15a 79.8 ± 4.33a 1.79 ± 5.15a Vitamin E 67.9 ± 7.18a 62.3 ± 6.06a 60.4 ± 4.01a 77.4 ± 5.42a 1.77 ± 4.01a Vitamins C + E 65.3 ± 4.20a 60.0 ± 4.00a 58.6 ± 3.01a 72.6 ± 6.11a 1.73 ± 3.01a EFV 190.8 ± 9.13b 194.7 ± 9.34b 186.0 ± 7.55b 200.9 ± 9.95b 4.89 ± 7.55b EFV + NAC 80.5 ± 6.08c 82.7 ± 5.00c 80.7 ± 5.00c 85.7 ± 6.72c 1.75 ± 5.00a EFV + Vitamin C 130.6 ± 7.14d 130.2 ± 8.21d 127.6 ± 9.13d 130.6 ± 9.68d 2.99 ± 9.13c EFV + Vitamin E 125.4 ± 8.05d 127.5 ± 9.15d 120.3 ± 9.24d 125.3 ± 8.65d 2. 73 ± 9.24c EFV + Vitamins C + E 84.2 ± 7.28c 85.6 ± 7.26c 85.5 ± 6.01c 87.9 ± 7.32c 1.70 ± 6.01d Table 4. Effects of n-acetylcysteine, vitamins C and E on liver oxidative stress indices of efavirenz-treated albino rats Treatment MDA (µmol/mg protein) CAT (U/mg protein) SOD (U/mg protein) GSH (µg/mg protein) GPX (µg/mg protein) Control 0.38 ± 0.05a 30.1 ± 3.12a 20.2 ± 1.19a 8.47 ± 0.17a 15.2 ± 1.14a NAC 0.30 ± 0.06a 34.8 ± 2.05a 23.3 ± 1.05a 8.79 ± 0.05a 16.0 ± 1.32a Vitamin C 0.36 ± 0.02a 31.5 ± 3.23a 21.7 ± 1.20a 8.50 ± 0.26a 15.5 ± 1.00a Vitamin E 0.35 ± 0.01a 32.2 ± 4.23a 21.3 ± 1.27a 8.55 ± 0.09a 15.8 ± 1.75a Vitamins C + E 0.31 ± 0.02a 34.2 ± 2.15a 23.5 ± 0.21a 8.71 ± 0.18a 17.8 ± 1.63a EFV 0.98 ± 0.02b 10.7 ± 1.23b 6.77 ± 0.24b 1.56 ± 0.08b 5.32 ± 0.53b EFV + NAC 0.48 ± 0.09c 25.3 ± 2.23c 15.9 ± 0.23c 8.27 ± 0.23a 14.0 ± 1.03a EFV + Vitamin C 0.69 ± 0.06d 15.7 ± 1.20d 10.5 ± 0.13d 3.22 ± 0.15c 8.42 ± 1.22c EFV + Vitamin E 0.61 ± 0.07d 17.0 ± 1.29d 11.8 ± 0.11d 3.59 ± 0.04c 8.99 ± 0.56c EFV + Vitamins C + E 0.49 ± 0.01c 23.5 ± 1.15c 15.5 ± 0.17c 8.19 ± 0.13a 13.9 ± 1.17a NAC, n-acetylcysteine; EFV, efavirenz. Data expressed as mean ± SD, n = 5. Values with different superscripts on the same column differ significantly at P < 0.05 ANOVA. Table 1. Effects of n-acetylcysteine, vitamin C and E on body and liver weights of efavirenz- treated albino rats Treatments Initial BW (g) Final BW (g) BW gain (g) Liver W (g) Relative liver W(%) Control 220 ± 12.0 250 ± 13.3 18.0 ± 2.00 6.95 ± 0.31 2.78 ± 0.18 EFV 230 ± 11.2 248 ± 10.6 18.0 ± 2.11 7.00 ± 0.19 2.82 ± 0.47 EFV +NAC 234 ± 12.7 253 ± 12.9 19.0 ± 1.00 6.70 ± 0.10 2.64 ± 0.84 EFV+Vitamin C 225 ± 12.0 243 ± 14.2 18.0 ± 3.00 7.05 ± 0.37 2.90 ± 0.51 EFV+Vitamin E 240 ± 11.7 260 ± 10.5 20.0 ± 3.13 6.90 ± 0.23 2.65 ± 0.24 EFV+Vitamins C + E 248 ± 9.66 268 ± 9.06 21.0 ± 2.20 7.01 ± 0.42 2.61 ± 0.89 W,weight; BW,Body weight; Data expresses ad Mean ± SD, n = 5. Table 2. Effects of n-acetylcysteine, vitamins C and E on serum liver function indices of efavirenz-treated albino rats Treatments ALT (U/L) AST (U/L) ALP (U/L) LDH (U/L) GGT (U/L) TB (g/dL) CB (g/dL) Control 38.8 ± 2.09a 32.7 ± 4.12a 34.2 ± 3.38a 50.2 ± 5.22a 0.85 ± 0.08a 9.20 ± 1.44a 4.78 ± 0.08a NAC 35.3 ± 3.09a 28.3 ± 3.09a 29.3 ± 3.00a 46.9 ± 3.11a 0.77 ± 0.06a 8.79 ± 0.09a 4.00 ± 0.12a Vitamin C 36.7 ± 5.06a 30.9 ± 3.00a 32.3 ± 0.23a 49.3 ± 4.20a 0.83 ± 0.08a 8.92 ± 0.31a 4.53 ± 0.06a Vitamin E 35.6 ± 3.04a 31.4 ± 3.06a 31.4 ± 2.11a 48.0 ± 5.20a 0.82 ± 0.05a 8.85 ± 0.22a 4.27 ± 0.01a Vitamins C + E 33.8 ± 4.20a 28.3 ± 2.00a 30.6 ± 3.01a 46.3 ± 4.35a 0.78 ± 0.01a 8.75 ± 0.05a 4.09 ± 0.05a EFV 114.8 ± 7.13b 130.0 ± 9.34b 120.7 ± 8.55b 160.1 ± 9.23b 3.97 ± 0.02b 21.1 ± 0.11b 14.9 ± 1.15b EFV + NAC 40.3 ± 3.09a 38.3 ± 3.09c 40.3 ± 3.09c 55.6 ± 5.40a 0.90 ± 0.01c 10.3 ± 1.52a 5.31 ± 0.09c EFV + Vitamin C 69.8 ± 5.14c 70.2 ± 6.21d 75.6 ± 9.13d 110.6 ± 9.07c 1.73 ± 0.06d 15.6 ± 1.21c 10.7 ± 0.13d EFV + Vitamin E 67.4 ± 4.05c 62.5 ± 5.15d 70.3 ± 7.24d 100.1 ± 8.22c 1.56 ± 0.05d 15.1 ± 1.31c 10.3 ± 0.24d EFV + Vitamins C + E 41.2 ± 2.08 a 38.6 ± 4.06c 37.5 ± 4.01c 57.7 ± 6.14a 0.87 ± 0.06a 10.7 ± 1.05c 575 ± 0.01c NAC, n-acetylcysteine; EFV, efavirenz. Data expressed as mean ± SD, n = 5. Values with different superscripts on the same column differ significantly at P < 0.05 ANOVA. Elias Adikwu and Bonsome Bokolo 42 J Contemp Med Sci | Vol. 4, No. 1, Winter 2018: 39–44 Antioxidants ameliorate efavirenz-induced liver damage Research GSH, GPX and MDA levels when compared to individual doses. However effects observed in rats pretreated with com- bined doses of vitamins C and E were not significantly (p > 0.05) different when compared to pretreatment with NAC (Table 4). Furthermore, microscopic examination of the H and E stained sections of the liver of control rat and rats adminis- tered with NAC, vitamins C and E showed normal histology (Fig 1A-E). Liver of rats treated with EFV shows necrosis of hepatocyte (Fig 1.F). However, the liver of rats pretreated with NAC, vitamins C and E and combined doses of vitamin C and E showed normal histology (Fig 1. G-J) Discussion Oxidative stress, a consequence of free radicals’ generation is a common mechanism underlying hepatotoxicity caused by drugs and toxicants.36 Antioxidants are chemical agents that scavenge, neutralize and mop-up free radicals, thereby preventing oxida- tive stress.37 Therefore, the present study evaluated the benefits of NAC, vitamins C and E on EFV-induced liver toxicity in albino rats. In the current study, treatment with NAC, vitamins C and E did not produce significant effects on AST, ALT, ALP, GGT, LDH, CB, TB, SOD, GSH, and CAT levels. These observa- tions are in conjunction with previous reports.38,39 This study did not observe significant effects on the body and liver weights in EFV-treated rats, however; levels of AST, ALT, ALP, GGT, LDH, CT, TB and MDA were elevated whereas SOD, GSH, CAT and GPX levels were reduced significantly. In addition, alterations in hepatic morphology were observed in EFV-treated rats. These finding are consistent with previous reports.40-42 AST and ALT are enzymes involved in the transfer of amino groups of aspartate and alanine to ketoglutaric acid.43 AST, ALT and ALP are present in the liver but, ALT is pri- marily located in the liver, and thus is a more specific marker of hepatocellular cell injury.44-46 Bilirubin is produced by the normal breakdown of pigment-containing proteins, especially haemoglobin from senescent red blood cells and myoglobin from muscle breakdown.47 AST, ALT, ALP, GGT, LDH and bilirubin are clinically used as fundamental indices for the assemement of the functionality of the liver. Elevations in the levels of these parameters typify hepatocellular injury.48 There- fore, the observed elevations in the levels of these parameters in EFV- treated rats indicate hepatocellular injury. In various forms of liver disease, serum levels of numerous cytosolic, mitochondrial and membrane associated enzymes are increased. The detailed mechanism by which enzymes released from the cytosol and mitochondria of hepatocytes into the blood stream is not completely known. Clinical observations and experimental studies have shown that subtle membrane changes are sufficient to allow passage of intracellular enzymes to the intracellular space.49 A very large concentration gradient between the hepatocytes and the sinusoidal space usually exists for enzymes. Cell damage increases membrane permea- bility, causing cytosolic iso-enzymes to spill into the sinusoids and from there into the peripheral blood.50 Oxidative stress has been considered as a pathological mechanism that characterises the initiation and progression of drug-induced liver injury. A network of antioxidant defence which include SOD, GAT and GSH is structured in the liver of mammals for cellular response to down-regulate oxidative stress under physiological condition and to maintain redox homeostasis in the liver.51 However, the stimulatory produc- tion of excessive free radicals from oxygen and nitrogen could alter liver redox homeostasis leading to antioxidant deple- tions.51-53 Hence, the observed decreases in the liver levels of SOD, CAT, GSH and GPX in EFV-treated rats are evidence of hepatic oxidative stress. Oxidative stress can cause hepatic damage by inducing antioxidants depletion, irretrievable alteration of lipids, proteins and DNA contents and more importantly, modulates pathways that control normal hepatic biological functions.54,55 MDA has been widely used for many years as a convenient biomarker for lipid peroxidation which is a free-radical-mediated chain of reactions. Its level is always elevated in a state of established oxidative stress and lipid per- oxidation.6,57 Therefore, the observed elevated liver levels of MDA in EFV-treated rats suggest lipid peroxidation which A Fig 1. (A) Liver of the control rat showing normal architecture. (B–E) Liver of rat administered with 20 mg/kg of n-acetylcysteine, 20 mg/kg of vitamin C, 20 mg/kg vitamin E and combined doses of vitamins C and E, respectively for 15 days showing normal architecture. (E) Liver of rat administered with 60 mg/kg/day of efavirenz (EFV) showing hepatocyte and centrilobular necrosis. (F–I) Liver of rats pre- treated with 20 mg/kg of n-acetylcysteine, 20 mg/kg of vitamin C, 20 mg/kg vitamin E and combined doses of vitamins C and E respectively prior to treatment with 60 mg/kg of EFV for 15 days showing normal architecture (H and EX200). B GF C H D I E J A Elias Adikwu and Bonsome Bokolo 43J Contemp Med Sci | Vol. 4, No. 1, Winter 2018: 39–44 Research Antioxidants ameliorate efavirenz-induced liver damage might have resulted in oxidative deterioration of hepatic poly- unsaturated lipids and increase membrane permeability. Furthermore, in the present study pretreatment with NAC, vitamins C and E ameliorate EFV-induced hepatic damage. More so, ameliorative effects were most observed in rats pretreated with combined doses of vitamins C and E than their individual doses. The hepatoprotective effects of antioxi- dants observed in the present study correlate with findings by Ebuehi et al (2012)58 who reported the palliative effects of vita- mins C and E on lead-induced hepatotoxicity in rats. Similarly Awodele and co-researchers reported the ameliorative effects of vitamins C and E on nevirapine-induced hepatotoxicity in rats.59 Also, Naglaa et al., 201560 reported the beneficial effect of NAC on acetaminophen-induced hepatotoxicity in rats. In the current study, these antioxidants might have pro- tected the liver by inhibiting the chain reactions of EFV-gener- ated free radicals or scavenged the free radicals before they reached their hepatic targets. Also, these antioxidants might have inhibited the depletion of endogenous antioxidants or facilitate their regeneration. NAC is a direct and indirect anti- oxidant that scavenges, neutralizes and chelates free radicals, thereby inhibiting oxidative destruction of tissues and organs.61 It can maintain –SH groups of enzymes and membrane pro- teins in their reduced state.62 NAC can reduce extracellular cystine to cysteine, or produce SH required for the synthesis of hepatic GSH. It can prevent drug-induced hepatic GSH deple- tion as well; up-regulate the activity of hepatic GSH. Also, NAC can enhance hepatic glutathione-S-transferase, SOD, and CAT activities, and promote detoxification mechanisms.63 Vitamin C is a water soluble antioxidant that scavenges free radicals in extracellular fluid, trapping radicals and pro- tecting biological membrane from oxidative damage.64 It is an important source of electrons and it easily donates electrons to free radicals such as hydroxyl radicals and super oxide radi- cals, thereby down-regulating their activities.65 Also, vitamin C can up-regulate the levels and activities of some endogenous antioxidants.66 Vitamin E is an essential lipophilic antioxidant that can inhibit oxidative radical-induced damage to polyun- saturated fatty acids and act as a membrane-stabilizing agent that can prevent damage to phospholipids.67 It is mainly found in the hydrocarbon part of membrane lipid bilayer towards the membrane interface and in close proximity to oxidise enzymes which can initiate the production of free radicals.68,69 Studies have shown that it can inhibit peroxidation of membrane lipids by scavenging lipid peroxyl radicals, as a consequence of which it is converted into a tocopheroxyl radical a potent anti- oxidant.70 Also, it can prevent xenobiotic-induced hepatic mitochondria oxidative stress by down-regulating the activi- ties of oxidative radicals.71 In conclusion, the present study gives an insight on the possible use of antioxidants as recipes for efavirenz-induced liver toxicity in rats. 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