ORIGINAL RESEARCH International Journal of Surgery 12 (2014) 213e218
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Original research
Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats _ lu b, Ug ur Fırat c, Ilker Öngüç Aycan a, *, Adnan Tüfek a, Orhan Tokgöz a, Osman Evliyaog ur Yüksel a Gönül Ölmez Kavak a, Hüseyin Turgut d, Mustafa Ug a
Department of Anesthesiology, Medical Faculty, Dicle University, Diyarbakir, Turkey Department of Biochemistry, Medical Faculty, Dicle University, Diyarbakir, Turkey Department of Pathology, Medical Faculty, Dicle University, Diyarbakir, Turkey d Department of Anesthesiology, Women Healthy and Gynocology Hospital, Diyarbakir, Turkey b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 September 2013 Received in revised form 10 December 2013 Accepted 20 December 2013 Available online 2 January 2014
Aim: In this study, we aimed to examine the efficacy of thymoquinone (TQ) treatment in acetaminophen-induced liver toxicity in rats. Methods: Forty Wistar Albino rats were used for the study (four groups, with 10 rats for each group). Animals in the control group were not given any medication. In the thymoquinone (TQ) group, animals were given three times 5 mg/kg oral thymoquinone for every six hours, which equals to a total dose of 15 mg/kg. In the acetaminophen (APAP) group, animals were given APAP at a single dose of 500 mg/kg orally. In the APAP þ TQ group, animals were given 500 mg/kg APAP orally followed by three doses of TQ at a 15 mg/kg total dose in an 18-h time interval. All animals were sacrificed at the 24th hour. Alanine amino transferase (ALT), aspartat amino transferase (AST), superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione peroxides (GSH-Px), and malondialdehyde (MDA) activities were measured in rat blood. Histopathological examination was also performed. Results: Serum ALT, AST levels, GSSG, and SOD activity as well as the serum and tissue MDA levels were found to be higher in the APAP group than in the control group (p 0.001). Likewise, serum GSH-Px activity was found to be lower in the APAP group (p 0.001). In contrast, in the APAP þ TQ group, serum ALT, AST levels, GSSG, SOD activity and the serum and tissue MDA levels were found to be lower compared to that of the APAP group. This difference was statistically significant (p 0.001). In the APAP þ TQ group, the GSH-Px activity was found to be significantly higher compared to the APAP group (p < 0.05). In contrast to this finding, the GSH-Px activity in the APAP þ TQ group was found to be lower than that of the control group (p 0.001). Histopathological analysis revealed significant liver necrosis and toxicity with a high dose of APAP where TQ treatment was related with significantly lower liver injury scores. Conclusion: TQ treatment may have an important therapeuthic effect via the upregulation of antioxidant systems in the APAP-induced liver hepatotoxicity in rats. Ó 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
Keywords: Acetaminophen Thymoquinone Hepatotoxicity Antioxidant
1. Introduction Acetaminophen (APAP) is one of the most commonly used analgesic and antipyretic drugs. It is considered as a safe choice when administered at therapeutic doses. In overdoses, acetaminophen can induce hepatic necrosis and death.1 Oxidative stress is a factor that leads to tissue damage. It is one of the most important factors that exacerbate damage by APAP. Following the oral administration of APAP, acetaminophen is metabolically activated * Corresponding author. Tel.: þ90 412 248 8004; fax: þ90 412 2488523. _ E-mail address: [email protected] (I.Ö. Aycan).
by cytochrome P450 to form a toxic reactive metabolite N-acetyl-pbenzoquinoneimine (NAPQI).2 NAPQI is detoxified by glutathione (GSH) to form an acetaminophen-GSH conjugate. After a toxic dose of acetaminophen, total hepatic GSH is depleted by as much as 90%. As a result, centrilobular necrosis and cell death occur.2e4 When the development of NAPQI and the depletion of hepatic glutathione store to a critical level, hepatotoxicity occurs. Majority of patients exposed to toxicity develop elevation in the levels of alanine amino transferase (ALT) and aspartat amino transferase (AST) within 24 h of ingestion.5 Antioxidant agents have therapeutic potential in drug-induced toxicity.6 N-acetyl cystein (NAC) is being used as a standard
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treatment agent in APAP toxicity. The side effect profile of NAC is well studied and allergic reactions may have occurred with NAC. In cases of drug allergy, no established alternative agent has been discovered until now. Thymoquinone (TQ) is the most potent component of Nigella sativa.7 Protective effects of orally administered TQ were established in doxorubicin,5 carbon tetrachloride,8 cisplatin,9 ethanol10 and aflotoxin11 induced oxidative damage. In addition, anti-inflammatory,12 anti-tumoral,13 anti-microbial,14 anti-histaminic10 and immuno-modulatory15 effects of TQ have been reported. Moreover, it has been suggested that TQ may act as an antioxidant agent and prevent the membrane lipid peroxidation in hepatocytes.16 In this study, we aimed to examine the efficacy of TQ treatment in APAP-induced hepatotoxicity in rats. 2. Materials and methods Our study included 40 Wistar-Albino rats, each weighing 200e 250 g. The rats were obtained from the Dicle University Health Sciences Application and Research Center. The study protocol was approved (2011e38) by the Committee of Experimental Animals of Dicle University. All experimental procedures complied with the committee’s Guide for the Care and Use of Laboratory Animals. Rats were housed under standard conditions in an airconditioned room with 12 h light and dark cycles and with constant temperature (22 2 C). The rats were housed in cages and allowed free access to standard rat chow and water before the experiments. The animals were fasted overnight the day before the study, but had access to water ad libitum. 2.1. Study design Acetaminophen (APAP) was purchased from Merck (Germany). It was dissolved in warm saline (65 C) and cooled to room temperature before oral administration. Thymoquinone (TQ) was purchased from SigmaeAldrich (Catalog no.: 274666). It was dissolved in warm drinking water (65 C) and cooled to room temperature before oral administration. Forty Wistar-Albino rats were divided into four groups (n ¼ 10). Group I (Control): no drug was given. Group II (TQ): TQ was given at a triple dose (0, 6, 12) of 5 mg/kg via the oral route.17e20 Group III (Acetaminophen, APAP): APAP was given at a single dose of 500 mg/kg via the oral route. Group IV (APAP þ TQ): APAP was given at a single dose of 500 mg/kg via the oral route and TQ was given at a triple dose1,6,12 of 5 mg/kg via the oral route. After a period of 24 h, the rats were anesthetized with 50 mg/kg ketamine hydrochloride (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun; Bayer AG, Leverkusen, Germany) via intramuscular injection and sacrificed by taking blood from the heart for biochemical analysis. Likewise, liver tissues were removed for histopathological examinations. Alanine amino transferase (ALT), aspartat amino transferase (AST), superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione peroxides (GSH-Px), and malondialdehyde (MDA) activities were measured in rat blood. Histopathological examination was also undertaken. 2.2. Biochemical analysis Each collected blood sample was immediately centrifuged at 4000 rpm for 10 min and then transferred into an eppendorf tube. Samples were transferred on ice and kept in 80 C deep freeze until the completion of the study.
Malonyldialdehyde (MDA) content was measured spectrophotometrically as described previously.17 GSH-Px (U/L) and Glutatyonin-GSSG (mmol/L) levels were determined with colorimetric assay kit (Cayman Ch. Co. AnnArbo. MI, USA). SOD activity was measured according to the method described by Fridovich.18 Plasma aspartat transaminase (AST) and alanine transaminase (ALT) levels were measured by photometric method with an automated analyzer (Architect c16000; Abbott Laboratories, Abbott Park, IL, USA). 2.3. Histopathological evaluation The liver tissues were taken for histopathological evaluation. Foreign tissue residues and blood were removed, washed with saline, and put into plastic containers holding 10% formaldehyde solution. Tissue specimens were fixed in 10% formalin for 48 h, then embedded in paraffin and cut into 5 mm sections. Slides were stained with hematoxylin-eosin. The sections were examined under a light microscope (Nikon ECLIPSE 80i) using 200 magnification to assess the degree of liver injury by a liver pathologist blinded to the grouping of the animals. Hepatic injury was graded as follows: grade 0: minimal or no evidence of injury; grade 1: mild injury characterized by cytoplasmic vacuolization and focal nuclear pyknosis; grade 2: moderate injury exhibiting cytoplasmic vacuolization, confluent areas of hepatocyte ballooning but no frank necrosis, sinusoidal dilatation and congestion, and blurring of intercellular borders; grade 3: moderate to severe injury with areas of coagulative necrosis, cytoplasmic hypereosinophilia, extensive sinusoidal dilatation and congestion; grade 4: severe injury consisting of severe confluent coagulative necrosis and disintegration of and hemorrhage into hepatic chords leading to loss of tissue architecture.19,20 2.4. Statistical analysis Statistical analysis was performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA). Data were presented as mean standard deviation (SD) values. Groups were compared using the nonparametric KruskaleWallis test. The ManneWhitney U test was used for binary comparisons. A p value of less than 0.05 was considered significant. 3. Results The liver enzyme levels of all groups were shown in Table 1. ALT and AST levels were found higher in the APAP group compared to that of the control group (p < 0.001). The mean ALT and AST levels of rats treated with APAP þ TQ were found to be lower compared to those treated with APAP only (p < 0.001). Both means of the two enzyme levels of the TQ group were similar to that of the control group (p > 0.05) (Fig. 1). Table 1 The distribution of liver enzymes between the groups.
ALT (U/L) AST (U/L)
Group I (control)
Group II (TQ)
Group III (APAP)
Group IV (APAP þ TQ)
43.3 10.7 155.6 30.3
59.1 15.4 155.7 50.4
131 32.8a 309.4 109.5a
52.9 10.3b 185.6 58.3b
Data were given as mean SD. TQ: Thymoquinone, APAP: Acetaminophene, ALT: alanine aminotransferase, AST: aspartat aminotransferase. a Significantly different when compared with control group, (p 0.001). b Significantly different when compared with APAP group, (p 0.001).
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Serum GSSG, SOD activities, and the MDA levels in both serum and liver tissue of the APAP group were significantly elevated compared to that of the control group (p 0.001) (Shown in Table 2) (Figs. 2e5 respectively). In contrast to the APAP only group, the serum GSSG, SOD activities, and MDA levels in both the serum and liver tissue of the APAP þ TQ group were found to be significant (p 0.001). When the serum SOD activities, and serum and tissue MDA levels of the control, TQ, and APAP þ TQ groups were compared, no significant difference was found between groups (p > 0.05). In addition to these findings, the serum GSSG activity in the TQ group was found to be lower than that of the control group (p 0.001). In terms of mean serum GSH-Px activity, the APAP group had significantly lower levels compared to the control group (p 0.001). Although the mean GSH-Px activity in the APAP þ TQ group was found to be higher than that of the APAP only group, the activity level was found to be significantly lower than that of the control group (p < 0.05 vs p 0.001). The difference in the GSH-Px activities of the control and TQ groups were found to be similar (p > 0.05) (Fig. 6). Histopathological changes in the TQ group were found to be similar with the histopathological changes in the control group (p > 0.05). In contrast to the TQ group, significant tissue damage was established in the APAP group compared with the control group (p 0.001). Significant lower scores were determined in the APAP þ TQ group when compared with the APAP group (p 0.001), while higher scores were obtained when compared with the control group (p 0.001) (Fig. 7). Histopathological examination results are shown in Table 3. 4. Discussion Acetaminophen (N-acetyl-p-aminophenol [APAP]) is a widely used medication that can cause fatal hepatotoxicity hepatic necrosis, renal toxicity and, rarely, death when administered at toxic doses. Children are particularly at risk for accidental overdose. APAP is also often used as a suicidal agent in the elderly. The majority of the therapeutic doses (80e85%) of APAP is glucuronidated or sulfated and then excreted. A small percentage (10e15%) is metabolized by cytochrome P45 0 enzymes (CYP) to the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI), which is readily detoxified by conjugation with glutathione (GSH). In cases of APAP overdose, induction of CYP enzymes, decrease in glutathione stores, and inhibition of glucronidation result in elevated levels of toxic APAP metabolite.2,21 Inadequate detoxification of NAPQI leads to intracellular structural damage followed by centrilobular necrosis. The early phase of toxication resembles that
Fig. 1. The distribution of liver enzymes between the groups.
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Table 2 Inter-group comparison of biochemical markers in serum and liver tissue.
GSSG (mmol/L) GSH-Px (U/L) SOD (U/L) MDA (nmol/L) Liver-MDA (nmol/g prt)
Group I (control)
Group II (TQ)
Group III (APAP)
Group IV (APAP þ TQ)
264 45.2 56.5 12.2 5.5 0.5 133.1 29.8 0.37 0.08
179 49.8a 57.4 14.9 5.8 0.4 119.7 26.6 0.33 0.07
429.3 128.3a,b 21.5 5.2a,b 7.9 1.1a,b 253.4 56.6a,b 0.71 0.15a,b
192.5 56.8c 37.1 8b,d,e 5.7 0.4c 162.5 39.3c 0.45 0.11c
Data were given as mean SD. GSSG: Okside glutatyon, GSH-Px: glutatyon peroksidaz, SOD: süperoksit dismutaz, MDA: malondialdehyde, TQ: Thymoquinone, APAP: Acetaminophene. a Significantly different when compared with control group, (p 0.001). b Significantly different when compared with TQ group, (p 0.001). c Significantly different when compared with APAP group, (p 0.001). d Significantly different when compared with control group, (p < 0.01). e Significantly different when compared with APAP group, (p < 0.05).
of mitochondrial disfunction, while the late phase includes mitochondrial collapse, oncotic necrosis and, subsequently, in vivo cell death.2,4 It is well known that oxidative stress plays an important role in APAP toxicity. Increased NAPQI levels in APAP overdose results in the increased formation of O2∙- hydrogen peroxide (H2O2). The high oxidative capacity of NAPQI leads to thiol oxidation, which is thought to be the major factor behind liver toxicity. Increased levels of NAPQI are associated with increased GSH/GSSG ratio and decreased NADPH/NADP þ ratio. Decreased GSH concentrations result in reduced activity of GPx enzyme function.22 N-acetylcysteine (NAC) is the standard clinical antidote of choice in treating acetaminophen-induced hepatotoxicity. The role of Nacetylcysteine in the treatment of acetaminophen toxicity is to replace stores of intracellular hepatic glutathione, a free radical scavenger, binding NAPQI directly and increasing microcirculatory oxygenation.23 However, it has several adverse effects, including nausea and vomiting, which are the most common adverse effects of NAC.24 Although NAC is a safe and effective antidote for APAP toxicity, in terms of adverse effect experiences, an alternative agent may be useful. Thus, alternative agents that are capable of reducing oxidative stressdthe main mechanism in APAP toxicitydhave to be developed. Nagi et al. reported protective effects on the prophilactic use of orally administered TQ in APAP-induced hepatotoxicity via antioxidant mechanisms.6 Our study is the first study in literature that
Fig. 2. Serum Glutathion (GSSG) levels.
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Fig. 3. Serum Superoxide dismutase (SOD) levels. Fig. 5. Liver Tissue Malondialdehyde (Liver-MDA) levels.
investigates the therapeutical effects of TQ after APAP overdose exposure. In our study, we have shown a remarkable reduction in APAP-induced ALT, AST levels, oxidative stress, and tissue damage. Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is the bioactive component of N. sativa seeds and it has various pharmacological effects.25 It is reported that thymoquinone possess strong antioxidant properties and protect several organs against oxidative damage induced by free radical-generating agents.6,26,27 In this study, APAP administration induced significantly high MDA levels compared to the control group. Several studies showed 40e120% elevated MDA levels compared to the control.6 GSH is the major intracellular defense molecule against ROS and electrophilic xenobiotics. Excessive exposure to xenobiotics reduces hepatic GSH concentrations.27 GSH is essential in protecting thiol and other nucleophilic groups from the toxic metabolite of APAP. Therefore, it is concluded that the key factor in determining the extension of APAP-induced liver damage is intracellular GSH levels. In our study, we found that APAP administration resulted in remarkably reduced GSH levels. Several studies investigated different doses and durations of APAP toxicity and reported similar findings with our study.21 Meotti et al. reported a significant reduction in GSH levels four hours following APAP administration, while no significant change in GSH levels 24 h after toxic dose.28 Authors suggested that the adaptive response of hepatocytes might be responsible for the initial measurements, while ROS detoxification could be the reason for late results. We have administered a higher APAP dose in this study and GSH levels were
Fig. 4. Serum Malondialdehyde (MDA) levels.
found to be significantly lower in the control at the end of the 24th hour. This finding correlated with the findings of previous studies. Antioxidant enzymes like SOD and GSH-Px are important for the elimination of ROS. It has been suggested that the tissue levels of SOD and GSH-Px may reflect ROS levels.27 In addition, MDA levels can be a reliable indicator of lipid peroxidation and oxidative stress.29 Thus, ROS can be evaluated indirectly with the determination of MDA and the levels of some antioxidant enzyme activities like SOD or GSH-Px in tissue.30 Vaquero et al. showed the antioxidant effects of TQ via the reduction of diphenylpcriylhydracyl (DPPH), an oxidative radical, by TQ.31 Another study showed the protective effects of TQ in doxorubicin-induced cardio toxicity that resulted from ROS formation and oxidative stress.5 APAP-induced hepatotoxicity resulted in elevated superoxide and hydrogen peroxide levels and decreased GSH/GSSG ratio.3,32 As a consequence of impairment in antioxidant defense systems, ROS and lipid peroxidation increases. In our study, the APAP group demonstrated an increase in MDA and GSSG levels in addition to decreased GSH-Px activity. ROS attacks membrane lipids and results to lipid peroxidation. MDA is the end-product of lipid peroxidation. It serves as an index of lipid peroxidation. In our study, the increase of MDA in liver tissue and in serum has improved in the rats’ exposure to APAP as a result of TQ treatment (p 0.001). This decrease in the level of MDA suggests that TQ may be effective in the prevention of lipid
Fig. 6. Serum Glutathion peroxidase (GSH-PX) levels.
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elevated resistance against APAP-induced hepatotoxicity in transgenic animals that have excessively high GSH-Px levels.36 We suggest that TQ treatment may have a role in the regulation of antioxidant mechanisms. In conclusion, we have shown the therapeutic effect of TQ in terms of the regulation of antioxidant activity in APAP-induced hepatotoxicity. Our findings suggest that TQ may be an alternative therapeutic agent to NAC in APAP toxicity. There are requirements for future studies. Ethical approval The study protocol was approved (2011e38) by the Committee of Experimental Animals of Dicle University. Funding This work is supported by grant number 11-TF-54 from Dicle University Scientific Research Project Coordination Unit (DUBAP), Turkey.
Fig. 7. Rat liver tissues histopathological changes.
peroxidation. We assessed the effect of TQ on lipid peroxidation, which was measured in terms of MDA, a stable metabolite of the free radical-mediated lipid peroxidation cascade. Similar studies have shown the protective effect of TQ in carbon tetrachloride-induced hepatotoxicity via the protection of lipid peroxidation.8 Another interesting finding in our study is the correlated MDA levels in both APAP þ TQ and control groups. Similar MDA levels in these groups indicate the protective effect of TQ in oxidative stress. Superoxide dismutase (SOD) is the major anti-oxidant enzymereducing superoxide.33 We found that similar SOD levels in the control and TQ groups indicates that TQ has positive effects on oxidative stress. In our study, SOD levels in the APAP group increased in association with increased free radicals. An interesting finding in our study is the decreased SOD levels in the APAP þ TQ treated group, which is similar with the control group. In contrast to our study, previous studies showed a decreased SOD enzyme activity in APAP hepatotoxicity.34,35 We suggest that this finding occurred due to the decreased formation of free radicals followed by TQ treatment. Moreover, decreased GSSG levels showed decreased glutathione oxidation. The elevated enzyme activity of SOD and the reduction of GSH-Px in the APAP group indirectly showed an increase in the number of ROS in hepatic tissue. The elevated SOD enzyme activity is one of the parts of the antioxidant defense system. Oxidative stress at the first level will be encountered by increased antioxidant enzyme activities. Interestingly, statistically significant decreases in SOD enzyme activity was seen in the APAP þ TQ group when compared to the APAP group. The exact mechanism of TQ on the enzyme activities is not yet known; however, it can be speculated that TQ may affect the transcriptional and/or translational pathways of these antioxidant enzymes. Another explanation for this effect of TQ may be that it prevents the induction of the enzymes (GSH-Px) by the inhibition of toxic oxidative products. Mirochnitchenko et al. showed a remarkably
Table 3 The comparison of histopathological evaluation scores of rat liver tissue between the groups.
mean SD minemax
Group I (Control)
Group II (TQ)
Group III (APAP)
Group IV (APAP þ TQ)
0.2 0.4 0e1
0.3 0.4 0e1
3 0.6a,b 2e4
1.5 0.8a,b,c 0e3
TQ: Thymoquinone, APAP: Acetaminophene. a Significantly different when compared with control group, (p 0.001). b Significantly different when compared with TQ group, (p 0.001). c Significantly different when compared with APAP group, (p 0.001).
Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgment This work is supported by grant number 11-TF-54 from Dicle University Scientific Research Project Coordination Unit (DUBAP), Turkey. References 1. Bonkovsky HL, Kane RE, Jones DP, Galinsky RE, Banner B. Acute hepatic and renal toxicity from low doses of acetaminophen in the absence of alcohol abuse or malnutrition: evidence for increased susceptibility to drug toxicity due to cardiopulmonary and renal insufficiency. Hepatology 1994 May;19(5):1141e8. 2. Bromer MQ, Black M. Acetaminophen hepatotoxicity. Clin Liver Dis 2003 May;7(2):351e67. 3. Gibson JD, Pumford NR, Samokyszyn VM, Hinson JA. Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress. Chem Res Toxicol 1996 ApreMay;9(3):580e5. 4. Vaquero J, Belanger M, James L, et al. Mild hypothermia attenuates liver injury and improves survival in mice with acetaminophen toxicity. Gastroenterology 2007 Jan;132(1):372e83. 5. Nagi MN, Mansour MA. Protective effect of thymoquinone against doxorubicininduced cardiotoxicity in rats: a possible mechanism of protection. Pharmacol Res 2000 Mar;41(3):283e9. 6. Nagi MN, Almakki HA, Sayed-Ahmed MM, Al-Bekairi AM. Thymoquinone supplementation reverses acetaminophen-induced oxidative stress, nitric oxide production and energy decline in mice liver. Food Chem Toxicol 2010 Auge Sep;48(8e9):2361e5. 7. Kapan M, Tekin R, Onder A, et al. Thymoquinone ameliorates bacterial translocation and inflammatory response in rats with intestinal obstruction. Int J Surg 2012;10(9):484e8. 8. Kanter M, Coskun O, Budancamanak M. Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats. World J Gastroenterol 2005 Nov 14;11(42):6684e8. 9. Badary OA, Nagi MN, al-Shabanah OA, al-Sawaf HA, al-Sohaibani MO, alBekairi AM. Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity. Can J Physiol Pharmacol 1997 Dec;75(12):1356e61. 10. Kanter M, Coskun O, Uysal H. The antioxidative and antihistaminic effect of Nigella sativa and its major constituent, thymoquinone on ethanol-induced gastric mucosal damage. Arch Toxicol 2006 Apr;80(4):217e24. 11. Nili-Ahmadabadi A, Tavakoli F, Hasanzadeh G, Rahimi H, Sabzevari O. Protective effect of pretreatment with thymoquinone against Aflatoxin B(1) induced liver toxicity in mice. Daru 2011;19(4):282e7. 12. Chehl N, Chipitsyna G, Gong Q, Yeo CJ, Arafat HA. Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells. HPB Oxf 2009 Aug;11(5):373e81. 13. Peng L, Liu A, Shen Y, et al. Antitumor and anti-angiogenesis effects of thymoquinone on osteosarcoma through the NF-kappaB pathway. Oncol Rep 2013 Feb;29(2):571e8. 14. Kouidhi B, Zmantar T, Jrah H, et al. Antibacterial and resistance-modifying activities of thymoquinone against oral pathogens. Ann Clin Microbiol Antimicrob 2011;10:29.
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15. Salem ML. Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. Int Immunopharmacol 2005 Dec;5(13e4):1749e70. 16. Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM. Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell Biochem Funct 2002 Jun;20(2):143e51. 17. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186: 407e21. 18. Fridovich I. Superoxide dismutases. Adv Enzymol Relat Areas Mol Biol 1974;41(0):35e97. 19. Camargo Jr CA, Madden JF, Gao W, Selvan RS, Clavien PA. Interleukin-6 protects liver against warm ischemia/reperfusion injury and promotes hepatocyte proliferation in the rodent. Hepatology 1997 Dec;26(6):1513e20. 20. Tufek A, Tokgoz O, Aliosmanoglu I, et al. The protective effects of dexmedetomidine on the liver and remote organs against hepatic ischemia reperfusion injury in rats. Int J Surg 2013;11(1):96e100. 21. Yapar K, Kart A, Karapehlivan M, et al. Hepatoprotective effect of L-carnitine against acute acetaminophen toxicity in mice. Exp Toxicol Pathol 2007 Oct;59(2):121e8. 22. Bessems JG, Vermeulen NP. Paracetamol (acetaminophen)-induced toxicity: molecular and biochemical mechanisms, analogues and protective approaches. Crit Rev Toxicol 2001 Jan;31(1):55e138. 23. Chandrasekaran VR, Chien SP, Hsu DZ, Liu MY. Anti-hepatotoxic effects of 3,4methylenedioxyphenol and N-acetylcysteine in acutely acetaminophenoverdosed mice. Hum Exp Toxicol 2011 Oct;30(10):1609e15. 24. Whyte IM, Francis B, Dawson AH. Safety and efficacy of intravenous N-acetylcysteine for acetaminophen overdose: analysis of the Hunter Area Toxicology Service (HATS) database. Curr Med Res Opin 2007 Oct;23(10):2359e 68. 25. Ali BH, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 2003 Apr;17(4):299e305.
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International Journal of Surgery journal homepage: www.journal-surgery.net
Original research
Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats _ lu b, Ug ur Fırat c, Ilker Öngüç Aycan a, *, Adnan Tüfek a, Orhan Tokgöz a, Osman Evliyaog ur Yüksel a Gönül Ölmez Kavak a, Hüseyin Turgut d, Mustafa Ug a
Department of Anesthesiology, Medical Faculty, Dicle University, Diyarbakir, Turkey Department of Biochemistry, Medical Faculty, Dicle University, Diyarbakir, Turkey Department of Pathology, Medical Faculty, Dicle University, Diyarbakir, Turkey d Department of Anesthesiology, Women Healthy and Gynocology Hospital, Diyarbakir, Turkey b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 September 2013 Received in revised form 10 December 2013 Accepted 20 December 2013 Available online 2 January 2014
Aim: In this study, we aimed to examine the efficacy of thymoquinone (TQ) treatment in acetaminophen-induced liver toxicity in rats. Methods: Forty Wistar Albino rats were used for the study (four groups, with 10 rats for each group). Animals in the control group were not given any medication. In the thymoquinone (TQ) group, animals were given three times 5 mg/kg oral thymoquinone for every six hours, which equals to a total dose of 15 mg/kg. In the acetaminophen (APAP) group, animals were given APAP at a single dose of 500 mg/kg orally. In the APAP þ TQ group, animals were given 500 mg/kg APAP orally followed by three doses of TQ at a 15 mg/kg total dose in an 18-h time interval. All animals were sacrificed at the 24th hour. Alanine amino transferase (ALT), aspartat amino transferase (AST), superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione peroxides (GSH-Px), and malondialdehyde (MDA) activities were measured in rat blood. Histopathological examination was also performed. Results: Serum ALT, AST levels, GSSG, and SOD activity as well as the serum and tissue MDA levels were found to be higher in the APAP group than in the control group (p 0.001). Likewise, serum GSH-Px activity was found to be lower in the APAP group (p 0.001). In contrast, in the APAP þ TQ group, serum ALT, AST levels, GSSG, SOD activity and the serum and tissue MDA levels were found to be lower compared to that of the APAP group. This difference was statistically significant (p 0.001). In the APAP þ TQ group, the GSH-Px activity was found to be significantly higher compared to the APAP group (p < 0.05). In contrast to this finding, the GSH-Px activity in the APAP þ TQ group was found to be lower than that of the control group (p 0.001). Histopathological analysis revealed significant liver necrosis and toxicity with a high dose of APAP where TQ treatment was related with significantly lower liver injury scores. Conclusion: TQ treatment may have an important therapeuthic effect via the upregulation of antioxidant systems in the APAP-induced liver hepatotoxicity in rats. Ó 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
Keywords: Acetaminophen Thymoquinone Hepatotoxicity Antioxidant
1. Introduction Acetaminophen (APAP) is one of the most commonly used analgesic and antipyretic drugs. It is considered as a safe choice when administered at therapeutic doses. In overdoses, acetaminophen can induce hepatic necrosis and death.1 Oxidative stress is a factor that leads to tissue damage. It is one of the most important factors that exacerbate damage by APAP. Following the oral administration of APAP, acetaminophen is metabolically activated * Corresponding author. Tel.: þ90 412 248 8004; fax: þ90 412 2488523. _ E-mail address: [email protected] (I.Ö. Aycan).
by cytochrome P450 to form a toxic reactive metabolite N-acetyl-pbenzoquinoneimine (NAPQI).2 NAPQI is detoxified by glutathione (GSH) to form an acetaminophen-GSH conjugate. After a toxic dose of acetaminophen, total hepatic GSH is depleted by as much as 90%. As a result, centrilobular necrosis and cell death occur.2e4 When the development of NAPQI and the depletion of hepatic glutathione store to a critical level, hepatotoxicity occurs. Majority of patients exposed to toxicity develop elevation in the levels of alanine amino transferase (ALT) and aspartat amino transferase (AST) within 24 h of ingestion.5 Antioxidant agents have therapeutic potential in drug-induced toxicity.6 N-acetyl cystein (NAC) is being used as a standard
1743-9191/$ e see front matter Ó 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijsu.2013.12.013
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treatment agent in APAP toxicity. The side effect profile of NAC is well studied and allergic reactions may have occurred with NAC. In cases of drug allergy, no established alternative agent has been discovered until now. Thymoquinone (TQ) is the most potent component of Nigella sativa.7 Protective effects of orally administered TQ were established in doxorubicin,5 carbon tetrachloride,8 cisplatin,9 ethanol10 and aflotoxin11 induced oxidative damage. In addition, anti-inflammatory,12 anti-tumoral,13 anti-microbial,14 anti-histaminic10 and immuno-modulatory15 effects of TQ have been reported. Moreover, it has been suggested that TQ may act as an antioxidant agent and prevent the membrane lipid peroxidation in hepatocytes.16 In this study, we aimed to examine the efficacy of TQ treatment in APAP-induced hepatotoxicity in rats. 2. Materials and methods Our study included 40 Wistar-Albino rats, each weighing 200e 250 g. The rats were obtained from the Dicle University Health Sciences Application and Research Center. The study protocol was approved (2011e38) by the Committee of Experimental Animals of Dicle University. All experimental procedures complied with the committee’s Guide for the Care and Use of Laboratory Animals. Rats were housed under standard conditions in an airconditioned room with 12 h light and dark cycles and with constant temperature (22 2 C). The rats were housed in cages and allowed free access to standard rat chow and water before the experiments. The animals were fasted overnight the day before the study, but had access to water ad libitum. 2.1. Study design Acetaminophen (APAP) was purchased from Merck (Germany). It was dissolved in warm saline (65 C) and cooled to room temperature before oral administration. Thymoquinone (TQ) was purchased from SigmaeAldrich (Catalog no.: 274666). It was dissolved in warm drinking water (65 C) and cooled to room temperature before oral administration. Forty Wistar-Albino rats were divided into four groups (n ¼ 10). Group I (Control): no drug was given. Group II (TQ): TQ was given at a triple dose (0, 6, 12) of 5 mg/kg via the oral route.17e20 Group III (Acetaminophen, APAP): APAP was given at a single dose of 500 mg/kg via the oral route. Group IV (APAP þ TQ): APAP was given at a single dose of 500 mg/kg via the oral route and TQ was given at a triple dose1,6,12 of 5 mg/kg via the oral route. After a period of 24 h, the rats were anesthetized with 50 mg/kg ketamine hydrochloride (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun; Bayer AG, Leverkusen, Germany) via intramuscular injection and sacrificed by taking blood from the heart for biochemical analysis. Likewise, liver tissues were removed for histopathological examinations. Alanine amino transferase (ALT), aspartat amino transferase (AST), superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione peroxides (GSH-Px), and malondialdehyde (MDA) activities were measured in rat blood. Histopathological examination was also undertaken. 2.2. Biochemical analysis Each collected blood sample was immediately centrifuged at 4000 rpm for 10 min and then transferred into an eppendorf tube. Samples were transferred on ice and kept in 80 C deep freeze until the completion of the study.
Malonyldialdehyde (MDA) content was measured spectrophotometrically as described previously.17 GSH-Px (U/L) and Glutatyonin-GSSG (mmol/L) levels were determined with colorimetric assay kit (Cayman Ch. Co. AnnArbo. MI, USA). SOD activity was measured according to the method described by Fridovich.18 Plasma aspartat transaminase (AST) and alanine transaminase (ALT) levels were measured by photometric method with an automated analyzer (Architect c16000; Abbott Laboratories, Abbott Park, IL, USA). 2.3. Histopathological evaluation The liver tissues were taken for histopathological evaluation. Foreign tissue residues and blood were removed, washed with saline, and put into plastic containers holding 10% formaldehyde solution. Tissue specimens were fixed in 10% formalin for 48 h, then embedded in paraffin and cut into 5 mm sections. Slides were stained with hematoxylin-eosin. The sections were examined under a light microscope (Nikon ECLIPSE 80i) using 200 magnification to assess the degree of liver injury by a liver pathologist blinded to the grouping of the animals. Hepatic injury was graded as follows: grade 0: minimal or no evidence of injury; grade 1: mild injury characterized by cytoplasmic vacuolization and focal nuclear pyknosis; grade 2: moderate injury exhibiting cytoplasmic vacuolization, confluent areas of hepatocyte ballooning but no frank necrosis, sinusoidal dilatation and congestion, and blurring of intercellular borders; grade 3: moderate to severe injury with areas of coagulative necrosis, cytoplasmic hypereosinophilia, extensive sinusoidal dilatation and congestion; grade 4: severe injury consisting of severe confluent coagulative necrosis and disintegration of and hemorrhage into hepatic chords leading to loss of tissue architecture.19,20 2.4. Statistical analysis Statistical analysis was performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA). Data were presented as mean standard deviation (SD) values. Groups were compared using the nonparametric KruskaleWallis test. The ManneWhitney U test was used for binary comparisons. A p value of less than 0.05 was considered significant. 3. Results The liver enzyme levels of all groups were shown in Table 1. ALT and AST levels were found higher in the APAP group compared to that of the control group (p < 0.001). The mean ALT and AST levels of rats treated with APAP þ TQ were found to be lower compared to those treated with APAP only (p < 0.001). Both means of the two enzyme levels of the TQ group were similar to that of the control group (p > 0.05) (Fig. 1). Table 1 The distribution of liver enzymes between the groups.
ALT (U/L) AST (U/L)
Group I (control)
Group II (TQ)
Group III (APAP)
Group IV (APAP þ TQ)
43.3 10.7 155.6 30.3
59.1 15.4 155.7 50.4
131 32.8a 309.4 109.5a
52.9 10.3b 185.6 58.3b
Data were given as mean SD. TQ: Thymoquinone, APAP: Acetaminophene, ALT: alanine aminotransferase, AST: aspartat aminotransferase. a Significantly different when compared with control group, (p 0.001). b Significantly different when compared with APAP group, (p 0.001).
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Serum GSSG, SOD activities, and the MDA levels in both serum and liver tissue of the APAP group were significantly elevated compared to that of the control group (p 0.001) (Shown in Table 2) (Figs. 2e5 respectively). In contrast to the APAP only group, the serum GSSG, SOD activities, and MDA levels in both the serum and liver tissue of the APAP þ TQ group were found to be significant (p 0.001). When the serum SOD activities, and serum and tissue MDA levels of the control, TQ, and APAP þ TQ groups were compared, no significant difference was found between groups (p > 0.05). In addition to these findings, the serum GSSG activity in the TQ group was found to be lower than that of the control group (p 0.001). In terms of mean serum GSH-Px activity, the APAP group had significantly lower levels compared to the control group (p 0.001). Although the mean GSH-Px activity in the APAP þ TQ group was found to be higher than that of the APAP only group, the activity level was found to be significantly lower than that of the control group (p < 0.05 vs p 0.001). The difference in the GSH-Px activities of the control and TQ groups were found to be similar (p > 0.05) (Fig. 6). Histopathological changes in the TQ group were found to be similar with the histopathological changes in the control group (p > 0.05). In contrast to the TQ group, significant tissue damage was established in the APAP group compared with the control group (p 0.001). Significant lower scores were determined in the APAP þ TQ group when compared with the APAP group (p 0.001), while higher scores were obtained when compared with the control group (p 0.001) (Fig. 7). Histopathological examination results are shown in Table 3. 4. Discussion Acetaminophen (N-acetyl-p-aminophenol [APAP]) is a widely used medication that can cause fatal hepatotoxicity hepatic necrosis, renal toxicity and, rarely, death when administered at toxic doses. Children are particularly at risk for accidental overdose. APAP is also often used as a suicidal agent in the elderly. The majority of the therapeutic doses (80e85%) of APAP is glucuronidated or sulfated and then excreted. A small percentage (10e15%) is metabolized by cytochrome P45 0 enzymes (CYP) to the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI), which is readily detoxified by conjugation with glutathione (GSH). In cases of APAP overdose, induction of CYP enzymes, decrease in glutathione stores, and inhibition of glucronidation result in elevated levels of toxic APAP metabolite.2,21 Inadequate detoxification of NAPQI leads to intracellular structural damage followed by centrilobular necrosis. The early phase of toxication resembles that
Fig. 1. The distribution of liver enzymes between the groups.
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Table 2 Inter-group comparison of biochemical markers in serum and liver tissue.
GSSG (mmol/L) GSH-Px (U/L) SOD (U/L) MDA (nmol/L) Liver-MDA (nmol/g prt)
Group I (control)
Group II (TQ)
Group III (APAP)
Group IV (APAP þ TQ)
264 45.2 56.5 12.2 5.5 0.5 133.1 29.8 0.37 0.08
179 49.8a 57.4 14.9 5.8 0.4 119.7 26.6 0.33 0.07
429.3 128.3a,b 21.5 5.2a,b 7.9 1.1a,b 253.4 56.6a,b 0.71 0.15a,b
192.5 56.8c 37.1 8b,d,e 5.7 0.4c 162.5 39.3c 0.45 0.11c
Data were given as mean SD. GSSG: Okside glutatyon, GSH-Px: glutatyon peroksidaz, SOD: süperoksit dismutaz, MDA: malondialdehyde, TQ: Thymoquinone, APAP: Acetaminophene. a Significantly different when compared with control group, (p 0.001). b Significantly different when compared with TQ group, (p 0.001). c Significantly different when compared with APAP group, (p 0.001). d Significantly different when compared with control group, (p < 0.01). e Significantly different when compared with APAP group, (p < 0.05).
of mitochondrial disfunction, while the late phase includes mitochondrial collapse, oncotic necrosis and, subsequently, in vivo cell death.2,4 It is well known that oxidative stress plays an important role in APAP toxicity. Increased NAPQI levels in APAP overdose results in the increased formation of O2∙- hydrogen peroxide (H2O2). The high oxidative capacity of NAPQI leads to thiol oxidation, which is thought to be the major factor behind liver toxicity. Increased levels of NAPQI are associated with increased GSH/GSSG ratio and decreased NADPH/NADP þ ratio. Decreased GSH concentrations result in reduced activity of GPx enzyme function.22 N-acetylcysteine (NAC) is the standard clinical antidote of choice in treating acetaminophen-induced hepatotoxicity. The role of Nacetylcysteine in the treatment of acetaminophen toxicity is to replace stores of intracellular hepatic glutathione, a free radical scavenger, binding NAPQI directly and increasing microcirculatory oxygenation.23 However, it has several adverse effects, including nausea and vomiting, which are the most common adverse effects of NAC.24 Although NAC is a safe and effective antidote for APAP toxicity, in terms of adverse effect experiences, an alternative agent may be useful. Thus, alternative agents that are capable of reducing oxidative stressdthe main mechanism in APAP toxicitydhave to be developed. Nagi et al. reported protective effects on the prophilactic use of orally administered TQ in APAP-induced hepatotoxicity via antioxidant mechanisms.6 Our study is the first study in literature that
Fig. 2. Serum Glutathion (GSSG) levels.
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Fig. 3. Serum Superoxide dismutase (SOD) levels. Fig. 5. Liver Tissue Malondialdehyde (Liver-MDA) levels.
investigates the therapeutical effects of TQ after APAP overdose exposure. In our study, we have shown a remarkable reduction in APAP-induced ALT, AST levels, oxidative stress, and tissue damage. Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is the bioactive component of N. sativa seeds and it has various pharmacological effects.25 It is reported that thymoquinone possess strong antioxidant properties and protect several organs against oxidative damage induced by free radical-generating agents.6,26,27 In this study, APAP administration induced significantly high MDA levels compared to the control group. Several studies showed 40e120% elevated MDA levels compared to the control.6 GSH is the major intracellular defense molecule against ROS and electrophilic xenobiotics. Excessive exposure to xenobiotics reduces hepatic GSH concentrations.27 GSH is essential in protecting thiol and other nucleophilic groups from the toxic metabolite of APAP. Therefore, it is concluded that the key factor in determining the extension of APAP-induced liver damage is intracellular GSH levels. In our study, we found that APAP administration resulted in remarkably reduced GSH levels. Several studies investigated different doses and durations of APAP toxicity and reported similar findings with our study.21 Meotti et al. reported a significant reduction in GSH levels four hours following APAP administration, while no significant change in GSH levels 24 h after toxic dose.28 Authors suggested that the adaptive response of hepatocytes might be responsible for the initial measurements, while ROS detoxification could be the reason for late results. We have administered a higher APAP dose in this study and GSH levels were
Fig. 4. Serum Malondialdehyde (MDA) levels.
found to be significantly lower in the control at the end of the 24th hour. This finding correlated with the findings of previous studies. Antioxidant enzymes like SOD and GSH-Px are important for the elimination of ROS. It has been suggested that the tissue levels of SOD and GSH-Px may reflect ROS levels.27 In addition, MDA levels can be a reliable indicator of lipid peroxidation and oxidative stress.29 Thus, ROS can be evaluated indirectly with the determination of MDA and the levels of some antioxidant enzyme activities like SOD or GSH-Px in tissue.30 Vaquero et al. showed the antioxidant effects of TQ via the reduction of diphenylpcriylhydracyl (DPPH), an oxidative radical, by TQ.31 Another study showed the protective effects of TQ in doxorubicin-induced cardio toxicity that resulted from ROS formation and oxidative stress.5 APAP-induced hepatotoxicity resulted in elevated superoxide and hydrogen peroxide levels and decreased GSH/GSSG ratio.3,32 As a consequence of impairment in antioxidant defense systems, ROS and lipid peroxidation increases. In our study, the APAP group demonstrated an increase in MDA and GSSG levels in addition to decreased GSH-Px activity. ROS attacks membrane lipids and results to lipid peroxidation. MDA is the end-product of lipid peroxidation. It serves as an index of lipid peroxidation. In our study, the increase of MDA in liver tissue and in serum has improved in the rats’ exposure to APAP as a result of TQ treatment (p 0.001). This decrease in the level of MDA suggests that TQ may be effective in the prevention of lipid
Fig. 6. Serum Glutathion peroxidase (GSH-PX) levels.
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elevated resistance against APAP-induced hepatotoxicity in transgenic animals that have excessively high GSH-Px levels.36 We suggest that TQ treatment may have a role in the regulation of antioxidant mechanisms. In conclusion, we have shown the therapeutic effect of TQ in terms of the regulation of antioxidant activity in APAP-induced hepatotoxicity. Our findings suggest that TQ may be an alternative therapeutic agent to NAC in APAP toxicity. There are requirements for future studies. Ethical approval The study protocol was approved (2011e38) by the Committee of Experimental Animals of Dicle University. Funding This work is supported by grant number 11-TF-54 from Dicle University Scientific Research Project Coordination Unit (DUBAP), Turkey.
Fig. 7. Rat liver tissues histopathological changes.
peroxidation. We assessed the effect of TQ on lipid peroxidation, which was measured in terms of MDA, a stable metabolite of the free radical-mediated lipid peroxidation cascade. Similar studies have shown the protective effect of TQ in carbon tetrachloride-induced hepatotoxicity via the protection of lipid peroxidation.8 Another interesting finding in our study is the correlated MDA levels in both APAP þ TQ and control groups. Similar MDA levels in these groups indicate the protective effect of TQ in oxidative stress. Superoxide dismutase (SOD) is the major anti-oxidant enzymereducing superoxide.33 We found that similar SOD levels in the control and TQ groups indicates that TQ has positive effects on oxidative stress. In our study, SOD levels in the APAP group increased in association with increased free radicals. An interesting finding in our study is the decreased SOD levels in the APAP þ TQ treated group, which is similar with the control group. In contrast to our study, previous studies showed a decreased SOD enzyme activity in APAP hepatotoxicity.34,35 We suggest that this finding occurred due to the decreased formation of free radicals followed by TQ treatment. Moreover, decreased GSSG levels showed decreased glutathione oxidation. The elevated enzyme activity of SOD and the reduction of GSH-Px in the APAP group indirectly showed an increase in the number of ROS in hepatic tissue. The elevated SOD enzyme activity is one of the parts of the antioxidant defense system. Oxidative stress at the first level will be encountered by increased antioxidant enzyme activities. Interestingly, statistically significant decreases in SOD enzyme activity was seen in the APAP þ TQ group when compared to the APAP group. The exact mechanism of TQ on the enzyme activities is not yet known; however, it can be speculated that TQ may affect the transcriptional and/or translational pathways of these antioxidant enzymes. Another explanation for this effect of TQ may be that it prevents the induction of the enzymes (GSH-Px) by the inhibition of toxic oxidative products. Mirochnitchenko et al. showed a remarkably
Table 3 The comparison of histopathological evaluation scores of rat liver tissue between the groups.
mean SD minemax
Group I (Control)
Group II (TQ)
Group III (APAP)
Group IV (APAP þ TQ)
0.2 0.4 0e1
0.3 0.4 0e1
3 0.6a,b 2e4
1.5 0.8a,b,c 0e3
TQ: Thymoquinone, APAP: Acetaminophene. a Significantly different when compared with control group, (p 0.001). b Significantly different when compared with TQ group, (p 0.001). c Significantly different when compared with APAP group, (p 0.001).
Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgment This work is supported by grant number 11-TF-54 from Dicle University Scientific Research Project Coordination Unit (DUBAP), Turkey. References 1. Bonkovsky HL, Kane RE, Jones DP, Galinsky RE, Banner B. Acute hepatic and renal toxicity from low doses of acetaminophen in the absence of alcohol abuse or malnutrition: evidence for increased susceptibility to drug toxicity due to cardiopulmonary and renal insufficiency. Hepatology 1994 May;19(5):1141e8. 2. Bromer MQ, Black M. Acetaminophen hepatotoxicity. Clin Liver Dis 2003 May;7(2):351e67. 3. Gibson JD, Pumford NR, Samokyszyn VM, Hinson JA. Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress. Chem Res Toxicol 1996 ApreMay;9(3):580e5. 4. Vaquero J, Belanger M, James L, et al. Mild hypothermia attenuates liver injury and improves survival in mice with acetaminophen toxicity. Gastroenterology 2007 Jan;132(1):372e83. 5. Nagi MN, Mansour MA. Protective effect of thymoquinone against doxorubicininduced cardiotoxicity in rats: a possible mechanism of protection. Pharmacol Res 2000 Mar;41(3):283e9. 6. Nagi MN, Almakki HA, Sayed-Ahmed MM, Al-Bekairi AM. Thymoquinone supplementation reverses acetaminophen-induced oxidative stress, nitric oxide production and energy decline in mice liver. Food Chem Toxicol 2010 Auge Sep;48(8e9):2361e5. 7. Kapan M, Tekin R, Onder A, et al. Thymoquinone ameliorates bacterial translocation and inflammatory response in rats with intestinal obstruction. Int J Surg 2012;10(9):484e8. 8. Kanter M, Coskun O, Budancamanak M. Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats. World J Gastroenterol 2005 Nov 14;11(42):6684e8. 9. Badary OA, Nagi MN, al-Shabanah OA, al-Sawaf HA, al-Sohaibani MO, alBekairi AM. Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity. Can J Physiol Pharmacol 1997 Dec;75(12):1356e61. 10. Kanter M, Coskun O, Uysal H. The antioxidative and antihistaminic effect of Nigella sativa and its major constituent, thymoquinone on ethanol-induced gastric mucosal damage. Arch Toxicol 2006 Apr;80(4):217e24. 11. Nili-Ahmadabadi A, Tavakoli F, Hasanzadeh G, Rahimi H, Sabzevari O. Protective effect of pretreatment with thymoquinone against Aflatoxin B(1) induced liver toxicity in mice. Daru 2011;19(4):282e7. 12. Chehl N, Chipitsyna G, Gong Q, Yeo CJ, Arafat HA. Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells. HPB Oxf 2009 Aug;11(5):373e81. 13. Peng L, Liu A, Shen Y, et al. Antitumor and anti-angiogenesis effects of thymoquinone on osteosarcoma through the NF-kappaB pathway. Oncol Rep 2013 Feb;29(2):571e8. 14. Kouidhi B, Zmantar T, Jrah H, et al. Antibacterial and resistance-modifying activities of thymoquinone against oral pathogens. Ann Clin Microbiol Antimicrob 2011;10:29.
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