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Page 1 of 21
Protective role of thymoquinone against liver damage induced by tamoxifen in female rats
Ghada M. Suddek* Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
*
Corresponding author
Address: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. Postal code: 35516 Tel.: 0020101266745 Fax: 0020502247496 E-mail address: [email protected]
- 1-
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Abstract
One of the major causes of clinical trial termination is the liver toxicity induced by chemotherapy. Tamoxifen (TAM) is an antiestrogen used in treatment and prevention of hormone-dependent breast cancer. Tamoxifen therapy may be accompanied with hepatic injury. Thymoquinone (TQ), an active principle of Nigella sativa seed, has been used in folk medicine for diverse ailments. It is reported to possess anticancer and hepatoprotective effects. In this study, the protective effects of TQ against TAM-induced hepatotoxicity in female rats were evaluated. Four groups of rats were used: control; TAM; TQ; TAM+TQ. TAM (45 mg/kg/day, i.p., for 10 consecutive days) resulted in elevation of serum ALT, AST, ALP, LDH, total bilirubin and γGT, depletion of liver GSH and accumulation of LPO. Also, TAM treatment resulted in inhibition of hepatic SOD activity. Further, it raised liver TNF-α level and induced histopathological changes. Pretreatment with TO (50 mg/kg/day; p.o., for 20 consecutive days, starting 10 days before TAM injection) significantly prevented elevation in serum activity of the assessed enzymes. TQ significantly inhibited TAM-induced hepatic GSH depletion and LPO accumulation. Consistently, TQ normalized the activity of SOD, inhibited the rise in TNF-α and ameliorated the histopathological changes. In conclusion, TQ protects against TAM-induced hepatotoxicity.
- 2-
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Key words: Tamoxifen, thymoquinone, antioxidant, anti-inflammatory, rats, liver damage.
Introduction Thymoquinone, 2-isopropyl-5-methyl-1, 4-benzoquinone, is the major active component of N. sativa and constitutes about 30% of its seed extract (Houghton et al. 1995). It has been demonstrated that the N. sativa and its chemical components produce a variety of pharmacological actions such as anticancer, anti-inflammatory and antioxidant properties and many of these effects are due to TQ (Ali and Blunden 2003; Valizadeh et al. 2010). The beneficial medical effects of N. sativa and TQ have been related to their radical scavenging activities (Burits and Bucar 2000). Tamoxifen citrate, 2-[4-[(Z)-1,2Diphenylbut-1-enyl] phenoxy]-N,N dimethylethanamine, is a non-steroidal antiestrogen used for the treatment of breast cancer and, more recently, as a chemopreventive agent in healthy women at high risk of developing breast cancer (Dunn et al. 2005; Jordan 2003). On the other hand, TAM is a potent hepatocarcinogen in rats, with both tumor-initiating and tumor-promoting properties (Ahotupa et al. 1994; Caballero et al. 2001; Dragan et al. 1996; Williams et al. 1997) which is due to oxygen radical overproduction that occurs during TAM metabolism. Imaging studies documented the development of fatty liver in approximately 33% of patients who took TAM (Nagaie et al. 2005). More sophisticated reports explored the relation between elevated transaminases
- 3-
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Page 4 of 21
and the diagnosis of steatohepatitis in patients with breast cancer (Liu et al. 2006) and the effects of TAM in patients with pre-existing steatohepatitis (Elefsiniotis et al. 2004).This hepatotoxic effect of TAM raises issues bearing on the prophylactic chronic administration to healthy women. The present study aimed at studying the impact of TQ supplementation on TAM-induced liver toxicity.
Materials and methods Animals Adult female Sprague-Dawley rats weighing 150-200 g were used. They were obtained from Urology and Nephrology Center of Mansoura University, Mansoura, Egypt. The animals were maintained under standard conditions of temperature 24 ± 1°C and 55 ± 5% relative humidity with regular 12 h light/12 h dark cycles. They were allowed free access to standard laboratory food and water. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and were approved by the Ethical Committee of Faculty of Pharmacy, Mansoura University, Egypt.
Drugs and chemicals Tamoxifen citrate was obtained as pharmaceutical drug (Nolvadex). Thymoquinone was obtained from (Sigma Chemical Company, St. Louis, MO,
- 4-
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Page 5 of 21
USA).
Ellman’s
reagent,
pyrogallol,
reduced
glutathione,
1,1,3,3-
tetramethoxypropane and tris (hydroxymethyl) amino-methane were purchased from Sigma Aldrich Chemical Co (St. Louis, MO, USA). n-Butanol was purchased from El-Nasr Chemical Co. (Abou-Zaabal, Cairo, Egypt). Thiobarbituric acid (TBA) was purchased from Fluka (Chemie, Switzerland). Trichloroacetic acid (TCA) was purchased from Winlab (Leicestershire, UK). All other chemicals used in this study were of high purity and purchased locally.
Experimental protocol The animals were divided at random into four groups of 6 rats each (Fig. 1). The first group (normal control). The second group, TAM intoxicated rats: Rats were treated with TAM at a dose of 45 mg/kg/day in 0.1 ml dimethylsulfoxide (DMSO) and normal saline, i.p., for 10 successive days (Albukhari et al., 2009). The third group, TQ-treated rats: Rats were orally administered 50 mg TQ/kg body weight in 0.5% carboxymethyl cellulose (CMC) daily for 20 successive days. The fourth group, TAM-TQ rats: Rats were orally administered 50 mg TQ/kg body weight daily for 10 successive days before and 10 days with TAM intoxication. At the end of the experiment, animals were subjected to light ether anaesthesia and blood samples were collected in centrifuge tubes and centrifuged to obtain serum. Animals were killed by cervical dislocation, the abdomen was excised and the liver was removed immediately by dissection, washed in ice-cold isotonic saline and blotted between two filter papers. A 10% - 5-
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(w/v) liver homogenate was prepared in ice-cold 0.1 M potassium phosphate buffer, pH 7.
Estimation of liver injury Serum was used to assess hepatic profile enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (γGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) according to the standard procedures given along with the kits purchased. Serum total bilirubin was also determined. Kits from BioMed-Diagnostics, Egy-Chem Co., Egypt were used. Fresh aliquots from liver homogenate were used to estimate lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and tumor necrosis factor-alpha (TNF-α).
Estimation of antioxidant profile LPO was measured as malondialdehyde (MDA), the end product of lipid peroxidation, according to the method of Ohkawa et al. (1979). MDA solution (made freshly by the hydrolysis of 1,1,3,3-tetramethoxypropane) was used as the standard. The absorbance was determined at 532 nm spectrophotometrically and the concentrations were expressed as nmol/g wet tissue. The level of acidsoluble thiols, mainly GSH, in the liver was assayed colorimetrically, based on its reaction with Ellman’s reagent [5,5′ -dithio-bis(2-nitrobenzoic acid)] according to the method earlier described by Ellman (1959) after protein precipitation with TCA. The absorbance was measured at 412 nm and the concentrations were expressed as µmol/g wet tissue. The enzymatic activity of - 6-
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SOD was assessed according to Marklund (1985). SOD activity was expressed as U/g wet tissue. One unit of SOD activity is defined as the amount of the enzyme causing 50% inhibition of auto-oxidation of pyrogallol.
Determination of TNF-α Enzyme-linked immunosorbent assays (ELISAs) were used to detect TNF-α level in rat liver homogenate according to the manufacturer’s manual (Quantikine R&D system Inc, Minneapolis, MN). This assay employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for rat TNF-α has been pre-coated onto a microplate. Standards, controls, and samples are pipetted into the wells and any rat TNF-α present is bound by the immobilized antibody. A biotinylated polyclonal antibody specific for rat to TNF-α is added which binds to rat TNF-α captured by the first antibody. Following incubation, unbound biotinylated TNF-α antibody is removed during a wash step. Streptavidin conjugated to horseradish peroxidase (HRP) is added and binds to biotin-conjugated anti-rat TNF-α antibody. Following incubation, unbound Streptavidin-HRP is removed during a wash step. A substrate solution reactive with HRP is added to the wells. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the color measured is proportional to the amount of rat TNF-α bound in the initial step. TNF-α concentration was calculated from standard curve and expressed as pg/g of liver.
- 7-
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Histopathologic examination Livers from all rats were rinsed with ice-cold saline, and a small cross section of the liver was obtained. Liver specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (4 to 5 µm) and stained with hematoxylin and eosin (H&E). The tissues were examined under a microscope in a random order and without knowledge of animal or group.
Statistical Analysis Data are expressed as mean ± standard deviation (SD). (Significance was calculated at p
Page 1 of 21
Protective role of thymoquinone against liver damage induced by tamoxifen in female rats
Ghada M. Suddek* Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
*
Corresponding author
Address: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. Postal code: 35516 Tel.: 0020101266745 Fax: 0020502247496 E-mail address: [email protected]
- 1-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 06/01/14 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.
Page 2 of 21
Abstract
One of the major causes of clinical trial termination is the liver toxicity induced by chemotherapy. Tamoxifen (TAM) is an antiestrogen used in treatment and prevention of hormone-dependent breast cancer. Tamoxifen therapy may be accompanied with hepatic injury. Thymoquinone (TQ), an active principle of Nigella sativa seed, has been used in folk medicine for diverse ailments. It is reported to possess anticancer and hepatoprotective effects. In this study, the protective effects of TQ against TAM-induced hepatotoxicity in female rats were evaluated. Four groups of rats were used: control; TAM; TQ; TAM+TQ. TAM (45 mg/kg/day, i.p., for 10 consecutive days) resulted in elevation of serum ALT, AST, ALP, LDH, total bilirubin and γGT, depletion of liver GSH and accumulation of LPO. Also, TAM treatment resulted in inhibition of hepatic SOD activity. Further, it raised liver TNF-α level and induced histopathological changes. Pretreatment with TO (50 mg/kg/day; p.o., for 20 consecutive days, starting 10 days before TAM injection) significantly prevented elevation in serum activity of the assessed enzymes. TQ significantly inhibited TAM-induced hepatic GSH depletion and LPO accumulation. Consistently, TQ normalized the activity of SOD, inhibited the rise in TNF-α and ameliorated the histopathological changes. In conclusion, TQ protects against TAM-induced hepatotoxicity.
- 2-
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Key words: Tamoxifen, thymoquinone, antioxidant, anti-inflammatory, rats, liver damage.
Introduction Thymoquinone, 2-isopropyl-5-methyl-1, 4-benzoquinone, is the major active component of N. sativa and constitutes about 30% of its seed extract (Houghton et al. 1995). It has been demonstrated that the N. sativa and its chemical components produce a variety of pharmacological actions such as anticancer, anti-inflammatory and antioxidant properties and many of these effects are due to TQ (Ali and Blunden 2003; Valizadeh et al. 2010). The beneficial medical effects of N. sativa and TQ have been related to their radical scavenging activities (Burits and Bucar 2000). Tamoxifen citrate, 2-[4-[(Z)-1,2Diphenylbut-1-enyl] phenoxy]-N,N dimethylethanamine, is a non-steroidal antiestrogen used for the treatment of breast cancer and, more recently, as a chemopreventive agent in healthy women at high risk of developing breast cancer (Dunn et al. 2005; Jordan 2003). On the other hand, TAM is a potent hepatocarcinogen in rats, with both tumor-initiating and tumor-promoting properties (Ahotupa et al. 1994; Caballero et al. 2001; Dragan et al. 1996; Williams et al. 1997) which is due to oxygen radical overproduction that occurs during TAM metabolism. Imaging studies documented the development of fatty liver in approximately 33% of patients who took TAM (Nagaie et al. 2005). More sophisticated reports explored the relation between elevated transaminases
- 3-
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Page 4 of 21
and the diagnosis of steatohepatitis in patients with breast cancer (Liu et al. 2006) and the effects of TAM in patients with pre-existing steatohepatitis (Elefsiniotis et al. 2004).This hepatotoxic effect of TAM raises issues bearing on the prophylactic chronic administration to healthy women. The present study aimed at studying the impact of TQ supplementation on TAM-induced liver toxicity.
Materials and methods Animals Adult female Sprague-Dawley rats weighing 150-200 g were used. They were obtained from Urology and Nephrology Center of Mansoura University, Mansoura, Egypt. The animals were maintained under standard conditions of temperature 24 ± 1°C and 55 ± 5% relative humidity with regular 12 h light/12 h dark cycles. They were allowed free access to standard laboratory food and water. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and were approved by the Ethical Committee of Faculty of Pharmacy, Mansoura University, Egypt.
Drugs and chemicals Tamoxifen citrate was obtained as pharmaceutical drug (Nolvadex). Thymoquinone was obtained from (Sigma Chemical Company, St. Louis, MO,
- 4-
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Page 5 of 21
USA).
Ellman’s
reagent,
pyrogallol,
reduced
glutathione,
1,1,3,3-
tetramethoxypropane and tris (hydroxymethyl) amino-methane were purchased from Sigma Aldrich Chemical Co (St. Louis, MO, USA). n-Butanol was purchased from El-Nasr Chemical Co. (Abou-Zaabal, Cairo, Egypt). Thiobarbituric acid (TBA) was purchased from Fluka (Chemie, Switzerland). Trichloroacetic acid (TCA) was purchased from Winlab (Leicestershire, UK). All other chemicals used in this study were of high purity and purchased locally.
Experimental protocol The animals were divided at random into four groups of 6 rats each (Fig. 1). The first group (normal control). The second group, TAM intoxicated rats: Rats were treated with TAM at a dose of 45 mg/kg/day in 0.1 ml dimethylsulfoxide (DMSO) and normal saline, i.p., for 10 successive days (Albukhari et al., 2009). The third group, TQ-treated rats: Rats were orally administered 50 mg TQ/kg body weight in 0.5% carboxymethyl cellulose (CMC) daily for 20 successive days. The fourth group, TAM-TQ rats: Rats were orally administered 50 mg TQ/kg body weight daily for 10 successive days before and 10 days with TAM intoxication. At the end of the experiment, animals were subjected to light ether anaesthesia and blood samples were collected in centrifuge tubes and centrifuged to obtain serum. Animals were killed by cervical dislocation, the abdomen was excised and the liver was removed immediately by dissection, washed in ice-cold isotonic saline and blotted between two filter papers. A 10% - 5-
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Page 6 of 21
(w/v) liver homogenate was prepared in ice-cold 0.1 M potassium phosphate buffer, pH 7.
Estimation of liver injury Serum was used to assess hepatic profile enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (γGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) according to the standard procedures given along with the kits purchased. Serum total bilirubin was also determined. Kits from BioMed-Diagnostics, Egy-Chem Co., Egypt were used. Fresh aliquots from liver homogenate were used to estimate lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and tumor necrosis factor-alpha (TNF-α).
Estimation of antioxidant profile LPO was measured as malondialdehyde (MDA), the end product of lipid peroxidation, according to the method of Ohkawa et al. (1979). MDA solution (made freshly by the hydrolysis of 1,1,3,3-tetramethoxypropane) was used as the standard. The absorbance was determined at 532 nm spectrophotometrically and the concentrations were expressed as nmol/g wet tissue. The level of acidsoluble thiols, mainly GSH, in the liver was assayed colorimetrically, based on its reaction with Ellman’s reagent [5,5′ -dithio-bis(2-nitrobenzoic acid)] according to the method earlier described by Ellman (1959) after protein precipitation with TCA. The absorbance was measured at 412 nm and the concentrations were expressed as µmol/g wet tissue. The enzymatic activity of - 6-
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SOD was assessed according to Marklund (1985). SOD activity was expressed as U/g wet tissue. One unit of SOD activity is defined as the amount of the enzyme causing 50% inhibition of auto-oxidation of pyrogallol.
Determination of TNF-α Enzyme-linked immunosorbent assays (ELISAs) were used to detect TNF-α level in rat liver homogenate according to the manufacturer’s manual (Quantikine R&D system Inc, Minneapolis, MN). This assay employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for rat TNF-α has been pre-coated onto a microplate. Standards, controls, and samples are pipetted into the wells and any rat TNF-α present is bound by the immobilized antibody. A biotinylated polyclonal antibody specific for rat to TNF-α is added which binds to rat TNF-α captured by the first antibody. Following incubation, unbound biotinylated TNF-α antibody is removed during a wash step. Streptavidin conjugated to horseradish peroxidase (HRP) is added and binds to biotin-conjugated anti-rat TNF-α antibody. Following incubation, unbound Streptavidin-HRP is removed during a wash step. A substrate solution reactive with HRP is added to the wells. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the color measured is proportional to the amount of rat TNF-α bound in the initial step. TNF-α concentration was calculated from standard curve and expressed as pg/g of liver.
- 7-
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Histopathologic examination Livers from all rats were rinsed with ice-cold saline, and a small cross section of the liver was obtained. Liver specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (4 to 5 µm) and stained with hematoxylin and eosin (H&E). The tissues were examined under a microscope in a random order and without knowledge of animal or group.
Statistical Analysis Data are expressed as mean ± standard deviation (SD). (Significance was calculated at p
