Biomedical and Biotechnology Research Journal (BBRJ)

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 4  |  Issue : 1  |  Page : 69--75

Protective effect of lycopene against tamoxifen-induced hepatotoxicity in albino rats


Elias Adikwu, Nelson Clemente Ebinyo, Ozeke Benalayefa 
 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Bayelsa State, Nigeria

Correspondence Address:
Dr. Elias Adikwu
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Bayelsa State
Nigeria

Abstract

Background: Tamoxifen citrate (TAM) is a drug of choice for the treatment of hormone-dependent breast cancer, but its use has been associated with frequent hepatotoxicity. Aim: This study evaluated the protective effect of lycopene (LYP) against hepatotoxicity induced by TAM in albino rats. Methods: Adult female albino rats (200–250 g) were divided into eight groups of n = 5. Group 1 (control) received normal saline (0.2mL) intraperitoneally (i.p.), daily for 7 days. Groups 2–4 were treated daily with LYP (10 mg/kg, 20 mg/kg, and 40 mg/kg) (i.p.) for 7 days, respectively. Group 5 was treated daily with TAM (45 mg/kg) i.p. for 7 days. Groups 6–8 were supplemented daily with LYP (10 mg/kg, 20 mg/kg, and 40 mg/kg) before treatment with TAM (45 mg/kg) i.p. for 7 days, respectively. At the end of treatment, the rats were anesthetized and blood samples were collected and assessed for serum markers of liver function. Liver samples were harvested for histology and biochemical assessments. Results: Significant (P < 0.001) elevations in serum and liver aminotransferases, lactate dehydrogenase, gamma glutamyl transferase, alkaline phosphatase, total bilirubin, and conjugated bilirubin levels were observed in TAM-treated rats when compared to control. Furthermore, significant (P < 0.001) elevations in liver malondialdehyde levels with significant (P < 0.001) decreases in glutathione peroxidase, superoxide dismutase, glutathione, and catalase levels were observed in TAM-treated rats when compared to control. Vascular congestion with necrotic materials and lipoid necrosis were observed in TAM-treated rats. However, TAM-induced hepatotoxicity was significantly attenuated in a dose-dependent fashion in rats supplemented with LYP 10 mg/kg (P < 0.05), 20 mg/kg (P < 0.01), and 40 mg/kg (P < 0.001) when compared to TAM. Conclusion: LYP may have clinical application in hepatotoxicity caused by tamoxifen.



How to cite this article:
Adikwu E, Ebinyo NC, Benalayefa O. Protective effect of lycopene against tamoxifen-induced hepatotoxicity in albino rats.Biomed Biotechnol Res J 2020;4:69-75


How to cite this URL:
Adikwu E, Ebinyo NC, Benalayefa O. Protective effect of lycopene against tamoxifen-induced hepatotoxicity in albino rats. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 Sep 19 ];4:69-75
Available from: https://www.bmbtrj.org/text.asp?2020/4/1/69/280880


Full Text



 Introduction



Tamoxifen citrate (TAM) is a nonsteroidal antiestrogen that is used as an agent of choice for the prevention and treatment of hormone-dependent breast cancer. It has shown notable curative effect in estrogen-positive breast cancer patients and has reduced the risk of developing breast cancer in women.[1] It inhibits estrogen receptor, therefore preventing the supply of estrogen required for the growth of cancerous estrogen receptor-positive cells. Clinically, it has decreased recurrence and mortality rate associated with breast cancer by as much as 50%.[2] TAM is a pro drug that has a very high affinity for hepatic tissues. It is bioactivated by liver cytochrome P450 enzymes, resulting in the formation of active metabolites, including 4-hydroxy-tamoxifen and endoxifen.[3] The clinical use of TAM in breast cancer patients has shown very high incidence of liver impairment which includes hepatic steatosis, hepatitis, hepatic necrosis, cirrhosis, and hepatocellular carcinoma.[4] The mechanism by which TAM causes hepatotoxicity is not well understood, however speculations include hepatic suppression of fatty acid β-oxidation and dicarboxylic acid formation through the activation of alternative fatty acid oxidation pathway leading to mitochondria damage.[5] Mitochondrial damage can stimulate reactive oxygen species (ROS) production causing the peroxidation of fatty acids (lipid peroxidation [LPO]), leading to liver biomolecular damage.[6] In addition, the formation of TAM-DNA adducts in the liver has been associated with TAM-induced hepatotoxicity.[7]

Lycopene (LYP) is a red pigmented, cyclic, and polar carotenoid. It is present in fruits and red-colored vegetables.[8] It has essential biological activities attributed to its large array of conjugated double bonds and acyclic structure. It has antioxidant effect characterized by the scavenging of ROS which include peroxyl radicals, superoxide radicals, hydroxyl radicals, and singlet oxygen and the inhibition of thiobarbituric acid-reactive substance formation.[9],[10] Recent studies showed that it can regulate redox signaling pathways responsible for cell regulatory function such as the downregulation of excess activities of ROS-producing enzymes and can increase the expression of endogenous antioxidants. It also modulates cross cell talk, hormones, immune system, and metabolic pathways.[11] LYP has anti-inflammatory activity; it can decrease inflammation by inhibiting the production of pro-inflammatory cytokines and chemokines in macrophages.[12] Furthermore, LYP has shown potential health benefits in numerous animal models of pathologies such as cancer, diabetes, and hypertension. In addition, LYP supplementation has shown protective effects against various forms of tissue injuries in animal models caused by toxic insults.[13] The present study examined the protective effect of LYP against tamoxifen-induced hepatotoxicity in albino rats, which has not been assessed.

 Methods



Drugs and chemicals

TAM used for this study was manufactured bySun Pharmaceutical Industries Ltd, India, whereas LYP was manufactured by Puritan Pride Inc., Holbrook (NY 11741, USA).

Animals

Healthy adult female albino rats of weight 200–250 g were obtained from the animal breeding facility of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Nigeria. The rats were housed at a temperature of 27°C–30°C and allowed to acclimatize for 2 weeks with ad libitum access to food and water.

Ethical consideration

The study protocol was approved by the Research Ethics Committee of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Nigeria.

Grouping of animals and treatment

The rats were divided into eight groups (1-8) of five rats each. Group 1 (control) was treated daily with normal saline (0.2 mL) intraperitoneally (i.p.) for 7 day. Groups 2-4 were treated daily with LYP (10 mg/kg, 20 mg/ kg, and 40 mg/kg) in normal saline [14] i.p. for 7 days, respectively. Group 5 was treated daily with TAM (45 mg/kg) in normal saline [15] i.p. for 7 days. Groups 6-8 were treated daily with LYP 10 mg/kg + TAM 45 mg/kg, LYP 20 mg/kg + TAM 45 mg/kg, and LYP 40 mg/kg + TAM 45 mg/kg i.p. for 7 days respectively.

Blood sample collection

The rats were sacrificed with diethyl ether, and blood samples were collected from the heart in a plain sample containers and allowed to clot. Serum samples were extracted from the clots by centrifugation at 1200 rpm for 15 min and used for biochemical investigations.

Preparation of liver homogenate

Liver samples were excised, rinsed in cold saline and homogenized in 10% w/v ice-cold 0.1 M Tris-HCl buffer (pH = 7.4). The homogenates were centrifuged at 3000 rpm for 15 min, and the supernatants were collected and used for biochemical analyses.

Evaluation of liver function parameters

Serum and liver alkaline phosphate (ALP), conjugated bilirubin (CB), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and total bilirubin (TB) levels were measured using commercial test kits according to manufacturer's specification.

Evaluation of oxidative stress markers

Malondialdehyde (MDA) was measured using the method of Buege and Aust.[16] Reduced glutathione (GSH) was measured using the method of Sedlak and Lindsay,[17] whereas superoxide dismutase (SOD) was measured as reported by Sun and Zigma.[18] Catalase (CAT) was measured using the method of Aebi, 1984,[19] whereas GSH peroxidase (GPx) was measured according to Rotruck et al, 1973.[20]

Histopathological examination

Liver samples taken from different groups were cleaned and fixed in 10% neutral buffered formaldehyde. The samples were rinsed in ascending concentrations of ethanol, processed and embedded in paraffin at 56°C in hot air oven for 24 h. Paraffin tissue blocks were sectioned (4 μm thicknesses) with the aid of a sledge microtome. The tissue sections were mounted on glass slides, deparaffinized and stained for histological examination with hematoxylin and eosin (H and E). The slides were examined for histopathology with the aid of a light microscope.

Data analysis

Data are represented as the mean ± standard error of the mean and were analyzed using one-way analysis of variance followed by the Tukey's test. Statistical significance was set at P < 0.05, 0.01, and 0.001.

 Results



Effects on liver and serum biochemical parameters

Serum ALT, AST, ALP, ALP, GGT, LDH, CB, and TB levels were normal (P > 0.05) in rats treated with LYP when compared to control. Hepatic perturbation characterized by significant (P < 0.001) elevations in the serum activities of ALT, AST, ALP, ALP, GGT, LDH, CB, and TB were observed in TAM-treated rats when compared to control [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]. However, the activities of the aforementioned hepatic markers were significantly reduced in a dose-dependent fashion in rats treated with LYP 10 mg/kg + TAM 45 mg/kg (P < 0.05), LYP 20 mg/kg + TAM 45 mg/kg (P < 0.01), and LYP 40 mg/kg + TAM 45 mg/kg (P < 0.001), when compared to TAM-treated rats [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]. Furthermore, the liver contents of AST, ALT, ALP, GGT, and LDH were normal (P > 0.05) in LYP-treated rats when compared to control [Table 1]. In contrast, the liver activities of the aforementioned parameters were significantly (P < 0.001) elevated in TAM-treated rats when compared to control [Table 1]. However, significant decreases in AST, ALT, ALP, GGT, and LDH activities in a dose-dependent fashion occurred in rats treated with LYP 10 mg/kg + TAM 45 mg/kg (P < 0.05), LYP 20 mg/kg + TAM 45 mg/kg (P < 0.01), and LYP 40 mg/kg + TAM 45 mg/kg (P < 0.001), when compared to TAM-treated rats [Table 1].{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}{Figure 7}{Table 1}

Effects on liver oxidative stress markers and histology

The liver activities of CAT, SOD, GSH, GPx, and MDA were normal (P > 0.05) in rats treated with LYP when compared to control [Table 2]. Impairments in antioxidant activities characterized by significant (P < 0.001) decreases in liver CAT, SOD, GSH, and GPx levels with significant (P < 0.001) increases in MDA levels were observed in TAM-treated rats when compared to control [Table 2]. However, the activities of CAT, SOD, GSH, and GPx were significantly increased, whereas the activities of MDA were significantly decreased in a dose-dependent fashion in rats treated with LYP 10 mg/kg + TAM 45 mg/kg (P < 0.05), LYP 20 mg/kg + TAM 45 mg/kg (P < 0.01), and LYP 40 mg/kg + TAM 45 mg/kg (P < 0.001), when compared to TAM-treated rats [Table 2]. Furthermore, the liver of control rat showed normal hepatocytes [Figure 8]a. In contrast, vascular congestion, necrotic materials [Figure 8]b, and lipoid necrosis [Figure 8]c were observed in the liver of TAM-treated rats. However, the liver of rats treated with LYP 10 mg/kg + TAM 45 mg/kg showed lipoid cells [Figure 8]d. Furthermore, the liver of rats treated with LYP 20 mg/kg + TAM 45 mg/kg (P < 0.01) and LYP 40 mg/kg + TAM 45 mg/kg (P < 0.001) showed inflammatory cell infiltrations [Figure 8]e and [Figure 8]f.{Table 2}{Figure 8}

 Discussion



TAM has been the most widely used hormonal therapy for breast cancer in women for more than two decades. It has successfully decreased mortality associated with hormone-dependent breast cancer in women to an appreciable level.[21] Nonetheless, the successful impact of TAM on the fight against breast cancer has been tainted by the frequent occurrence of hepatotoxicity, which may require dose adjustment or complete withdrawal.[22] There is an acute lack of effective drugs that can protect the liver from damage or help regenerate hepatic cells with the advent of hepatotoxicity caused by TAM.[23] This study assessed the ability of LYP to protect against TAM-induced alterations in liver function and structure in a model of rat. The magnitude of liver damage in the experimental groups was assessed by estimating serum and liver ALT, CB, AST, TB ALP, LDH, and GGT levels. The serum levels of the aforementioned parameters are clinically used to ascertain the physiologic status of the liver. The activities of these parameters can be upregulated beyond the acceptable benchmark with the advent of hepatic assaults by chemical substances. Normal levels of ALT, CB, AST, TB ALP, LDH, and GGT were observed in rats treated with LYP. In contrast, the serum levels of the aforementioned parameters were upregulated in TAM-treated rats. This observation is a vivid sign of hepatic perturbation, which is in agreement with previous reports.[24] TAM might have damaged liver parenchyma, leading to the release of ALT, AST and ALP, GGT, and LDH into the blood. Furthermore, it might have decreased the ability of the liver to excrete normal amount of bilirubin or its excretory ducts obstructed.[25],[26] Nonetheless, LYP supplementation restored the activities of serum and liver ALT, AST, ALP, GGT, LDH, CB, and TB in a dose-dependent fashion. This shows that LYP has hepatoprotective activity, probably by healing hepatic parenchyma and regenerating hepatocytes.

SOD, CAT, GSH, and GPx are antioxidants that safeguard cells from the nefarious oxidative activities of ROS. GSH in collaboration with GPx and GST collectively establish a network for the termination of the actions of lipid peroxides. CAT and SOD are anti-peroxidative enzymes, which collectively safeguard cells against the detrimental activities of superoxide radicals and hydrogen peroxide. The hepatic antioxidant activities of SOD, CAT, GSH, and GPx can easily be overwhelmed and decreased by the excess activities of ROS, which may render hepatocytes vulnerable to toxic insults.[27] The present study observed normal liver SOD, CAT, GSH, and GPx levels in LYP-treated rats. On the other hand, impairments in liver antioxidant activities marked by decreases in SOD, CAT, GSH, and GPx levels were observed in TAM-treated rats. This observation has been previously reported, and it lays credence to the involvement of oxidative stress in TAM-induced hepatotoxicity.[28] Nonetheless, LYP supplementation prior to treatment with TAM increased the hepatic activities of SOD, CAT, GSH, and GPx in a dose-dependent fashion.

LPO is a consequence of ROS-mediated degradation of polyunsaturated fatty acid culminating in the production of injurious by-products. MDA is a by-product of LPO; its concentration gives a vivid picture of the occurrence and magnitude of LPO in tissues.[29] This study observed normal liver MDA activity in LYP-treated rats. In contrast, the activity of MDA was upregulated in the liver of TAM-treated rats, which is consistent with previous report.[30] Studies have shown that lipids are responsible for maintaining the integrity of cellular membranes. TAM might have caused LPO leading to the production of lipid radicals, thereby impairing assembly, composition, structure, and dynamics of hepatocyte lipid membrane.[31]

Furthermore, this study assessed hepatic morphology in the control and the experimental groups. Normal hepatocytes were observed in rats treated with LYP. In contrast, vascular congestion with necrotic materials and lipoid necrosis were observed in TAM-treated rats. TAM-induced hepatotoxicity has been characterized by the accumulation of hepatocyte lipids, especially triglycerides, which are formed from the esterification of free fatty acids and glycerol.[32] However, the observed liver morphologic changes in TAM-treated rats were ameliorated in LYP-supplemented rats. The mechanisms by which TAM causes hepatotoxicity have been proposed to include ROS-induced OS, mitochondrial dysfunction, and the peroxidation of fatty acid. In addition, the induction of inflammation through the production of inflammatory cytokines such as tumor necrosis factor-α and interleukin 6 has been speculated.[33] The downregulation of the hepatotoxic effect of TAM by LYP observed in this study might have occurred through the antioxidant and anti-inflammatory activities of LYP. LYP is an antioxidant that scavenges free radicals, inhibits OS, reduces LPO, and enhances endogenous antioxidant functions.[34] It might have reacted with peroxy radicals, produced in the propagation phase of LPO to form carbon-centered radical which carry radicals, but are stable than ROS, thus preventing LPO.[35] Furthermore, LYP might have stabilized and strengthened cellular membrane, thereby inhibiting enzyme leakage from the liver and preserved its function in protein biosynthesis.[36]

 Conclusion



LYP may be useful in preventing or treating hepatotoxicity caused by tamoxifen.

Acknowledgments

The authors appreciate the support of Mr. Cosmos Obi of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Nigeria, for animal handling.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Kramer R, Brown P. Should tamoxifen be used in breast cancer prevention? Drug Saf 2004;27:979-89.
2Klein DJ, Thorn CF, Desta Z, Flockhart DA, Altman RB, Klein TE. PharmGKB summary: Tamoxifen pathway, pharmacokinetics. Pharmacogenet Genomics 2013;23:643-7.
3de Vries Schultink AH, Zwart W, Linn SC, Beijnen JH, Huitema AD. Effects of Pharmacogenetics on the Pharmacokinetics and Pharmacodynamics of Tamoxifen. Clin Pharmacokinet 2015;54:797-810.
4Nishino M, Hayakawa K, Nakamura Y, Morimoto T, Mukaihara S. Effects of tamoxifen on hepatic fat content and the development of hepatic steatosis in patients with breast cancer: High frequency of involvement and rapid reversal after completion of tamoxifen therapy. AJR Am J Roentgenol 2003;180:129-34.
5Miyamura M, Yokota J, Saibara T. Drug-induced nonalcoholic steatohepatitis. Yakugaku Zasshi 2016;136:579-82.
6Miele L, Liguori A, Marrone G, Biolato M, Araneo C, Vaccaro FG, et al. Fatty liver and drugs: The two sides of the same coin. Eur Rev Med Pharmacol Sci 2017;21:86-94.
7Hellmann-Blumberg U, Taras TL, Wurz GT, DeGregorio MW. Genotoxic effects of the novel mixed antiestrogen FC-1271a in comparison to tamoxifen and toremifene. Breast Cancer Res Treat 2000;60:63-70.
8Jiang W, Guo MH, Hai X. Hepatoprotective and antioxidant effects of lycopene on non-alcoholic fatty liver disease in rat. World J Gastroenterol 2016;22:10180-8.
9Stahl W, Sies H. Carotenoids and flavonoids contribute to nutritional protection against skin damage from sunlight. Mol Biotechnol 2007;37:26-30.
10Pirayesh Islamian J, Mehrali H. Lycopene as a carotenoid provides radioprotectant and antioxidant effects by quenching radiation-induced free radical singlet oxygen: An overview. Cell J 2015;16:386-91.
11Palozza P, Catalano A, Simone R, Cittadini A. Lycopene as a guardian of redox signalling. Acta Biochim Pol 2012;59:21-5.
12Marcotorchino J, Romier B, Gouranton E, Riollet C, Gleize B, Malezet-Desmoulins C, et al. Lycopene attenuates LPS-induced TNF-α secretion in macrophages and inflammatory markers in adipocytes exposed to macrophage-conditioned media. Mol Nutr Food Res 2012;56:725-32.
13Story EN, Kopec RE, Schwartz SJ, Harris GK. An update on the health effects of tomato lycopene. Annu Rev Food Sci Technol 2010;1:189-210.
14Cavusoglu K, Oruc E, Yapar K, Yalcin E. Protective effect of lycopene against mercury-induced cytotoxicity in albino mice: Pathological evaluation. J Environ Biol 2009;30:807-14.
15Albukhari AA, Gashlan HM, El-Beshbishy HA, Nagy AA, Abdel-Naim AB. Caffeic acid phenethyl ester protects against tamoxifen-induced hepatotoxicity in rats. Food Chem Toxicol 2009;47:1689-95.
16Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-10.
17Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968;25:192-205.
18Sun M, Zigman S. An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation. Anal Biochem 1978;90:81-9.
19Aebi H. Catalase in vitro. In: Colowick SP, Kaplane NO, editors. Method in Enzymology. New York, USA: Academic Press; 1984.
20Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179:588-90.
21Moerkens M, Zhang Y, Wester L, van de Water B, Meerman JH. Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation. BMC Cancer 2014;14:283.
22Suddek GM. Protective role of thymoquinone against liver damage induced by tamoxifen in female rats. Can J Physiol Pharmacol 2014;92:640-4.
23Wakawa HY, Musa H. Protective effect of Erythrina senegalensis (DC) leaf extract on CCl4-induced liver damage in rats. Asian J Bio Sci 2013;6:234-8.
24Sorour SM. Cardamonin attenuates tamoxifen induced hepatotoxicity in rats through modulation of inflammatory mediators oxidative stress and apoptosis. Egypt J Basic Clin Pharm 2017;7:56-9.
25Jain R, Nandakumar K, Srivastava1 V, Vaidya SK, Patet S, Kumar P. Hepatoprotective activity of ethanolic and aqueous extract of Terminalia belerica in rats. Pharmacologyonline 200;2:411-27.
26Gaw A, Cowan RA, O'Reilly DS, Stewart MJ, Shepherd J. Clinical Biochemistry an Illustrated Colour Text. Edinburgh: Harcourt Brace; 1999. p. 165.
27Sheik Abdulazeez S, Thiruvengadam D. Effect of lycopene on oxidative stress induced during D-galactosamine/lipopolysaccharide-sensitized liver injury in rats. Pharm Biol 2013;51:1592-9.
28El-Beshbishy HA, Mohamadin AM, Nagy AA, Abdel-Naim AB. Amelioration of tamoxifen-induced liver injury in rats by grape seed extract, black seed extract and curcumin. Indian J Exp Biol 2010;48:280-8.
29Comporti M. Lipid peroxidation and cellular damage in toxic liver injury. Lab Invest 1985;53:599-623.
30Mahboub FA. The effect of green tea (Camellia sinensis) Extract against Hepato-toxicity induced by tamoxifen in rats. J Food Process Technol 2016;10:1-5.
31Gaschler MM, Stockwell BR. Lipid peroxidation in cell death. Biochem Biophys Res Commun 2017;482:419-25.
32Zhao F, Xie P, Jiang J, Zhang L, An W, Zhan Y. The effect and mechanism of tamoxifen-induced hepatocyte steatosis in vitro. Int J Mol Sci 2014;15:4019-30.
33Hassanein NA, Ali AA, El-Den, El-Khawaga AM. Methylseleninic acid and grape seed extract alleviate tamoxifen induce hepatotoxicity in rats. Toxicol and Environ Health Sci 2018;10:278-87.
34Rao AV, Rao LG. Carotenoids and human health. Pharmacol Res 2007;55:207-16.
35Aggarwal S, Singh K, Nagpal M, Kaur A, Ahluwalia P. Studies on the effect of lycored supplementation (lycopene) on lipid peroxidation and reduced glutathione in pregnancy induced hypertensive patients. Biomed Res 2009;20:51-4.
36Abdel-Rahman HG, Abdelrazek HA, Zeidan DW, Mohamed RM, Abdelazim AM. Lycopene: Hepatoprotective and antioxidant effects toward bisphenol a-induced toxicity in female wistar rats. Oxid Med Cell Longev 2018;2018:8.