RESEARCH ARTICLE
Melatonin Reduces Oxidative Stress and Restores Mitochondrial Function in the Liver of Rats Exposed to Chemotherapeutics P. MADHU1,2, K. PRATAP REDDY1,2, 2 AND P. SREENIVASULA REDDY * 1
Department of Biotechnology, Sri Venkateswara University, Tirupati, India Department of Zoology, Sri Venkateswara University, Tirupati, India
2
ABSTRACT
This study was undertaken to investigate whether administration of melatonin protects PVBInduced oxidative and metabolic toxicity in the liver of Wistar rats. Adult male Wistar rats were intraperitoneally injected with either melatonin or PVB (cisplatin, vinblastine, and bleomycin) alone or combination for a period of 9 weeks. A significant increase in lipid peroxidation levels and decrease in catalase and superoxide dismutase activity levels were observed in the liver mitochondria of rats treated with PVB indicating increased oxidative stress. PVB treatment significantly decreased the succinate dehydrogenase activity with a significant increase in lactate dehydrogenase, glucose-6-phosphate dehydrogenase, aspartate aminotransaminase, alanine aminotransaminase, and glutamate dehydrogenase activities indicating deranged hepatic metabolism. Melatonin administration, on the other hand was found to significantly improve PVB-Induced biochemical changes, bringing them closer to the controls. The results from the study provide evidence that treatment with PVB affects hepatic metabolism in rats by inducing oxidative stress followed by decreasing mitochondrial oxidation and also point towards the clinical potential of melatonin as an adjuvant therapy to conventional chemotherapeutic regimens. J. Exp. Zool. 323A:301–308, 2015. © 2015 Wiley Periodicals, Inc.
J. Exp. Zool. 323A:301–308, 2015
How to cite this article: MADHU P, REDDY KP, REDDY PS. 2015. Melatonin reduces oxidative stress and restores mitochondrial function in the liver of rats exposed to chemotherapeutics. J. Exp. Zool. 323A:301–308.
INTRODUCTION Incidence of cancer has been on rise all over the world. According to American Cancer Society (2014), most common cancers among adolescents are Hodgkin’s lymphoma (15%), thyroid carcinoma (11%), brain (10%), and testicular germ cell tumors (8%). The most commonly used chemotherapeutics are doxorubicin, bleomycin, vinblastine, and dacarbazine for Hodgkin’s lymphoma (Meyer et al., 2012), cabozantinib for thyroid carcinoma (Traynor, 2013), and temozolomide for brain cancer (Pouratian et al., 2007). In case of testicular germ cell tumors, combinational therapy of cisplatin (P), bleomycin (B), and vinblastine (V) or etoposide (E) is commonly used chemotherapeutic regimen. The prognosis of testicular cancer has improved considerably in the past three decades; hence approximately 70–80% of patients with meta-
static disease will achieve a durable complete remission after 3–4 cycles of a combination therapy with PVB followed by secondary surgery. The improved prognosis of testicular cancer has brought
Conflicts of interest: None. Correspondence to: P. Sreenivasula Reddy, Department of Zoology, S. V. University, Tirupati-517502, India. E-mail: [email protected] Received 15 October 2014; Revised 16 December 2014; Accepted 6 January 2015 DOI: 10.1002/jez.1917 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
© 2015 WILEY PERIODICALS, INC.
302 the long-term toxicity of the treatment and life-quality after treatment into focus. Unfortunately, therapeutic potential of these drugs is limited by its ototoxicity, neurotoxicity, nephrotoxicity, and hepatotoxicity (Church et al., 2004; Steeg and Theodorescu, 2008; Ciftci et al., 2011). Earlier, it has been reported that simultaneous administration of some antiemetic drugs exaggerated the hepatic toxicity induced by cisplatin (Lu and Cederbaum, 2006). Thus ameliorating chemotherapeutic drug(s) toxicity on non-target tissues is an important health-related goal. The liver is an important detoxifying organ that decomposes endogenous and exogenous substances; therefore, liver diseases are considered a serious health problem (Yang et al., 2008). PVB activates hepatic microsomal cytochrome P450, which has a high capacity to generate ROS, resulting in oxidative stress, including changes in redox state, mitochondrial damage, and decreased antioxidant enzyme levels (Partibha et al., 2006). Under abnormal conditions, generation of excess ROS damages cellular lipids, proteins, or DNA, and finally leading to cell injury (Wong-ekkabut et al., 2007). These toxic side effects adversely affect the patient’s quality of life. Hydrogen peroxide, superoxide anion, and hydroxyl radicals are thought to be produced during oxidative stress. Superoxide dismutase, catalase, and glutathione peroxidase are endogenous antioxidant enzymes which play a key role in prevention of oxidative injury (Movahed et al., 2012). Numerous studies have reported that natural antioxidants are effective against oxidative stress-related pathology by increasing intracellular antioxidant enzyme activities (Giriwono et al., 2010; Lee et al., 2010; Yeh et al., 2012). Chronic low doses of cisplatin exposure may induce hepatotoxicity due to drug accumulation in liver (Uozumi et al., '93). One mechanism of cisplatin’s antitumor action involves its binding to DNA, resulting in a cascade of events that culminate in cell death. Another mechanism of cisplatin’s toxicity involves the induction of reactive oxygen species (ROS) and the possible depletion of antioxidants (Jordan and Carmo-Fonseca, 2000; Koc et al., 2005) resulting in mitochondrial dysfunction (Saad et al., 2004). Bleomycin a glycopeptide antineoplastic drug can induce breaks in single and double stranded DNA. Bleomycin is often used as combination chemotherapy for lymphoma and testicular carcinoma in humans and causes morbidity and mortality (de Haas et al., 2008). Vinca alkaloids and adriamycin are frequently used for the haematological malignancies. Since vinblastine is generally metabolized by the liver, dosage adjustments in patients with hepatic dysfunction is essential to control the drug accumulation (Zhou-Pan et al., '93; Benjamin et al., '77). Melatonin is the principal secretary product produced by pineal gland. Melatonin is present ubiquitously, including bacteria, invertebrates, and vertebrates (Reiter, '91). It is now well established that melatonin is not only identified in the pineal J. Exp. Zool.
MADHU ET AL. gland, but also in many other organs and tissues including retina, skin, Harderian glands, gut, ovary, testes, bone marrow, lens, intestine, salivary glands, and blood platelets (Tan et al., 2014). Melatonin mainly involves in the regulation of circadian rhythms, seasonal reproduction, and inhibition of cancer initiation (Karbownik and Reiter, 2000). Melatonin plays an important role in scavenging free radicals and stimulating antioxidant enzymes (Reiter et al., 2003; Rodriguez et al., 2004). Several reports have shown that melatonin protects DNA, lipids, and proteins against the harmful effects of free radicals (Reiter et al., 2000). In normal cells melatonin inhibits apoptotic processes whereas in cancer cells it could promote apoptosis (Blask et al., 2002). Melatonin also acts as oncostatic molecule (Dragojevic et al., 2014). The purpose of this study was to ascertain the influence that melatonin exert on the hepatotoxicity caused by PVB in rats.
MATERIALS AND METHODS Procurement and Maintenance of Experimental Animals Three month old male Wistar strain albino rats (body weight 190 20 g) were obtained from an authorized vendor (M/S Raghavendra Enterprises, Bengaluru, India). The animals were housed (four per cage) in sterile polypropylene cages, under standard laboratory conditions (temperature 24 2°C; light and dark 12:12 h). The rats were allowed free access to a standard pellet chow (Purchased from Sai Durga Agencies, Bengaluru, India) and water ad libitum. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 2003). The experimental protocol was reviewed and approved by the Institutional Animal Ethical Committee at Sri Venkateswara University, Tirupati, India (Resolution No. 02/2011-12/(i)/a/ CPCSEA/IAEC/SVU/PSR-PM/Dt. 26-08-2011). Test Chemicals Cisplatin (purity 99%) (Cadila Pharmaceuticals Ltd. Ahmedabad, India), vinblastine (purity >99%) (United Biotech Pvt. Ltd., Bagbnia, India), and bleomycin (purity >99%) (Miracalus Pharma Pvt. Ltd., Mumbai, India) were purchased from local drug store and used as test chemicals. Thiobarbituric acid (purity 99%) and malondialdehyde (purity 98%) (MDA) were obtained from Merck, Darmstadt, Germany. NADP and NAD were purchased from Sigma Chemical Company (St Louis, MO, USA). Melatonin (purity 98.5) was purchased from MP Biomedicals, Inc., (Illkirch, France). All other chemicals used in the present study were of analytical grade and obtained from local commercial sources. Experimental Design Animals were randomly divided into four groups. Rats in group 1 (n ¼ 8) served as control and animals in group 2 (n ¼ 8) were
MELATONIN MITIGATES PVB-INDUCED HEPATO-TOXICITY intraperitoneally injected with PVB (cisplatin 3 mg/kg bw, vinblastine 0.15 mg/kg bw, and bleomyicn 0.5 mg/kg bw) on day 1 of the treatment. Vinblastine alone was injected on day 2 of experiment. Bleomycin alone was injected on days 8 and 15 to complete 1st cycle of treatment. This dose regimen was continued for three cycles per rat. Schematic representation of PVB treatment is presented in Figure 1. The combination of test chemicals and dose regimen were selected based on recommendations of Dr. T. Kannan, Medical oncologist, Sri Venkateswara Institute of Medical Sciences, Tirupati. The human doses for each drug were converted to rat doses by adjusting for body weight/ surface area ratio (Bachmann et al., '96). Rats in group 3 (n ¼ 8) were injected intraperitoneally with melatonin (Ml) (10 mg/kg bw) once in a week for a period of 9 weeks. Animals in group 4 (n ¼ 8) were treated with PVB and melatonin as in groups 2 and 3. The melatonin dose was selected on the basis of previous published studies (Ahmet et al., 2006; Ilbay et al., 2009) and our preliminary kinetic data (authors’ unpublished data). Necropsy The animals were fasted overnight and sacrificed by using anesthetic ether on the day following the last dosing. Liver was removed and cleared from adhering tissues and used for biochemical studies.
METHODS Different sub-cellular (mitochondrial and cytosolic) fractions were obtained by differential centrifugation using the method described by Frezza et al. (2007) and used for biochemical analysis. A 10% (w/v) homogenate was prepared in ice-cold 0.25 M sucrose solution with the help of a motor-driven glass Teflon homogeniser on crushed ice for a minute. The homogenate was centrifuged at 1,000 g for 10 min at 4°C to obtain the
303 nuclear pellet. Mitochondrial pellet was obtained by centrifuging the post-nuclear supernatant at 10,000 g for 10 min at 4°C. All mitochondrial fractions were washed thrice with ice-cold homogenization solution and dissolved in the 0.25 M sucrose solution (1 mg protein/0.1 mL). The cytosolic fraction was separated by centrifuging the post-mitochondrial supernatant at 25,000 g for 60 min. The sub-cellular fractions of mitochondria and cytosol were stored at 70°C until analysis. The levels of lipid peroxidation and activity levels of succinate dehydrogenase (SDH), catalase, and superoxide dismutase were determined in the mitochondrial fraction whereas the activities of other enzymes were determined in cytosolic fraction. The activity of SDH (EC: 1.3.99.1) was determined by the method of Nachlas et al. ('60) and lactate dehydrogenase (LDH) (EC: 1.1.1.27) by the method of Srikanthan and Krishnamurthy ('55). The activity levels of glucose-6-phosphate dehydrogenase (G-6-PDH) (EC: 1.1.1.49), aspartate aminotransaminase (AAT) (EC: 2.6.1.1), and alanine aminotransaminase (AlAT) (EC: 2.6.1.2) were determined by the method of Bergmeyer and Bernt ('65). The activity of glutamate dehydrogenase (GDH) (EC: 1.4.1.3) was determined by the method of Lee and Lardy ('65), catalase (EC: 1.11.1.6) by the method of Chance and Machly ('55), and superoxide dismutase (SOD) (E.C. 1.15.1.1) by the method of Misra and Fridovich ('72). The levels of lipid peroxidation were determined by measuring TBARS according to the method of Ohkawa et al. ('79). Protein concentrations in liver fractions were measured by the method of Lowry et al. ('51) using bovine serum albumin as the protein standard. Statistical Analysis Results were represented as mean SD of eight animals per group. The results were subjected to one-way analysis of variance (ANOVA) followed by Dunnet’s test using SPSS (student version 7.5, SPSS, Inc., UK) to analyze the difference. P < 0.05 were considered significant.
RESULTS
Figure 1. Experimental design for the administration of cisplatin, vinblastine, and bleomycin for a period of 9 weeks.
None of the animals in the control, melatonin, PVB alone, or PVB þ melatonin died during treatment and none of the rats were excluded from the experiment. Hepatic SOD and catalase activity levels in the PVB group decreased significantly (P < 0.05) when compared with those in the control group with an increase in MDA levels (Table 1). The levels of MDA and activity levels of SOD and catalase in the liver of rats treated with melatonin are comparable with control. Administration of melatonin into PVB treated rats resulted in significant decrease in hepatic MDA levels with an increase in SOD and catalase activities when compare to PVB alone treated rats. The activity levels of LDH, G6PDH, GDH, AAT, and AlAT increased significantly in the liver of rats exposed to PVB, whereas the activity of SDH decreased (Table 2). Administration J. Exp. Zool.
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Table 1. Changes in the levels of lipid peroxidation (m moles of malondialdehyde/g tissue) and activity levels of SOD (Units/mg protein/min) and catalase (n moles of H2O2 metabolized/mg protein/min) in the liver of rats exposed to PVB and melatonin (Ml) alone or in combination. Parameter Lipid peroxidation SOD Catalase
Control
PVB
Ml
PVBþMl
5.88a 1.65 3.06a 0.54 18.0a 2.0
16.15b 1.01 (174.8) 1.06b 0.16 (65.36) 7.0b 0.9 (61.11)
6.20a 0.90 (5.44) 3.63a 0.67 (18.62) 19.0a 3.0 (5.55)
11.01c 2.87 (87.24) 2.66a 0.33 (15.03) 9.5c 0.18 (47.22)
Values are mean S.D of 8 animals. Values in the parenthesis are % change from control. Values with same superscript in a row do not differ significantly from each other. P < 0.05.
of melatonin alone has no effect on these enzyme activities, whereas co-administration of melatonin and PVB resulted in significant decrease in LDH, G6PDH, GDH, AAT, and AlAT activities when compare to PVB alone treated rats. On contrary, SDH activity increased in the liver of rats treated with melatonin þ PVB when compared with PVB alone treated rats. However, SDH activity is significantly lower in melatonin þ PVB treated rats when compared to control (Table 2).
DISCUSSION This study produced two observations with clinically important implications. Specifically, PVB treatment was harmful to liver and melatonin treatment was beneficial in protecting against PVB-induced hepato-toxicity. The doses of chemotherapeutic drugs selected in this study were clinically relevant to human beings without the presence of testicular cancer in the rat model. In general drugs used for the cancer therapy were metabolized by liver. In the present study we investigated the effect of intraperitoneal administration of PVB on liver oxidative status and mitochondrial function. Several antitumor drugs, such as cisplatin, doxorubicin, mitomycin C, and bleomycin exhibit ROS dependent apoptotic cell death (Uozumi et al., '93; Partibha et al.,
2006; Yagmurca et al., 2007; El-Sayyad et al., 2009). Jie et al. ('98) have shown that cisplatin produce apoptotic liver cells. Koc et al. (2005) reported that cisplatin-induced apoptosis occurs in primary hepatocytes. Lipid peroxidation has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in humans and experimental animals (Yang et al., 2008). MDA is a major reactive aldehyde produced during the final stages of lipid peroxidation of membrane polyunsaturated fatty acids (Vaca et al., '88). The balance between oxidant and antioxidant system seemed to be disturbed in our study due to PVB treatment. In the present study, significant increases in MDA concentration were observed in the liver homogenates, indicating increased lipid peroxidation. Accumulation of ROS in mitochondria is associated with a decrease in electron transport chain activity that impairs cellular energy metabolism and viability (Orrenius et al., 2007). Antioxidant enzymes such as SOD and catalase protect mitochondria from oxidative stress (Reiter et al., 2006). The decreased SOD activity in the liver of PVB-treated rats suggests impaired dismutation of superoxide radicals resulting in accumulation of superoxide levels. It has been reported that high levels of superoxide radicals inhibits the catalase activity. In the present study, we observed a significant reduction in catalase activity in the liver of PVB treated rats. The loss in SOD and
Table 2. Changes in the activity levels of SDH, LDH, G-6-PDH, GDH, AAT and AlAT in the liver of rats exposed to PVB and melatonin (Ml) alone or in combination. Enzyme $
SDH LDH$ G-6-PDH$ GDH$ AAT# AlAT#
Control 4.09 0.49 0.46a 0.01 0.94a 0.06 0.32a 0.01 0.27a 0.04 0.24a 0.02 a
PVB 1.77 0.18 0.92b 0.06 2.39b 0.25 1.37b 0.13 0.54b 0.05 0.49b 0.02 b
(56.23) (100) (154.25) (328.12) (100) (104.17)
Ml
PVBþMl
3.98 0.7 (2.68) 0.42a 0.05 (8.69) 1.07a 0.09 (13.83) 0.42c 0.05 (31.25) 0.32a 0.03 (18.52) 0.27a 0.01 (12.50)
2.34 0.27 (42.79) 0.38c 0.04 (17.39) 1.64c 0.37 (74.47) 0.96d 0.06 (200.00) 0.38c 0.01 (40.74) 0.36c 0.01 (50.00)
a
Values (expressed as $mmoles of formazon formed or #mmoles of pyruvate formed/mg. protein/hr) are mean S.D of 8 animals. Values in the parenthesis are % change from control. Values with same superscript in a row do not differ significantly from each other. P < 0.05.
J. Exp. Zool.
c
MELATONIN MITIGATES PVB-INDUCED HEPATO-TOXICITY catalase activities in the liver of PVB-Treated rats might be a result of oxidative inactivation by ROS (Macmillan-Crow and Cruthirds, 2001). Our data clearly suggests that in PVB-Treated rats, the liver mitochondria experience a double jeopardy: increased generation of ROS and compromised antioxidant defense (decreased SOD and catalase activities) which may lead to mitochondrial dysfunction (Chiu et al., 2012). Meanwhile, the elevated SOD and catalase in PVB þ melatonin treated rats indirectly showed an increase in the number of free radicals after PVB administration and also reflected that these enzymes played important roles in clearing away excessive free radicals. Naziroglu et al. (2004) demonstrated that treatment with antioxidants such as selenium and vitamin E prevents cisplatin-induced high ROS production in the liver cells of rats. In a similar manner, cisplatin-induced high ROS level was decreased by erdosteine (Koc et al., 2005) or melatonin (Ilbay et al., 2009; Ohta et al., 2004) treatment in the hepatic cells of rats. Mitochondria contribute to a number of different processes in living cells, of which the most important is ATP synthesis by oxidative phosphorylation. Oxidative phosphorylation is a major source of endogenous, toxic free radicals, including hydrogen peroxide, superoxide radical, and hydroxyl radical, which are products of normal cellular respiration (Saraste, '99). Mitochondrial SDH, which catalyses the oxidation of succinate to fumarate in the Krebs cycle, is a vital mitochondrial antioxidant enzyme, controlling the superoxide-scavenging activity in the respiratory chain (Rustin et al., 2002). In our result, the SDH activity was decreased by PVB treatment indicating impaired mitochondrial oxidation during PVB treatment. Lactate dehydrogenase, which catalyses the conversion of pyruvic acid into lactic acid under anaerobic conditions, was significantly increased in the liver of PVB treated rats. The decrease in SDH and the increase of LDH, indicate anaerobic metabolism in the liver of adriamycin-treated rats. In support to this, Deepa and Varalakshmi (2003) have reported an increase in LDH activity and consequent impairment of oxidative metabolism in the hepatic tissue of PVB treated rats. Li-Ping et al. (2000) observed hypoxia in different organs including liver after administration of chemotherapeutics. It was reported earlier that the hypoxic zone is resistant to the chemotherapy and radiotherapy results in the treatment failures (Airley and Mobasheri, 2007). As a consequence of anaerobiosis, the oxidation of glucose was elevated in the hexose monophosphate (HMP) shunt pathway as evidenced from the significant increase in G-6-PDH activity in the liver of PVB treated rats. The increased G-6-PDH indicates oxidation of glucose through the HMP shunt pathway thereby facilitating the increased production of NADPH2 for the detoxification process. It seems that the operation of the HMP shunt pathway serves as an alternate route for glucose oxidation in the tissues during chemotherapy.
305 The coupled action of transaminases (AAT and AlAT) and GDH is an important step in the utilization of amino acids for oxidation/or gluconeogenesis in tissues (Knox and Greengard, '65). A significant increase in the activity levels of AAT, AlAT, and GDH may be suggestive of an increased utilization of amino acids through these reactions and such utilization indicates the channelling of keto acids to the Krebs cycle, in the event of blockage of glycolytic metabolism during PVB treatment. In support of this, Gaona-Gaona et al. (2011) reported increased transaminase activities in the tissues of rat exposed to cisplatin. Several investigations linked enhanced transaminases to tissue damage (Rouiller,'64) as a consequence of cisplatin exposure (Ramadan et al., 2001). Impaired mitochondrial function and deranged metabolism in the liver of rats exposed to PVB was restored by melatonin. The antioxidant effects of melatonin have been well described, and are known to include both direct and indirect effects with equal efficiency in nucleus, cytosol, and mitochondria of the cell (Chowdhury and Lieberman, 2008). It scavenges a variety of free radicals and ROS, such as hydroxyl radical (Tan et al., 2007), peroxy radical (Pieri et al., '94), nitric oxide (Tan et al., 2007), hydrogen peroxide (Barlow-Walden et al., '95), and singlet oxygen (Cagnoli et al. '95) and also up regulates certain antioxidant enzymes like SOD, catalase and glutathione peroxidise (Barlow-Walden et al., '95; Liu and Ng, 2000; Rodriguez et al., 2004; Reiter et al., 2006; Tan et al., 2007). Thus the antioxidant activity of melatonin might be related with combination of antioxidant enzyme modulation and free radicalscavenging activities. To our understanding, melatonin might be ambidextrous antioxidant having potential application for pharmaceutical supplements. The present investigation concludes that PVB reduces the oxidation of glucose through the Krebs cycle and enhances the HMP shunt pathway; this yields a larger number of reduced NADP molecules for detoxification. Also, the derangement in oxidative metabolism might be due to damage of the mitochondrial membrane during PVB treatment by generation of more ROS. The PVB-Treated rats appear to meet their energy requirements through anaerobic oxidation, as evidenced by elevated LDH during impaired mitochondrial oxidation. Elevated levels of AAT, AlAT, and GDH facilitate greater feeding of keto acids into citric acid cycle to meet impaired oxidative metabolism. This can be interpreted as a functional/physiological adaptation during PVB treatment and these enzymes may provide a good indicator for monitoring stress conditions in rats. In addition, the ability of melatonin to mitigate PVB-Induced oxidative toxicity and deranged metabolism in the liver of rat might be of therapeutic relevance. Therefore, melatonin might be used in conjunction with traditional chemotherapeutic drugs as a means to protect against drug-associated complications. At present, it is unknown whether the melatonin would influence the chemotherapeutic effect of PVB, although it has been suggested that melatonin does J. Exp. Zool.
306 not interfere with the antibiotic capacity of aminoglycosides such as gentamycin and tobramycin (Lopez-Gonzalez et al., '99). Studies are under way to clarify the specific role of melatonin in its protective action against hepato-toxicity in rats.
ACKNOWLEDGMENTS P. Madhu and K. Pratap Reddy are grateful to University Grants Commission, New Delhi for financial support in the form of UGCBSR (RFMS) fellowship. The authors declare that the experiments conducted comply with the current laws of their country and approved by the Institutional Animal Ethical Committee at S.V. University, Tirupati, India.
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Melatonin Reduces Oxidative Stress and Restores Mitochondrial Function in the Liver of Rats Exposed to Chemotherapeutics P. MADHU1,2, K. PRATAP REDDY1,2, 2 AND P. SREENIVASULA REDDY * 1
Department of Biotechnology, Sri Venkateswara University, Tirupati, India Department of Zoology, Sri Venkateswara University, Tirupati, India
2
ABSTRACT
This study was undertaken to investigate whether administration of melatonin protects PVBInduced oxidative and metabolic toxicity in the liver of Wistar rats. Adult male Wistar rats were intraperitoneally injected with either melatonin or PVB (cisplatin, vinblastine, and bleomycin) alone or combination for a period of 9 weeks. A significant increase in lipid peroxidation levels and decrease in catalase and superoxide dismutase activity levels were observed in the liver mitochondria of rats treated with PVB indicating increased oxidative stress. PVB treatment significantly decreased the succinate dehydrogenase activity with a significant increase in lactate dehydrogenase, glucose-6-phosphate dehydrogenase, aspartate aminotransaminase, alanine aminotransaminase, and glutamate dehydrogenase activities indicating deranged hepatic metabolism. Melatonin administration, on the other hand was found to significantly improve PVB-Induced biochemical changes, bringing them closer to the controls. The results from the study provide evidence that treatment with PVB affects hepatic metabolism in rats by inducing oxidative stress followed by decreasing mitochondrial oxidation and also point towards the clinical potential of melatonin as an adjuvant therapy to conventional chemotherapeutic regimens. J. Exp. Zool. 323A:301–308, 2015. © 2015 Wiley Periodicals, Inc.
J. Exp. Zool. 323A:301–308, 2015
How to cite this article: MADHU P, REDDY KP, REDDY PS. 2015. Melatonin reduces oxidative stress and restores mitochondrial function in the liver of rats exposed to chemotherapeutics. J. Exp. Zool. 323A:301–308.
INTRODUCTION Incidence of cancer has been on rise all over the world. According to American Cancer Society (2014), most common cancers among adolescents are Hodgkin’s lymphoma (15%), thyroid carcinoma (11%), brain (10%), and testicular germ cell tumors (8%). The most commonly used chemotherapeutics are doxorubicin, bleomycin, vinblastine, and dacarbazine for Hodgkin’s lymphoma (Meyer et al., 2012), cabozantinib for thyroid carcinoma (Traynor, 2013), and temozolomide for brain cancer (Pouratian et al., 2007). In case of testicular germ cell tumors, combinational therapy of cisplatin (P), bleomycin (B), and vinblastine (V) or etoposide (E) is commonly used chemotherapeutic regimen. The prognosis of testicular cancer has improved considerably in the past three decades; hence approximately 70–80% of patients with meta-
static disease will achieve a durable complete remission after 3–4 cycles of a combination therapy with PVB followed by secondary surgery. The improved prognosis of testicular cancer has brought
Conflicts of interest: None. Correspondence to: P. Sreenivasula Reddy, Department of Zoology, S. V. University, Tirupati-517502, India. E-mail: [email protected] Received 15 October 2014; Revised 16 December 2014; Accepted 6 January 2015 DOI: 10.1002/jez.1917 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
© 2015 WILEY PERIODICALS, INC.
302 the long-term toxicity of the treatment and life-quality after treatment into focus. Unfortunately, therapeutic potential of these drugs is limited by its ototoxicity, neurotoxicity, nephrotoxicity, and hepatotoxicity (Church et al., 2004; Steeg and Theodorescu, 2008; Ciftci et al., 2011). Earlier, it has been reported that simultaneous administration of some antiemetic drugs exaggerated the hepatic toxicity induced by cisplatin (Lu and Cederbaum, 2006). Thus ameliorating chemotherapeutic drug(s) toxicity on non-target tissues is an important health-related goal. The liver is an important detoxifying organ that decomposes endogenous and exogenous substances; therefore, liver diseases are considered a serious health problem (Yang et al., 2008). PVB activates hepatic microsomal cytochrome P450, which has a high capacity to generate ROS, resulting in oxidative stress, including changes in redox state, mitochondrial damage, and decreased antioxidant enzyme levels (Partibha et al., 2006). Under abnormal conditions, generation of excess ROS damages cellular lipids, proteins, or DNA, and finally leading to cell injury (Wong-ekkabut et al., 2007). These toxic side effects adversely affect the patient’s quality of life. Hydrogen peroxide, superoxide anion, and hydroxyl radicals are thought to be produced during oxidative stress. Superoxide dismutase, catalase, and glutathione peroxidase are endogenous antioxidant enzymes which play a key role in prevention of oxidative injury (Movahed et al., 2012). Numerous studies have reported that natural antioxidants are effective against oxidative stress-related pathology by increasing intracellular antioxidant enzyme activities (Giriwono et al., 2010; Lee et al., 2010; Yeh et al., 2012). Chronic low doses of cisplatin exposure may induce hepatotoxicity due to drug accumulation in liver (Uozumi et al., '93). One mechanism of cisplatin’s antitumor action involves its binding to DNA, resulting in a cascade of events that culminate in cell death. Another mechanism of cisplatin’s toxicity involves the induction of reactive oxygen species (ROS) and the possible depletion of antioxidants (Jordan and Carmo-Fonseca, 2000; Koc et al., 2005) resulting in mitochondrial dysfunction (Saad et al., 2004). Bleomycin a glycopeptide antineoplastic drug can induce breaks in single and double stranded DNA. Bleomycin is often used as combination chemotherapy for lymphoma and testicular carcinoma in humans and causes morbidity and mortality (de Haas et al., 2008). Vinca alkaloids and adriamycin are frequently used for the haematological malignancies. Since vinblastine is generally metabolized by the liver, dosage adjustments in patients with hepatic dysfunction is essential to control the drug accumulation (Zhou-Pan et al., '93; Benjamin et al., '77). Melatonin is the principal secretary product produced by pineal gland. Melatonin is present ubiquitously, including bacteria, invertebrates, and vertebrates (Reiter, '91). It is now well established that melatonin is not only identified in the pineal J. Exp. Zool.
MADHU ET AL. gland, but also in many other organs and tissues including retina, skin, Harderian glands, gut, ovary, testes, bone marrow, lens, intestine, salivary glands, and blood platelets (Tan et al., 2014). Melatonin mainly involves in the regulation of circadian rhythms, seasonal reproduction, and inhibition of cancer initiation (Karbownik and Reiter, 2000). Melatonin plays an important role in scavenging free radicals and stimulating antioxidant enzymes (Reiter et al., 2003; Rodriguez et al., 2004). Several reports have shown that melatonin protects DNA, lipids, and proteins against the harmful effects of free radicals (Reiter et al., 2000). In normal cells melatonin inhibits apoptotic processes whereas in cancer cells it could promote apoptosis (Blask et al., 2002). Melatonin also acts as oncostatic molecule (Dragojevic et al., 2014). The purpose of this study was to ascertain the influence that melatonin exert on the hepatotoxicity caused by PVB in rats.
MATERIALS AND METHODS Procurement and Maintenance of Experimental Animals Three month old male Wistar strain albino rats (body weight 190 20 g) were obtained from an authorized vendor (M/S Raghavendra Enterprises, Bengaluru, India). The animals were housed (four per cage) in sterile polypropylene cages, under standard laboratory conditions (temperature 24 2°C; light and dark 12:12 h). The rats were allowed free access to a standard pellet chow (Purchased from Sai Durga Agencies, Bengaluru, India) and water ad libitum. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 2003). The experimental protocol was reviewed and approved by the Institutional Animal Ethical Committee at Sri Venkateswara University, Tirupati, India (Resolution No. 02/2011-12/(i)/a/ CPCSEA/IAEC/SVU/PSR-PM/Dt. 26-08-2011). Test Chemicals Cisplatin (purity 99%) (Cadila Pharmaceuticals Ltd. Ahmedabad, India), vinblastine (purity >99%) (United Biotech Pvt. Ltd., Bagbnia, India), and bleomycin (purity >99%) (Miracalus Pharma Pvt. Ltd., Mumbai, India) were purchased from local drug store and used as test chemicals. Thiobarbituric acid (purity 99%) and malondialdehyde (purity 98%) (MDA) were obtained from Merck, Darmstadt, Germany. NADP and NAD were purchased from Sigma Chemical Company (St Louis, MO, USA). Melatonin (purity 98.5) was purchased from MP Biomedicals, Inc., (Illkirch, France). All other chemicals used in the present study were of analytical grade and obtained from local commercial sources. Experimental Design Animals were randomly divided into four groups. Rats in group 1 (n ¼ 8) served as control and animals in group 2 (n ¼ 8) were
MELATONIN MITIGATES PVB-INDUCED HEPATO-TOXICITY intraperitoneally injected with PVB (cisplatin 3 mg/kg bw, vinblastine 0.15 mg/kg bw, and bleomyicn 0.5 mg/kg bw) on day 1 of the treatment. Vinblastine alone was injected on day 2 of experiment. Bleomycin alone was injected on days 8 and 15 to complete 1st cycle of treatment. This dose regimen was continued for three cycles per rat. Schematic representation of PVB treatment is presented in Figure 1. The combination of test chemicals and dose regimen were selected based on recommendations of Dr. T. Kannan, Medical oncologist, Sri Venkateswara Institute of Medical Sciences, Tirupati. The human doses for each drug were converted to rat doses by adjusting for body weight/ surface area ratio (Bachmann et al., '96). Rats in group 3 (n ¼ 8) were injected intraperitoneally with melatonin (Ml) (10 mg/kg bw) once in a week for a period of 9 weeks. Animals in group 4 (n ¼ 8) were treated with PVB and melatonin as in groups 2 and 3. The melatonin dose was selected on the basis of previous published studies (Ahmet et al., 2006; Ilbay et al., 2009) and our preliminary kinetic data (authors’ unpublished data). Necropsy The animals were fasted overnight and sacrificed by using anesthetic ether on the day following the last dosing. Liver was removed and cleared from adhering tissues and used for biochemical studies.
METHODS Different sub-cellular (mitochondrial and cytosolic) fractions were obtained by differential centrifugation using the method described by Frezza et al. (2007) and used for biochemical analysis. A 10% (w/v) homogenate was prepared in ice-cold 0.25 M sucrose solution with the help of a motor-driven glass Teflon homogeniser on crushed ice for a minute. The homogenate was centrifuged at 1,000 g for 10 min at 4°C to obtain the
303 nuclear pellet. Mitochondrial pellet was obtained by centrifuging the post-nuclear supernatant at 10,000 g for 10 min at 4°C. All mitochondrial fractions were washed thrice with ice-cold homogenization solution and dissolved in the 0.25 M sucrose solution (1 mg protein/0.1 mL). The cytosolic fraction was separated by centrifuging the post-mitochondrial supernatant at 25,000 g for 60 min. The sub-cellular fractions of mitochondria and cytosol were stored at 70°C until analysis. The levels of lipid peroxidation and activity levels of succinate dehydrogenase (SDH), catalase, and superoxide dismutase were determined in the mitochondrial fraction whereas the activities of other enzymes were determined in cytosolic fraction. The activity of SDH (EC: 1.3.99.1) was determined by the method of Nachlas et al. ('60) and lactate dehydrogenase (LDH) (EC: 1.1.1.27) by the method of Srikanthan and Krishnamurthy ('55). The activity levels of glucose-6-phosphate dehydrogenase (G-6-PDH) (EC: 1.1.1.49), aspartate aminotransaminase (AAT) (EC: 2.6.1.1), and alanine aminotransaminase (AlAT) (EC: 2.6.1.2) were determined by the method of Bergmeyer and Bernt ('65). The activity of glutamate dehydrogenase (GDH) (EC: 1.4.1.3) was determined by the method of Lee and Lardy ('65), catalase (EC: 1.11.1.6) by the method of Chance and Machly ('55), and superoxide dismutase (SOD) (E.C. 1.15.1.1) by the method of Misra and Fridovich ('72). The levels of lipid peroxidation were determined by measuring TBARS according to the method of Ohkawa et al. ('79). Protein concentrations in liver fractions were measured by the method of Lowry et al. ('51) using bovine serum albumin as the protein standard. Statistical Analysis Results were represented as mean SD of eight animals per group. The results were subjected to one-way analysis of variance (ANOVA) followed by Dunnet’s test using SPSS (student version 7.5, SPSS, Inc., UK) to analyze the difference. P < 0.05 were considered significant.
RESULTS
Figure 1. Experimental design for the administration of cisplatin, vinblastine, and bleomycin for a period of 9 weeks.
None of the animals in the control, melatonin, PVB alone, or PVB þ melatonin died during treatment and none of the rats were excluded from the experiment. Hepatic SOD and catalase activity levels in the PVB group decreased significantly (P < 0.05) when compared with those in the control group with an increase in MDA levels (Table 1). The levels of MDA and activity levels of SOD and catalase in the liver of rats treated with melatonin are comparable with control. Administration of melatonin into PVB treated rats resulted in significant decrease in hepatic MDA levels with an increase in SOD and catalase activities when compare to PVB alone treated rats. The activity levels of LDH, G6PDH, GDH, AAT, and AlAT increased significantly in the liver of rats exposed to PVB, whereas the activity of SDH decreased (Table 2). Administration J. Exp. Zool.
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Table 1. Changes in the levels of lipid peroxidation (m moles of malondialdehyde/g tissue) and activity levels of SOD (Units/mg protein/min) and catalase (n moles of H2O2 metabolized/mg protein/min) in the liver of rats exposed to PVB and melatonin (Ml) alone or in combination. Parameter Lipid peroxidation SOD Catalase
Control
PVB
Ml
PVBþMl
5.88a 1.65 3.06a 0.54 18.0a 2.0
16.15b 1.01 (174.8) 1.06b 0.16 (65.36) 7.0b 0.9 (61.11)
6.20a 0.90 (5.44) 3.63a 0.67 (18.62) 19.0a 3.0 (5.55)
11.01c 2.87 (87.24) 2.66a 0.33 (15.03) 9.5c 0.18 (47.22)
Values are mean S.D of 8 animals. Values in the parenthesis are % change from control. Values with same superscript in a row do not differ significantly from each other. P < 0.05.
of melatonin alone has no effect on these enzyme activities, whereas co-administration of melatonin and PVB resulted in significant decrease in LDH, G6PDH, GDH, AAT, and AlAT activities when compare to PVB alone treated rats. On contrary, SDH activity increased in the liver of rats treated with melatonin þ PVB when compared with PVB alone treated rats. However, SDH activity is significantly lower in melatonin þ PVB treated rats when compared to control (Table 2).
DISCUSSION This study produced two observations with clinically important implications. Specifically, PVB treatment was harmful to liver and melatonin treatment was beneficial in protecting against PVB-induced hepato-toxicity. The doses of chemotherapeutic drugs selected in this study were clinically relevant to human beings without the presence of testicular cancer in the rat model. In general drugs used for the cancer therapy were metabolized by liver. In the present study we investigated the effect of intraperitoneal administration of PVB on liver oxidative status and mitochondrial function. Several antitumor drugs, such as cisplatin, doxorubicin, mitomycin C, and bleomycin exhibit ROS dependent apoptotic cell death (Uozumi et al., '93; Partibha et al.,
2006; Yagmurca et al., 2007; El-Sayyad et al., 2009). Jie et al. ('98) have shown that cisplatin produce apoptotic liver cells. Koc et al. (2005) reported that cisplatin-induced apoptosis occurs in primary hepatocytes. Lipid peroxidation has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in humans and experimental animals (Yang et al., 2008). MDA is a major reactive aldehyde produced during the final stages of lipid peroxidation of membrane polyunsaturated fatty acids (Vaca et al., '88). The balance between oxidant and antioxidant system seemed to be disturbed in our study due to PVB treatment. In the present study, significant increases in MDA concentration were observed in the liver homogenates, indicating increased lipid peroxidation. Accumulation of ROS in mitochondria is associated with a decrease in electron transport chain activity that impairs cellular energy metabolism and viability (Orrenius et al., 2007). Antioxidant enzymes such as SOD and catalase protect mitochondria from oxidative stress (Reiter et al., 2006). The decreased SOD activity in the liver of PVB-treated rats suggests impaired dismutation of superoxide radicals resulting in accumulation of superoxide levels. It has been reported that high levels of superoxide radicals inhibits the catalase activity. In the present study, we observed a significant reduction in catalase activity in the liver of PVB treated rats. The loss in SOD and
Table 2. Changes in the activity levels of SDH, LDH, G-6-PDH, GDH, AAT and AlAT in the liver of rats exposed to PVB and melatonin (Ml) alone or in combination. Enzyme $
SDH LDH$ G-6-PDH$ GDH$ AAT# AlAT#
Control 4.09 0.49 0.46a 0.01 0.94a 0.06 0.32a 0.01 0.27a 0.04 0.24a 0.02 a
PVB 1.77 0.18 0.92b 0.06 2.39b 0.25 1.37b 0.13 0.54b 0.05 0.49b 0.02 b
(56.23) (100) (154.25) (328.12) (100) (104.17)
Ml
PVBþMl
3.98 0.7 (2.68) 0.42a 0.05 (8.69) 1.07a 0.09 (13.83) 0.42c 0.05 (31.25) 0.32a 0.03 (18.52) 0.27a 0.01 (12.50)
2.34 0.27 (42.79) 0.38c 0.04 (17.39) 1.64c 0.37 (74.47) 0.96d 0.06 (200.00) 0.38c 0.01 (40.74) 0.36c 0.01 (50.00)
a
Values (expressed as $mmoles of formazon formed or #mmoles of pyruvate formed/mg. protein/hr) are mean S.D of 8 animals. Values in the parenthesis are % change from control. Values with same superscript in a row do not differ significantly from each other. P < 0.05.
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c
MELATONIN MITIGATES PVB-INDUCED HEPATO-TOXICITY catalase activities in the liver of PVB-Treated rats might be a result of oxidative inactivation by ROS (Macmillan-Crow and Cruthirds, 2001). Our data clearly suggests that in PVB-Treated rats, the liver mitochondria experience a double jeopardy: increased generation of ROS and compromised antioxidant defense (decreased SOD and catalase activities) which may lead to mitochondrial dysfunction (Chiu et al., 2012). Meanwhile, the elevated SOD and catalase in PVB þ melatonin treated rats indirectly showed an increase in the number of free radicals after PVB administration and also reflected that these enzymes played important roles in clearing away excessive free radicals. Naziroglu et al. (2004) demonstrated that treatment with antioxidants such as selenium and vitamin E prevents cisplatin-induced high ROS production in the liver cells of rats. In a similar manner, cisplatin-induced high ROS level was decreased by erdosteine (Koc et al., 2005) or melatonin (Ilbay et al., 2009; Ohta et al., 2004) treatment in the hepatic cells of rats. Mitochondria contribute to a number of different processes in living cells, of which the most important is ATP synthesis by oxidative phosphorylation. Oxidative phosphorylation is a major source of endogenous, toxic free radicals, including hydrogen peroxide, superoxide radical, and hydroxyl radical, which are products of normal cellular respiration (Saraste, '99). Mitochondrial SDH, which catalyses the oxidation of succinate to fumarate in the Krebs cycle, is a vital mitochondrial antioxidant enzyme, controlling the superoxide-scavenging activity in the respiratory chain (Rustin et al., 2002). In our result, the SDH activity was decreased by PVB treatment indicating impaired mitochondrial oxidation during PVB treatment. Lactate dehydrogenase, which catalyses the conversion of pyruvic acid into lactic acid under anaerobic conditions, was significantly increased in the liver of PVB treated rats. The decrease in SDH and the increase of LDH, indicate anaerobic metabolism in the liver of adriamycin-treated rats. In support to this, Deepa and Varalakshmi (2003) have reported an increase in LDH activity and consequent impairment of oxidative metabolism in the hepatic tissue of PVB treated rats. Li-Ping et al. (2000) observed hypoxia in different organs including liver after administration of chemotherapeutics. It was reported earlier that the hypoxic zone is resistant to the chemotherapy and radiotherapy results in the treatment failures (Airley and Mobasheri, 2007). As a consequence of anaerobiosis, the oxidation of glucose was elevated in the hexose monophosphate (HMP) shunt pathway as evidenced from the significant increase in G-6-PDH activity in the liver of PVB treated rats. The increased G-6-PDH indicates oxidation of glucose through the HMP shunt pathway thereby facilitating the increased production of NADPH2 for the detoxification process. It seems that the operation of the HMP shunt pathway serves as an alternate route for glucose oxidation in the tissues during chemotherapy.
305 The coupled action of transaminases (AAT and AlAT) and GDH is an important step in the utilization of amino acids for oxidation/or gluconeogenesis in tissues (Knox and Greengard, '65). A significant increase in the activity levels of AAT, AlAT, and GDH may be suggestive of an increased utilization of amino acids through these reactions and such utilization indicates the channelling of keto acids to the Krebs cycle, in the event of blockage of glycolytic metabolism during PVB treatment. In support of this, Gaona-Gaona et al. (2011) reported increased transaminase activities in the tissues of rat exposed to cisplatin. Several investigations linked enhanced transaminases to tissue damage (Rouiller,'64) as a consequence of cisplatin exposure (Ramadan et al., 2001). Impaired mitochondrial function and deranged metabolism in the liver of rats exposed to PVB was restored by melatonin. The antioxidant effects of melatonin have been well described, and are known to include both direct and indirect effects with equal efficiency in nucleus, cytosol, and mitochondria of the cell (Chowdhury and Lieberman, 2008). It scavenges a variety of free radicals and ROS, such as hydroxyl radical (Tan et al., 2007), peroxy radical (Pieri et al., '94), nitric oxide (Tan et al., 2007), hydrogen peroxide (Barlow-Walden et al., '95), and singlet oxygen (Cagnoli et al. '95) and also up regulates certain antioxidant enzymes like SOD, catalase and glutathione peroxidise (Barlow-Walden et al., '95; Liu and Ng, 2000; Rodriguez et al., 2004; Reiter et al., 2006; Tan et al., 2007). Thus the antioxidant activity of melatonin might be related with combination of antioxidant enzyme modulation and free radicalscavenging activities. To our understanding, melatonin might be ambidextrous antioxidant having potential application for pharmaceutical supplements. The present investigation concludes that PVB reduces the oxidation of glucose through the Krebs cycle and enhances the HMP shunt pathway; this yields a larger number of reduced NADP molecules for detoxification. Also, the derangement in oxidative metabolism might be due to damage of the mitochondrial membrane during PVB treatment by generation of more ROS. The PVB-Treated rats appear to meet their energy requirements through anaerobic oxidation, as evidenced by elevated LDH during impaired mitochondrial oxidation. Elevated levels of AAT, AlAT, and GDH facilitate greater feeding of keto acids into citric acid cycle to meet impaired oxidative metabolism. This can be interpreted as a functional/physiological adaptation during PVB treatment and these enzymes may provide a good indicator for monitoring stress conditions in rats. In addition, the ability of melatonin to mitigate PVB-Induced oxidative toxicity and deranged metabolism in the liver of rat might be of therapeutic relevance. Therefore, melatonin might be used in conjunction with traditional chemotherapeutic drugs as a means to protect against drug-associated complications. At present, it is unknown whether the melatonin would influence the chemotherapeutic effect of PVB, although it has been suggested that melatonin does J. Exp. Zool.
306 not interfere with the antibiotic capacity of aminoglycosides such as gentamycin and tobramycin (Lopez-Gonzalez et al., '99). Studies are under way to clarify the specific role of melatonin in its protective action against hepato-toxicity in rats.
ACKNOWLEDGMENTS P. Madhu and K. Pratap Reddy are grateful to University Grants Commission, New Delhi for financial support in the form of UGCBSR (RFMS) fellowship. The authors declare that the experiments conducted comply with the current laws of their country and approved by the Institutional Animal Ethical Committee at S.V. University, Tirupati, India.
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