Ameliorative effect of curcumin on hepatotoxicity induced by chloroquine phosphate

Environmental Toxicology and Pharmacology 30 (2010) 103–109

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Ameliorative effect of curcumin on hepatotoxicity induced by chloroquine phosphate J.J. Dattani, D.K. Rajput, N. Moid, H.N. Highland, L.B. George, K.R. Desai ∗ Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, Gujarat, India

a r t i c l e

i n f o

Article history: Received 1 October 2009 Received in revised form 16 April 2010 Accepted 19 April 2010 Available online 20 May 2010 Keywords: Chloroquine phosphate Hepatic toxicity Oxidative stress Curcumin

a b s t r a c t India is one of the most endemic areas, where malaria predominates and its control has become a formidable task. Chloroquine phosphate (CQ) on account of its rapid action on blood schizontocide of all the malarial parasite strains has become the most widely prescribed drug for prophylaxis and treatment of malaria. Toxicity of CQ is most commonly encountered at therapeutic and higher doses of treatment. Thus, the present study was undertaken to evaluate the protective effect of Curcumin, a herbal antioxidant obtained from Curcuma longa, on hepatic biochemical and histopathological status of CQ induced male mice. Swiss albino male mice were administered oral doses of CQ (100 mg/kg body wt., 200 mg/kg body wt. and 300 mg/kg body wt.) and CQ + curcumin (300 mg/kg body wt. + 80 mg/kg body wt.) for 45 days. A withdrawal of high dose treatment for 45 days was also studied. Administration of CQ brought about a significant decrease in Protein content with a decline in SDH, ATPase and ALKase activities, whereas ACPase activity was found to be significantly increased following CQ treatment. Antioxidant enzyme SOD registered a significant reduction as opposed to TBARS which was found to be elevated in a significant manner in the CQ treated groups as compared to control. Gravimetric indices (body weight and organ weight) declined significantly following CQ treatment. Administration of curcumin exhibited significant reversal of CQ induced toxicity in hepatic tissue. Protein content, SDH, ATPase, ALKase, ACPase, SOD, TBARS, body weight and organ weight were found to be comparable to that of control group after curcumin administration. Thus, obtained results led us to conclude the curative potential of curcumin against CQ induced hepatotoxicity. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Since India is a malaria prone endemic area, its control has proved to be a challenge. At the time of launching of the national malaria control programme in 1953, the estimated malaria incidence was 75 million cases and 0.8 million deaths in India per annum. Malaria can be treated by antimalarial drugs and the ones in common use come from five classes of compounds, viz. the quinolines, arylaminoalcohols, antifals, artemisinin derivatives, hydroxynaphtharuinoaes and antibacterial agents. Quinolines and arylaminoalcohols include chloroquine, quinine, quinidine, etc. (Ashley et al., 2006). Despite the introduction of many new antimalarial drugs, chloroquine is still the most widely prescribed drug for prophylaxis and treatment of malaria (WHO, 2003). Chloroquine is effective against Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and drug-sensitive Plasmodium falciparum and is used for treatment/prophylaxis by clinicians.

∗ Corresponding author. Tel.: +91 9426820843. E-mail addresses: [email protected] (J.J. Dattani), [email protected] (K.R. Desai). 1382-6689/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2010.04.001

Chloroquine by virtue of its weak base properties accumulates in the vacuole where it exerts antimalarial properties by inhibiting the process of heme polymerization and detoxification which eventually results in the death of parasites (Krogstad and Schlesinger, 1987; Loria et al., 1999). It is the attachment of the chlorine atom at the 7th position of the quinoline ring which confers the greatest antimalarial activity in both avian and human malaria (Ogunbayo et al., 2006). CQ is well absorbed from the gastrointestinal (GI) tract as well as from other sites on account of its tendency of tissue sequestration. This further leads to toxic implications both at higher dose and overdose. Curcumin is a naturally occurring polyphenolic compound derived from the root of Curcuma longa, Linn. a perennial herb belonging to ginger family Zingiberaceae. It is consumed in high quantities in India, where it is used as a spice and as an antiinflammatory compound in traditional medicine. Increasingly, its therapeutic effects are being investigated in well-defined models of disease, such as inflammatory hepatic and pancreatic diseases (Nanji et al., 2003; Gaddipati et al., 2003) and pulmonary models of inflammation-triggered fibrosis (Punithavathi et al., 2000; Wolf et al., 1999).

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Curcumin when administered orally has about 60% absorption and major part of it gets transformed to glucuronide and sulphate conjugates (Ravindranath and Chandrasekhara, 1980). While the intravenous or intraperitoneal administration in rodents gives higher absorption (Holder et al., 1978) it cannot be injected intravenously in long term experiments owing to its lipophilic nature. Further it has good absorption, metabolism and tissue distribution which is a requisite quality of a compound to be used as a therapeutic or mitigating agent. With reference to this it has been shown to have a dose-dependant chemopreventive effect in animal system (Huang et al., 1994). After a long term use in traditional Ayurvedic medicine, modern scientific community discovered that curcumin has beneficial effects on a variety of diseases and pathological condition (Wang et al., 1997). Curcumin has shown to possess anticancer effects by blocking transformation, tumor initiation, tumor promotion, invasion, angiogenesis and metastasis (Aggarwal et al., 2003). It has also been demonstrated to have a dose-dependant chemopreventive effect in animal systems of colon, duodenal, stomach, oesophageal and oral carcinogenesis (Huang et al., 1994). In addition to its anticancerous effects, curcumin has been effective against a variety of conditions in in vitro and in vivo preclinical studies (Shishodia et al., 2005). A wide range of biological and pharmacological activities of curcumin have been investigated (Okada et al., 2001). Hepatoprotective, antioxidant, antimutagenic and anticarcinogenic effects of curcumin have been shown by previous workers (Premkumar et al., 2004). Hence, in the present study the efficacy of curcumin was evaluated against chloroquine induced hepatotoxicity under in vivo conditions. It is hypothesized that the results of our study would provide novel insight about the hepatic ameliorative potential of curcumin.

2.3. Tissue collection After the termination of treatment period, animals were euthanized and dissected. Liver was dissected out carefully, blotted free of blood and weighed. Tissue was then processed and homogenate was prepared.

2.4. Protein estimation Protein estimation was done by using standard protocol of Lowry et al. (1951). Color development was read at 540 nm.

2.5. Energy metabolism estimation 2.5.1. Succinate dehydrogenase activity (SDH) SDH activity was measured by using the method of Beatty et al. (1966). Reaction mixture contained 1 ml of 0.2 M PO4 buffer, 1 ml INT, and 1 ml sodium succinate. 0.4 ml of tissue homogenate was added to the reaction mixture. It was then incubated at 37 ◦ C for 15 min. Reaction was terminated by adding 0.1 ml of 30% TCA followed by 7 ml of ethyl acetate. It was then centrifuged for 5 min at 2000 rpm. Upper layer containing the formazan was used to read absorbance at 420 nm. SDH activity was expressed as ␮g formazan formed/15 min/mg tissue wt.

2.5.2. Adenosine triphosphatase activity (ATPase) ATPase activity was determined by using standard protocol of Quinn and White (1968). 0.2 ml of tissue homogenate was added in the reaction mixture containing 0.1 ml of 3 mM MgCl2 , 0.1 ml of 30 mM KCl, 0.1 ml of 150 mM NaCl, 0.3 ml Substrate buffer (containing 18 mg disodium salt of ATP in 10 ml Tris–HCl buffer). It was then incubated at 37 ◦ C for 30 min. 10% TCA was added finally. Reaction mixture was then kept in refrigerator and centrifuged finally. 1.5 ml of supernatant was then taken and 1.0 ml of acidic ammonium molybdate, 0.5 ml of freshly prepared ANSA reagent and 7.5 ml of distilled water was added to it. Finally absorbance was read at 660 nm.

2.6. Acid phosphatase activity (ACPase) Activity of ACPase was assessed by using the technique of Bessey et al. (1946). Absorbance was measured at 410 nm and it was expressed as ␮moles of pnitrophenol/30 min/mg tissue wt.

2. Materials and methods 2.1. Animals and chemicals Healthy adult male albino mice, Mus musculus of Swiss strain weighing between 25 and 40 g were obtained from Cadila Pharaceuticals, Dholka, Gujarat. All the animals were acclimatized for 7 days prior to the commencement of treatment and were maintained under controlled condition with 12 h light and 12 h dark cycles at temperature of 26 ± 2 ◦ C and relative humidity 30–70%. Animals were randomized into control and experimental group and were caged separately. Standard chow (obtained from Amrut Laboratory, Baroda, India) and water was given ad libitum. Experiments were conducted in accordance with the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India and experimental protocols were approved by the institutional animal ethics committee (167/1999/CPCSEA). Chloroquine phosphate (99.30% pure) was generously gifted from IPCA Laboratories Ltd., India. Curcumin and other chemicals were procured from Himedia Laboratories, India and Sigma–Aldrich (UK). All the chemicals used were of AR grade.

2.2. Experimental design Stock solution of CQ was prepared in double distilled water and orally given to mice with feeding canula using a hypodermic syringe. All the doses for CQ were derived from its oral LD50 value (500 mg/kg; Walum, 1998). The dose for curcumin is based on earlier work done (Pari and Amali, 2005). Animals were divided into following seven groups: Group I: Control (given distilled water only). Group II: Given 80 mg/kg body wt. curcumin. Group III: Low dosage group (given 100 mg/kg body wt. CQ). Group IV: Moderate dosage group (given 200 mg/kg body wt. CQ). Group V: High dosage group (given 300 mg/kg body wt. CQ). Group VI: High dosage of CQ + curcumin (300 mg/kg body wt. + 80 mg/kg body wt.). Group VII: Withdrawal group (high dose treatment for 45 days followed by withdrawal period of 45 days). All the groups were treated for a 45-day period. At the end of each treatment, animals were weighed and sacrificed using light ether anesthesia.

2.7. Alkaline phosphatase activity (ALKase) ALKase activity was estimated using the method of Bessey et al. (1946). Absorbance was taken at 410 nm and it was expressed as ␮moles of pnitrophenol/30 min/mg tissue wt.

2.8. Determination of thiobarbituric acid-reactive substances (TBARS) Level of Lipid Peroxidation was measured by the method of Ohkawa et al. (1979). Briefly, reaction mixture consist of 0.2 ml 8% SDS, 1.5 ml 20% acetic acid and 0.6 ml distilled water. 0.2 ml of tissue homogenate was added to the reaction mixture. Reaction was initiated by adding 1.5 ml of 1%TBA and terminated by 10% TCA. It was the centrifuged and absorbance was read at 532 nm. LPO was expressed in terms of nmoles MDA formed/mg tissue using an extinction coefficient of 1.56 × 105 M−1 cm−1 .

2.9. Measurement of antioxidant enzyme activity 2.9.1. Superoxide dismutase activity (SOD) SOD activity in liver of mice were estimated by using the technique of Kakkar et al. (1984) based on inhibition of the formation of nicotinamide adenine dinucleotide, phenazine methosulphate (PMS) and nitro blue tetrazolium formazan (NBT). Briefly, to 0.2 ml of homogenate is added 2.4 ml of 0.052 M sodium pyrophosphate buffer (pH 8.3), 0.1 ml of 186 ␮M PMS and 0.3 ml of 300 ␮M NBT. Reaction was initiated by addition of 0.2 ml 780 ␮M NADH. After 1 min, 4 ml of glacial acetic acid followed by 40 ml of N-butanol. Absorbance was measured at 560 nm. Results were expressed as unit of SOD/min/mg protein.

2.10. Statistics All the data are presented as mean ± SEM. Statistical analysis was performed using the SPSS software package version 16.0 (USA). Comparison between groups was made by one-way analysis of variance (ANOVA) taking significance at P < 0.05 followed by Student’s t-test taking significance at ***P < 0.001, **P < 0.005 and *P < 0.01. Tukey’s honestly significance difference (HSD) post hoc test was used for comparison among different treatment groups (P < 0.05).

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Table 3 Activities of ACPase and ALKpase in testis of control and treated animals.

Table 1 Body weight and organ weight of control and treated animals. Groups

Treatment

Body weight (g)

Liver (g)

Groups

Treatment

ACPase

ALKpase

I II III IV V VI VII

Control Curcumin CQ L.D. CQ M.D. CQ H.D. CQ H.D. + curcumin Withdrawal 45 days ANOVA

42.20 ± 0.25 41.10 ± 0.49NS 36.50 ± 0.51* 34.41 ± 0.26*** 29.22 ± 0.64*** 37.43 ± 0.53NS 35.87 ± 0.25NS 73.56©

1.892 ± 0.33 1.890 ± 0.56NS 1.789 ± 0.39* 1.665 ± 0.53*** 1.533 ± 0.49*** 1.756 ± 0.51NS 1.765 ± 2.54NS 59.26©

I II III IV V VI VII

Control Curcumin CQ L.D. CQ M.D. CQ H.D. CQ H.D. + curcumin Withdrawal 45 days ANOVA

0.93 ± 0.03 0.96 ± 0.06NS 1.20 ± 0.54* 1.36 ± 0.02** 1.52 ± 0.04*** 0.99 ± 0.01NS 1.09 ± 0.25* 144.60©

0.45 ± 0.02 0.44 ± 0.04NS 0.40 ± 0.02NS 0.35 ± 0.06* 0.22 ± 0.07*** 0.44 ± 0.08NS 0.40 ± 0.02NS 103.59©

Values are mean ± S.E. * P < 0.01. *** P < 0.001. © Significant analysis of variance at P < 0.05. NS Non-significant.

Values are mean ± S.E. ACPase and ALKpase activities are expressed ␮moles of pnitrophenol released/mg protein. * P < 0.01. ** P < 0.005. *** P < 0.001. © Significant analysis of variance at P < 0.05. NS Non-significant.

3. Results

Table 4 Activities of SDH and ATPase in testis of control and treated animals.

3.1. Terminal body weight and tissue weight Reduction in body weight was observed in low dose (P < 0.01), moderate and high dose (P < 0.001) of chloroquine (CQ) treated mice compared to control mice. While low dose, high dose with mitigating agent (curcumin) and the withdrawal group showed insignificant reduction in terminal body weight as compared to control values (Table 1). Maximum reduction, in body weight was observed in CQ treated mice at a dose level of high (300 mg/kg body wt.). Hepatic tissue weight declined following CQ treatment in a dose dependent manner. The reduction in tissue was significant (P < 0.001) when moderate dose and high dose were administered. The terminal body weight and organ weight did not show any change in CQ + curcumin treated mice, when compared with control mice. Mice treated with high dose of CQ and kept for a withdrawal period of 45 days, showed recovery in terminal body weight as well as tissue weight (Table 1). 3.2. Biochemical analysis Table 2 represents the protein content in the liver of CQ treated and withdrawal groups. Chloroquine administration significantly decreased protein content in the low dose (P < 0.01), moderate dose (P < 0.001) and high dose (P < 0.001) groups. Curcumin supplementation regulated the protein content and the values were found to be comparable to the control group. Protein level did not resurge to control values after the withdrawal period of 45 days, revealing only partial recovery. ANOVA was found to be 9.31. Effect of CQ and curcumin on the activities of ACPase and ALKpase is shown in Table 3. ACPase activity was significantly (P < 0.01; P < 005; P < 0.001) elevated in all the doses (low, moderate and Table 2 Protein content of control and treated animals. Groups

Treatment

Liver

I II III IV V VI VII

Control Curcumin CQ L.D. CQ M.D. CQ H.D. CQ H.D. + curcumin Withdrawal 45 days ANOVA

20.31 ± 0.28 20.90 ± 0.46NS 18.54 ± 0.89* 16.32 ± 0.91*** 13.94 ± 0.48*** 19.02 ± 0.23NS 17.36 ± 0.25*** 09.31©

Values are mean ± S.E. Protein expressed mg/100 mg tissue weight. * P < 0.01. *** P < 0.001. © Significant analysis of variance at P < 0.05. NS Non-significant.

Groups

Treatment

SDH

ATPase

I II III IV V VI VII

Control Curcumin CQ L.D. CQ M.D. CQ H.D. CQ H.D. + curcumin Withdrawal 45 days ANOVA

17.41 ± 1.23 17.54 ± 1.12NS 14.02 ± 0.99** 12.49 ± 0.87*** 9.39 ± 0.83*** 17.24 ± 1.05NS 16.28 ± 1.25NS 23.03©

3.24 ± 0.02 3.23 ± 0.08NS 3.01 ± 0.01* 2.78 ± 0.05** 2.17 ± 0.06*** 3.22 ± 0.10NS 3.10 ± 0.25NS 9.88©

Values are mean ± S.E. SDH activity expressed ␮g formazan fomed/15 min/mg protein. ATPase activity expressed ␮moles i.p. released/30 min/mg protein (i.p. – inorganic phosphate). * P < 0.01. ** P < 0.005. *** P < 0.001. © Significant analysis of variance at P < 0.05. NS Non-significant.

high dose) respectively following CQ treatment. Curcumin when co-supplemented with high dose of CQ, brought down the activity comparable to that of control. Activity of ALKpase declined significantly (P < 0.001; P < 0.01) in the liver at high and moderate dose respectively but low dosed mice showed insignificant decline. Curcumin administration appeared to reverse the toxic effects. In withdrawal group, activity of ACPase did not show recovery but ALKpase activity was restored and was comparable to control. Table 4 demonstrates the activities of SDH and ATPase in liver of CQ treated mice. Activity of SDH was significantly (P < 0.005) decreased in low dose, while (P < 0.001) in moderate and high dose. ATPase activity in liver showed significant (P < 0.01; P < 0.005; P < 0.001) decline in dose dependent manner. Curcumin therTable 5 Activities of SOD and TBARS in testis of control and treated animals. Groups

Treatment

SOD

TBARS

I II III IV V VI VII

Control Curcumin CQ L.D. CQ M.D. CQ H.D. CQ H.D. + curcumin Withdrawal 45 days ANOVA

0.61 ± 0.03 0.62 ± 0.08NS 0.46 ± 0.06NS 0.38 ± 0.09** 0.23 ± 0.03*** 0.56 ± 0.02NS 0.59 ± 0.05NS 179.75©

155.13 ± 1.34 165.12 ± 1.56NS 185.29 ± 1.99* 215.74 ± 1.88*** 267.01 ± 1.23*** 174.66 ± 1.46NS 169.00 ± 2.54NS 4543.01©

Values are mean ± S.E. SOD activity expressed units/mg protein. TBARS expressed nM of MDA/100 mg tissue weight. * P < 0.01. ** P < 0.005. *** P < 0.001. © Significant analysis of variance at P < 0.05. NS Non-significant.

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Plate 1. Normal hepatic cells showing well defined cytoplasm, prominent nucleus and clearly observed central portal vein, sinusoid and hepatic ducts.

apy reversed the values of these enzymes towards control and established protection. Withdrawal group exhibited insignificant recovery with values comparable to control (ANOVA for SDH 23.03 and for ATPase 9.88). Free radicals generated by chloroquine administration caused modulation in SOD activity; the first line of antioxidant defense (Table 5). CQ diminished the antioxidant status by decreasing the activity of hepatic SOD in moderate (P < 0.005) and high dose (P < 0.001) groups, while low dosed mice registered an insignificant decline. Treatment of curcumin along with CQ restored the activities of SOD towards control. Protective effect of curcumin on CQ induced experimental peroxidative damage is shown in Table 5. Hepatic LPO was enhanced after chloroquine administration significantly (P < 0.01) in low dose, moderate and high dose (P < 0.001) with all the three doses. Curcumin therapy along with high dose of CQ inhibited LPO and reduced hepatic peroxidative stress. Hence, TBARS level was comparable to control. In withdrawal group, LPO of treated mice did not show recovery. 3.3. Histopathological analysis 3.3.1. Control Normal hepatic cells could be observed in the control group liver sections (Plate 1), with well defined cytoplasm, prominent nucleus

Plate 2. Treated group (high dose of CQ) showing loss of hepatic architecture with centrilobular hepatic necrosis and vacuolization.

Plate 3. Treated group (high dose of CQ + curcumin) showing recovery in hepatocyte morphology and nuclear integrity, but persistence of vacuolization.

and clearly observed central portal vein, sinusoid and hepatic ducts. Hepatic cells showed normal morphology with uniform, radially arranged orientation 3.3.2. Treated group (high dose of CQ) Loss of hepatic architecture with centrilobular hepatic necrosis, vacuolization was found in the sections of liver of this treated group (Plate 2). Hepato nuclear pycnosis was observed. Enlargement of portal vein with necrosis of cells and vacuolization 3.3.3. Treated group (high dose of CQ + curcumin) Recovery could be observed in hepatocyte morphology and nuclear integrity in sections obtained from the ameliorated group (Plate 3). Normal hepatic organization was seen. However, vacuolization persisted in certain areas of tissue. 3.3.4. Withdrawal group Withdrawal of treatment had a minor effect on the liver histology (Plate 4). The hepatic cells did not show normal organization. There was evidence of hepatic necrosis and cellular atrophy with few vacuoles.

Plate 4. Withdrawal group showing minor effect on the liver histology, evidence of hepatic necrosis and cellular atrophy with few vacuoles.

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4. Discussion The present study demonstrates the curative potential of curcumin by reversing chloroquine induced hepatic damage. Reduction in body weight, organ weight, protein content and alterations in the activities of metabolic enzymes viz. SDH, ATPase, ALKpase, ACPase indicated deterioration in the hepatic function and cellular damage due to toxic effects of CQ. The oxidative stress results into toxicity when the rate at which reactive oxygen species (ROS) are generated exceeds scavenging capacity of the cell. Enhancement of LPO and reduction in SOD implicated oxidative damage in liver. Histopathology was also consistent with CQ induced damage in the concerned organ (Plate 2). Treatment by curcumin restored normal hepatic histoarchitecture (Plate 3) as well as various diagnostic biochemical variables towards normal indicating reversal of CQ induced hepatotoxicity and conforming the free radical scavenging property of curcumin. Curcumin, the yellow pigment in turmeric, has received attention as a promising dietary supplement for the protection against fibrogenic insults (Chuang et al., 2000; Park et al., 2000). Zheng and Chen (2004), have previously demonstrated that curcumin dramatically induced gene expression of peroxisome proliferatoractivated receptor (PPAR-␥) in activated hepatic stellate cells (HSC), which facilitated its trans-activation activity, leading to the inhibition of HSC proliferation, the induction of apoptosis, and the suppression of extracellular matrix (ECM) production (Xu et al., 2003; Zheng and Chen, 2004). They also observed that curcumin interrupted transforming growth factor-␤ (TGF-␤) signaling in activated HSC likely by suppressing gene expression of TGF-␤ receptors (Zheng and Chen, 2004, 2006). Hepatic stellate cells (HSC) are the primary source of excessive production and deposition of ECM during hepatic fibrogenesis (Bissell, 1998; Friedman, 2000). HSC activation, characterized by enhanced cell growth and overproduction of ECM, is triggered by the release of fibrogenic TGF-␤ from Kupffer cells and activated HSC (Schuppan et al., 1995). This process is coupled with up-expression of type I and II TGF-␤ receptors (14). In addition, HSC activation coincides with a dramatic reduction in expression of the PPAR␥ (Galli et al., 2000; Marra et al., 2000; Miyahara et al., 2000). TGF-␤ signaling is initiated by binding of active TGF-␤1 to type II TGF-␤ receptor (T␤-RII), which leads to the phosphorylation and activation of type I TGF-␤ receptor (T␤-RI) (Baker and Harland, 1997; Massague and Chen, 2000). The latter, in turn, phosphorylates Smad2 or 3, which subsequently form a complex with Smad4 and migrate into the nucleus to regulate expression of target genes (Paradis et al., 1999, 2002). Recent studies showed different roles of Smad2 and Smad3 in deposition of ECM components and cell proliferation in rat HSC (Uemura et al., 2005). TGF-␤ signaling via Smad3 played an important role in the morphological and functional maturation of HSC (Uemura et al., 2005). In response to a variety of endogenous and exogenous agonists, the nuclear receptor PPAR-␥ forms heterodimers with the retinoid X receptor and binds to peroxisome proliferator response elements (PPRE) in gene promoters to regulate the transcription of target genes (Houseknecht et al., 2002). PPAR-␥ is highly expressed in quiescent HSC in the normal liver (Galli et al., 2000; Marra et al., 2000; Miyahara et al., 2000). However, the level of PPAR-␥ and its activity are dramatically reduced during HSC activation in vitro and in vivo (Galli et al., 2000; Marra et al., 2000; Miyahara et al., 2000). The stimulation of PPAR-␥ activity by its agonists inhibits HSC proliferation and ␣1 (I) collagen expression in vitro and in vivo (Galli et al., 2002; Miyahara et al., 2000). Forced expression of exogenous PPAR-␥ cDNA itself is sufficient to reverse the morphology of activated HSC to the quiescent phenotype (Hazra et al., 2003). Results of our experiments showed significant reduction in body weight of toxicant treated animals. A decline in liver weight fol-

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lowing CQ treatment was also evident in a dose dependent manner which is in accordance of earlier reports (Zahid and Abidi, 2003). Pari and Amali in 2005 have reported liver damage, a life threatening toxic hepatitis at higher dosage of CQ (970 mg/kg body wt.) as well as impaired protein metabolism. Present study also showed dose dependent decrease in protein content. This decreased protein level; post treatment could be attributed to the low food intake, altered physiology or impairment in protein synthesis. The available literature also suggests that CQ cause liver damage and severe life threatening toxic hepatitis at higher dosage (Koranda, 1981; Dass and Shah, 2000; Pari and Murugavel, 2004). Zahid and Abidi (2003) showed that the lysomotropic property of CQ induces its uptake in the lysosomes leading to an increase in the size and number of hepatic lysosomes. Further, secondary increase in the certain enzymatic activities along with the increase in autophagy of cells exposed to CQ was also reported. Hence, this could probably justify the observed elevation in ACPase activity in liver of CQ treated mice. The positive ameliorative effects were seen in the said enzyme activity with curcumin co-administration for 45 days duration along with high dose of CQ treatment. A wide range of industrial pollutants is known to cause adverse effects on phosphatase activities. Alkaline phosphatases are a group of enzymes which hydrolyse phosphate at alkaline pH. Alkaline phosphatase has ubiquitous distribution in all tissues of the body especially in cell membrane where it is associated with the transport of metabolite across the membrane. It is highly sensitive to different xenobiotics and its inhibition leads to disturbances in cellular functions (Thaker et al., 1997). The activity of ALKpase decreased in a dose dependent manner by CQ treatment, which was restored by curcumin co-administration. Withdrawal of high dose treatment for 45 days showed insignificant decrease in ALKpase activity revealing partial recovery as compared to the antidote treated group. Activities of succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase) declined significantly in liver of chloroquine treated mice. SDH is an oxidative enzyme involved in Krebs cycle, catalyzing the conversion of succinate to fumarate and can alter the rate of reactions in the Krebs cycle, thereby altering the energy metabolism of the tissue. Moreover, SDH and ATPase being a mitochondrial enzyme, a decline in their activity indicate a possible alteration in mitochondrial structure and function. Superoxide dismutase (SOD) has an antitoxic effect against the superoxide anion. SOD accelerates the dismutation of superoxide to H2 O2 which in turn is removed by catalase (Usoh et al., 2005). Thus, SOD can act as a primary defense against superoxide anion and prevents further generation of free radicals (El-Demerdash et al., 2009). The decreased SOD activity in hepatic tissue suggests that the accumulation of superoxide anion radical might be responsible for increased lipid peroxidation following CQ treatment, which is evident in our study. CQ acts directly or indirectly and alters antioxidant status that makes hepatocytes, more susceptible to oxidative stress. Several studies have shown that CQ causes increased lipid peroxidation and decreased enzymatic and non-enzymatic antioxidants, which is in agreement to the data obtained in our study (Magwere et al., 1997; Murugavel and Pari, 2004). Lipid peroxidation is a free radical mediated process that has been implicated in a variety of disease states. Lipid peroxidation involves the formation and propagation of lipid radicals, the uptake of oxygen and a rearrangement of the double and unsaturated lipids, resulting in a variety of degraded products that eventually cause destruction of membrane lipids. Increased peroxidation can result in changes in cellular metabolism of hepatic and extra hepatic tissues (Das et al., 2001). In the present study, lipid peroxidation was assayed as marker of oxidative damage in the liver of mice treated with CQ. Results of our investigation revealed a significant increase in the TBARS level in liver of treated mice. This suggests an

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increased peroxidation of lipids, with contaminant loss of cellular functions in this organ by CQ feeding. Curcumin a known scavenger of free radicals, when administered in the current study, efficiently lowered the peroxidation levels thus protecting tissues from oxidative stress. In agreement with Pari and Amali (2005), data obtained in the present study also showed that curcumin alone significantly decreased the levels of TBARS. Withdrawal of high dose treatment for a period of 45 days did not record reversal, as mice retained significant peroxidative effects even after the withdrawal period. In the present study treatment with curcumin along with CQ reduced toxic effect of CQ in liver as most of the biochemical indices and enzyme activity were comparable to the control showing the ameliorative role of curcumin. Metabolic biomarkers like SDH, ATPase were restored to normal values in liver. LPO level was reduced in liver of mice. Similarly enzymatic (SOD) component of defense system was revived in liver by curcumin co-supplementation. Curcumin alone did not show any adverse effects on all indices of metabolite and its value is comparable to the control. In accordance with our data several reports have indicated a protective effect of curcumin against many known toxicants. The antioxidant activity of curcumin was reported by Sharma as early as 1976. It acts as a scavenger of oxygen free radicals, it can protect haemoglobin from oxidation (Subramaniam et al., 1994; Unnikrishnan and Rao, 1995). It also lowers the production of ROS in vivo (Joe and Lokesh, 1994). Its derivatives, demethoxycurcumin and bis-demethoxycurcumin also have antioxidant effect (Unnikrishnan and Rao, 1995). Pulla Reddy and Lokesh (1994) have demonstrated decreased lipid peroxidation in rat liver microsomes, erythrocyte membranes and brain homogenates. The antioxidant mechanism of curcumin is attributed to its unique conjugated structure, which includes two methoxylated phenols and an enol form of ␤-diketone; the structure shows typical radical-trapping ability as a chain-breaking antioxidant (Sreejayan and Rao, 1994; Masuda et al., 2001). Masuda et al. (2001) further studied the antioxidant mechanism of curcumin using linoleate as an oxidizable polyunsaturated lipid and proposed that the mechanism involves oxidative coupling reaction at the 3 -position of the curcumin with the lipid. Priyadarshini et al. (2003) tested the antioxidant activity of curcumin and dimethoxy curcumin by radiation-induced lipid peroxidation in liver microsomes of rat. It is evident therefore that curcumin exerts significant protection against CQ induced toxicity due to its antioxidant activity, though a minor recovery was observed in withdrawal group, the recovery was not as significant as that observed in the animals treated with curcumin. Thus CQ can bring about irreversible toxic effect in tissue, hence use of strong antioxidants like curcumin should be recommended with CQ for treating malaria, so as to avoid the toxic influences of the above-mentioned drug. Conflicts of Interest None.

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