Mol Biol Rep DOI 10.1007/s11033-014-3292-5
Protective effect of ellagic acid against TCDD-induced renal oxidative stress: Modulation of CYP1A1 activity and antioxidant defense mechanisms Viswanadha Vijaya Padma • Palaniswamy Kalai Selvi Samadi Sravani
•
Received: 16 March 2013 / Accepted: 13 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) belongs to toxicologically important class of poly halogenated aromatic hydrocarbons and produce wide variety of adverse effects in humans. The present study investigated the protective effect of ellagic acid, a natural polyphenolic compound against TCDD-induced nephrotoxicity in Wistar rats. TCDD-induced nephrotoxicity was reflected in marked changes in the histology of kidney, increase in levels of kidney markers (serum urea, serum creatinine) and lipid peroxides. A significant increase in activity of phase I enzyme CYP1A1 with concomitant decline in the activities of phase II enzymes [non-enzymic antioxidant and various enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione-stransferase] was also observed. In addition, TCDD treated rats showed alterations in ATPase enzyme activities such as Na? K?-ATPase, Mg2? ATPase and Ca2? ATPase. Oral pre-treatment with ellagic acid prevented TCDD-induced alterations in levels of kidney markers. Ellagic acid pretreatment significantly counteracted TCDD-induced oxidative stress by decreasing CYP1A1 activity and enhancing the antioxidant status. Furthermore, ellagic acid restored TCDD-induced histopathological changes and alterations in ATPase enzyme activities. The results of the present study show that significant protective effect rendered by ellagic acid against TCDD-induced nephrotoxicity might be attributed to its antioxidant potential.
V. Vijaya Padma (&) P. Kalai Selvi S. Sravani Department of Biotechnology, School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641046, Tamil Nadu, India e-mail: [email protected]
Keywords ATPase CYP1A1 Oxidative stress Reactive oxygen species TCDD Abbreviations AhR Aryl hydrocarbon receptor ARNT Aryl hydrocarbon receptor nuclear translocator CAT Catalase EA Ellagic acid GPx Glutathione peroxidase GSH Glutathione GST Glutathione-s-transferase ROS Reactive oxygen species SOD Superoxide dismutase TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin XRE Xenobiotic responsive elements
Introduction 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic and the representative compound of dioxins. TCDD exposure results in adverse effects such as wasting syndrome, dermal toxicity, immunotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, reproductive disorders, endocrine disruption, neurotoxicity and numerous other biochemical alterations [1–3]. TCDD is unintentionally produced from various manufacturing industries and during incineration of municipal and industrial wastes. The released TCDD and their congeners, are found in the environment including air, food, and soil. The exposure of TCDD among the general population mainly occurs through dietary consumption of meat, dairy products and fish. Since, TCDD is lipophilic in nature it tends to bioaccumulate in the food
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chain. The half-life of TCDD is estimated to be 5.8–11.3 years in humans [4]. Adverse health effects induced by TCDD are mediated through aryl hydrocarbon receptor (AhR) signaling mechanism [5, 6]. The AHR is a ubiquitous cytosolic protein, which gets activated upon binding to TCDD and translocates into nucleus and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR/ARNT heterodimer binds to xenobiotic responsive elements (XREs) and activates transcription of numerous phase I and II xenobiotic-metabolizing genes. One of the most sensitive targets of this heterodimer is CYP1A1 gene [7]. Cytochrome P450 is a super family of microsomal hemoproteins involved in the process of chemically induced carcinogenesis involved in conversion of procarcinogens into ultimate carcinogens [8]. During such metabolic process it generates reactive oxygen species and initiates oxidative stress [9]. ROS mediates toxic responses by creating an atmosphere of oxidative stress by increased lipid peroxidation and decreased antioxidant status [10]. Ellagic acid (EA), a natural polyphenolic antioxidant found in high amounts in the fruits and seeds of raspberries, strawberries, blueberries, pomegranates, walnuts and other plant foods, is being hailed as a potent antioxidant, possessing broad chemo protective properties against a variety of different carcinogens [11]. EA has been found to possess cardioprotecive, hepatoprotective and nephroprotective properties against various conditions of oxidative stress [12– 15]. Recently Hseu et al. [16] have reported that Nrf-2 is believed to play a role in protective effect of EA against UVA induced oxidative stress and apoptosis. Previous reports have documented that EA exhibits better protection than vitamin E against TCDD- induced fetal growth redardation [17]. However TCDD-induced nephrotoxicity is sparsely reported in the literature and protection offered by antioxidants against these effects is not well understood. So the present study, aimed at studying TCDD-induced oxidative stress in kidney tissue and extent of protection offered by EA against these effects. Protection offered by EA was evaluated by determining kidney markers (serum urea and creatinine), antioxidant status (GSH, SOD, CAT, GST and GPx), lipid peroxidation levels, ATPase enzyme activities (Na? K? ATPase, Ca2? ATPase and Mg2? ATPase) and CYP1A1 activity.
Materials and methods Chemicals TCDD was a kind gift from Dr. Howard G. Shertzer, Professor, Director, Environmental Genetics and Molecular Toxicology Division, University of Cincinnati. Ellagic Acid (purity [99 %), 7-ethoxy resorufin, dicumarol, resorufin were purchased from Sigma Aldrich Private
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Limited, Bangalore, India. All other chemicals were purchased from Hi Media Laboratories, Mumbai, India. Animals Male albino wistar rats weighing between 150 and 200 g were procured from Kerala Agricultural University Mannuthy, Kerala, India. The study protocol was approved from the Institutional Animal Ethics Committee constituted in accordance with the rules and guidelines of the Committee for the purpose of control and supervision of experiments and animals (CPCSEA), India. The rats were maintained in a controlled environment with standard temperature (28 ± 2 °C) and humidity with an alternating light and dark cycle. The animals were fed with commercially available pelleted rat chow (Sai Durga Private Limited, Bangalore, India) and water ad libitum. After a week of acclimatization, animals were randomly divided into control and test groups with six rats maintained in each group. Treatment schedule Rats were given intra peritoneal injection of TCDD (15 lg/kg body weight) dissolved in 0.15 ml of corn oil) and EA (10 mg/kg body weight) dissolved in 0.15 ml of 50 % DMSO was administered orally. Experimental procedure The animals were divided into six groups with 6 rats in each group. The dosing protocol of TCDD (15 lg/kg body weight) was chosen from the previous reports of Shertzer et al. [18], which showed that such a dosing schedule intensifies TCDD inducible oxidative stress response. EA dose (10 mg/kg body weight) was fixed based on the reports of Turk et al. [19] and our preliminary studies also confirmed the same. Groups
Concentration and volume of toxin/ antioxidant/vehicle administered
Treatment period
Sacrifice
Group I (control)
–
–
On 21st day
Group II (DMSO— vehicle for EA)
0.15 ml of 50 % DMSO administered/day
From 1st– 10th day
On 21st day
Group III (corn oil—vehicle for TCDD)
0.15 ml of corn oil administered/day
From 11th– 13th day
On 21st day
Group IV (EA)
10 mg of EA/kg body weight dissolved in 0.15 ml of 50 % DMSO per day by oral administration
From 1st– 10th day
On 21st day
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Groups
Concentration and volume of toxin/ antioxidant/vehicle administered
Treatment period
Sacrifice
Group V (TCDD)
15 lg TCDD/kg body weight dissolved in 0.15 ml corn oil per day by intraperitoneal injection
From 11th– 13th day
On 21st day
10 mg of EA/kg body weight dissolved in 0.15 ml of 50 % DMSO per day by oral administration
From 1st– 10th day
15 lg TCDD/kg body weight dissolved in 0.15 ml corn oil per day by intraperitoneal injection
From 11th– 13th day
Group VI (pretreatment with EA followed by TCDD treatment)
On 21st day
After the treatment period the animals were fasted overnight, weighed and anesthetized by exposing to diethyl ether. Blood was collected from jugular vein and serum was separated and used for kidney marker enzyme assays. The kidney tissues were dissected out, washed in chilled physiological saline, patted dry and weighed. A small portion of the kidney tissue was stored in 10 % formalin for histopathological examination. The remaining tissue was stored at -80 °C for further analysis. About 100 mg kidney tissue was weighed and homogenized in chilled 0.1 M Tris–HCl buffer in Potter–Elvehjem Teflon homogenizer. The homogenates were centrifuged at 3,000 rpm for 15 min and supernatant was aliquoted and used for assessing oxidative stress parameters and ATPase enzyme activities. Serum kidney marker analysis Blood samples were allowed to clot at room temperature and then centrifuged at 1,200 g for 15 min. The clear serum was separated and used for biochemical assays. Renal function was assessed by measurement of serum urea [20] and serum creatinine levels [21]. Histopathology A small portion of kidney tissue from the experimental animals was fixed in 10 % neutral buffered formalin and processed by standard procedure for paraffin embedding
and serial sections of about 5 lm sizes were cut and were stained with hematoxylin and eosin (H and E) dyes and studied under light microscope. Assessment of oxidative stress status and ATPases activities Oxidative stress was measured by estimating the enzymic and non-enzymic antioxidants such as catalase (CAT) [22], superoxide dismutase (SOD) [23], glutathione-s-transferase (GST) [24], glutathione peroxidase (GPX) [25], reduced glutathione (GSH) [26] and thio barbituric acid reactive substances (TBARS) [27]. Membrane bound ATPase enzyme activities namely Na? K? ATPase [28], Ca2? ATPase [29] and Mg2? ATPase [30] were assayed in the tissue homogenate. Metabolic activation of TCDD by CYP1A1 Microsomes from kidney tissues were isolated by the method described by Walawalkar and Iyer, [31] and CYP1A1 activity (EROD) was determined by the method of Peters et al. [32]. Statistical analysis The data was analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.
Results Effect of EA on renal biomarkers and histopathological alterations induced by TCDD The biomarkers of renal function, serum urea and creatinine were estimated in the present study. The TCDD induced kidney toxicity was reflected in the significant increase (P \ 0.01) in levels of these two kidney markers when compared to control rats. Pre-treatment with EA followed by TCDD treatment significantly decreased (P \ 0.001) their levels in serum when compared to TCDD alone treated group. Rats treated with corn oil/DMSO/EA alone did not show any change in the serum kidney markers when compared to control rats (Fig. 1). Histological changes on TCDD treatment showed sclerosed glomerulus with lose of shape, shrinkage and increase in interstium (Fig. 2a, b) shows interstitial inflammation with dilated tubules. Histopathological examination of control (Fig. 3a) and rats which received DMSO (Fig. 3b)/corn oil (Fig. 3c)/EA (Fig. 3d) alone showed normal kidney tissue architecture with prominent shape of glomerules and tubules. Rats pre-treated with EA followed by TCDD
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Fig. 1 Effect of ellagic acid and TCDD on renal markers a serum creatinine b serum urea. Group I control, Group II DMSO (vehicle for EA), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDD, Group VI pre-treatment of EA followed by TCDD treatment. Each bar represents, mean ± standard error of mean (SEM) of each
group. **P \ 0.01, when compared to control. ###P \ 0.001, when compared to TCDD treated group. NS non significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison)
Fig. 2 Histopathological alterations induced by TCDD in kidney of wistar rats. a Sclerosed glomeruli with lose of shape, shrinkage and increase in interstium b interstitial inflammation, sclerosed glomeruli and dilated tubules. (H&E, magnification 9400)
Fig. 3 Histopathology of rats treated with ellagic acid and TCDD. a Control rats, b rats treated with DMSO (solvent control for ellagic acid), c rats treated with corn oil (solvent control for TCDD), d rats treated with ellagic acid (10 mg/kg body weight), showing typical
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kidney morphology with normal glomeruli and tubules. e Rats pretreated with ellagic acid (10 mg/kg body weight) followed by TCDD (15 lg/kg body weight) shows normal glomeruli with no signs of sclerosis and inflammation. (H&E, magnification 9400)
treatment (Fig. 3e) showed normal kidney architecture which was comparable to that of control rats. Protective effect of EA on oxidative stress induced by TCDD
##
P \ 0.01,
###
P \ 0.001, when compared to TCDD treated group
The results of the present study showed a significant increase (P \ 0.001) in the levels of TBARS with concomitant decrease (P \ 0.001) in the levels of non- enzymic antioxidant (GSH) in rats treated with TCDD when compared to control. The activities of various antioxidant enzymes like SOD, CAT, GST and GPX were found to be significantly decreased (P \ 0.001) in TCDD treated rats when compared to control. Pre-treatment with EA followed by TCDD treatment significantly increased the antioxidant status with SOD, GPX (P \ 0.001) and GSH, GST, CAT (P \ 0.01) whereas the levels of TBARS significantly decreased (P \ 0.001) when compared to TCDD treated rats. Rats treated with EA/corn oil/DMSO showed nonsignificant levels when compared to control rats (Table 1).
*** P \ 0.001, when compared to control.
2.4 ± 0.01 5.4 ± 0.05 5.4 ± 0.04 5.0 ± 0.05 GST
5.1 ± 0.04
59 ± 1.12 GPX
Group I control, Group II DMSO (vehicle for ellagic acid), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDD, Group VI pre-treatment of EA followed by TCDD treatment. Values are mean ± standard error of mean of each group. NS not significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison). Units are expressed as: LPO in nanomoles of TBA reactants/mg of protein, GSH in nanomoles of GSH/g of tissue, SOD, CAT, GST and GPx activities in Units/mg protein. SOD 1 U the amount of enzyme required to give 50 % inhibition of pyrogallol auto-oxidation. Catalase 1 U the amount of enzyme that consumes 1 nmol of H2O2/min, GST 1 U the amount of enzyme that conjugates 1 lmol CDNB/min, GPx 1 U the amount of enzyme that converts 1 l mole GSH to GSSG in the presence of H2O2/min
4.2 ± 0.02###
49 ± 0.96##
*** NS NS
0.030 ± 0.002## 0.050 ± 0.001 CAT
56 ± 1.28
59 ± 1.09
NS
58 ± 0.90
41 ± 1.65
***
0.010 ± 0.002 0.049 ± 0.002
NS NS
0.041 ± 0.003
98 ± 1.60### 119 ± 2.10 SOD
0.042 ± 0.001
NS
*** NS
32 ± 1.70 120 ± 2.10
45 ± 2.00 GSH
115 ± 1.90
NS
NS NS
111 ± 1.54
41 ± 1.30NS 42 ± 1.90NS
30.65 ± 1.50 32.00 ± 1.11 LPO
30.21 ± 1.17
NS
*** NS
37 ± 1.06## 22 ± 1.24*** 42 ± 1.12NS
39.20 ± 1.37### 31.45 ± 1.22
72.45 ± 1.45
***
Group V Group IV
NS NS
Group III Group II Group I Parameters
Table 1 Effect of ellagic acid on TCDD-induced oxidative stress and antioxidant status in experimental rats
NS
Group VI
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Effect of EA and TCDD on ATPases enzyme activities The rats treated with TCDD showed a significant decrease in both Na?/K? ATPase (P \ 0.001) and Mg2? ATPase (P \ 0.01) activities and significant increase in the Ca2? ATPase (P \ 0.001) activity when compared to control rats. In rats pre-treated with EA followed by TCDD treatment showed a marked increase in Na?/K? ATPase (P \ 0.01) and Mg2? ATPase activities (P \ 0.01) with significant decrease (P \ 0.01) in Ca2? ATPases activity when compared to TCDD alone treated rats. Whereas rats which received EA/corn oil/DMSO alone did not show any significant change in ATPases activities when compared to control rats (Fig. 4). Inhibition of metabolic activation of TCDD by EA EROD activity describes the rate of the CYP1A mediated de ethylation of the substrate 7-ethoxyresorufin (7-ER) to form the product resorufin. The catalytic activity towards this substrate is an indication of the amount of enzyme present and is measured as the concentration of resorufin produced [33]. The results are expressed as percentage fold induction of EROD activity with control values considered as 100 %. In the present study, the activities of CYP1A1 were found to be significantly increased (P \ 0.01) in TCDD treated rats when compared to control rats. In rats pre-treated with EA followed by TCDD treatment there was a significant (P \ 0.001) decrease in activity when compared to TCDD treated rats. Rats which received EA/Corn oil/DMSO alone did not show significant change in the activity when compared to control rats (Fig. 5).
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Mol Biol Rep Fig. 4 Effect of EA on TCDDinduced changes in membrane bound ATPase enzyme activities. Group I control, Group II DMSO (vehicle for DMSO), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDD, Group VI pre-treatment of EA followed by TCDD treatment. Each bar represents mean ± standard error of mean of each group. ***P \ 0.001, **P \ 0.01 when compared to control. ## P \ 0.01, when compared to TCDD treated group. NS non significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison)
Fig. 5 Effect of EA on TCDD-induced CYP1A1 activity. Group I control, Group II DMSO (vehicle for DMSO), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDDm, Group VI pretreatment of EA followed by TCDD treatment. Each bar represents mean ± standard error of mean of each group. ***P \ 0.001, when compared to control. ###P \ 0.001, when compared to TCDD treated group. NS non significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison)
Discussion TCDD is classified as a group I carcinogen by the International Agency for Research on Cancer [34]. TCDD is resistant to degradation which allows it to bioaccumulate in food chain [35]. Since liver is the target organ for detoxification of environmental toxins, most of the studies have focused on TCDD-induced liver toxicity. Recent reports show that TCDD also causes adverse effects in various
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extra hepatic tissues and sparse literature is available on TCDD induced nephrotoxicity and its protection by antioxidants. Lu et al. [36] has first demonstrated combined effect of TCDD and PCB congeners on kidney toxicity in rats. Ciftci et al. [37] have reported protective effect offered by quercetin and chrysin on TCDD induced renal damage by evaluating the antioxidant status. In present study the antioxidant ellagic acid was evaluated for its protective effect by assessing CYP1A1 activity (prime candidate involved in the generation of ROS), ATP ase enzyme activities and serum kidney markers in addition to oxidative stress parameters in TCDD-induced nephrotoxicity in male wistar rats. Phenolic antioxidants play a pivotal role in offering protection during various oxidative stress conditions. EA (gallic acid dimer), a naturally occurring polyphenolic compound possesses lipophilic and free radical scavenging property. EA exhibits antioxidant [11], anti-proliferative [38] and anti-mutagenic properties [39]. Studies on structure–function relationship of EA revealed that phenolic hydroxyl group and lactone are responsible for its protective action against various conditions of oxidative stress [40]. So, the present study evaluated the oxidative stress responses induced by TCDD in kidney of wistar rats and possible protective effect of EA on TCDD induced nephrotoxicity. Histopathological observation and levels of serum kidney markers reveal that TCDD causes severe damage to the kidney tissues. The rats treated with TCDD showed sclerosis in glomeruli with interstitial inflammation and dilation in distal tubules. The observed changes are consistent with earlier reports of Ciftci et al. [37] and Lu et al. [36]. Along with these changes the present study observed that
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TCDD also induced inflammation and damages to the proximal and distal tubules. Pre-treatment with EA rendered significant protection against the histological changes induced by TCDD by maintaining the near normal architecture of glomerulus and tubules. The results of the present study corroborates with the earlier report of Atessahin et al. [15], where EA offered protective effect by reducing the histopathological alterations against cisplatininduced nephrotoxicity. Oxidative stress results when production of reactive oxidative species exceeds the capacity of cellular antioxidant defenses to remove these toxic species. Oxidative stress conditions induced by various xenobiotics result from upstream signaling mechanisms and in the case of TCDD, AhR play an important role in mediating the toxicity. Upon TCDD binding to AhR, it gets translocated to the nucleus where it associates with ARNT. The whole complex then acts as a transcription factor that binds to DNA promoter sequences termed xenobiotic responsive elements, thereby enhancing the transcription of array of genes [41]. Out of which induction of CYP1A1 by AhR is of toxicological relevance. TCDD acts as substrate for CYP1A1 and the formed metabolites potentially initiates toxic responses by increasing oxidative stress status [42, 43]. The ROS generated can directly react with cellular membrane phospholipid moieties due to the presence of their methylene group [44]. Polyunsaturated fatty acid oxidation yields short-lived lipid hydroperoxide molecules known to further perpetuate ROS production [45]. Lipid peroxidation and oxidative stress have been indicated as factors which lead to acute toxicity of TCDD [46, 47]. The cells exhibit various non- enzymic and enzymic antioxidants which act as natural defense mechanisms to counteract the oxidative stress conditions. Of which, reduced glutathione (GSH) is a non protein thiol which is actively present inside the cells and acts as first defense against oxidative attack. It participates in a variety of detoxification reactions, in addition to providing protection against oxidative damage by virtue of its thiol group. Peroxide level and thiol status is indicative of oxidative stress conditions, since increase in lipid peroxidation causes depletion in GSH levels [48]. So, in the present study these two levels were measured to analyze the extent of oxidative stress caused by TCDD. In the present study there was a significant decrease in total GSH and with concomitant increase in lipid peroxide levels in TCDD treated rats. Earlier report by Stohs, [49] also showed decreased thiol levels and increased lipid peroxide levels in mitochondria of TCDD treated rats. In rats pre-treated with EA followed by TCDD treatment there was a significant increase in GSH levels with concomitant decrease in lipid peroxide levels. Thus EA by its potent free radical scavenging property and glutathione sparing action might have
attenuated the oxidative stress status invoked by TCDD. The free radical scavenging property of EA leads to the inhibition of LPO and 8-OGDG formation both in vitro and in vivo [50, 51]. Protection offered by EA by decreasing the levels of lipid peroxides on fetotoxicity induced by TCDD in C57BL/67 mice [17] and increasing the levels of GSH in N-nitrosodiethylamine induced lung tumorigenesis in mice [52] has been reported earlier which supports the present findings. Apart from GSH, various antioxidant enzymes are involved to overcome the conditions of oxidative stress. Optimum levels and proper balance among these enzymes may be critical for the prevention of cellular oxidative stress. In the present study, a significant decrease in the activities of the antioxidant enzymes (SOD, GSH, GPx, and CAT) reflects TCDD-induced oxidative stress in renal tissue. The observed decline in antioxidant status after TCDD treatment is in line with the previous reports of Jin et al. [53] who has demonstrated significant decrease in the activities of antioxidant enzymes in reproductive system of mouse treated with TCDD. In addition, a dose-dependent suppression of SOD activity in cerebral cortex and hippocampus regions following TCDD treatment has been reported earlier by Hassoun et al. [54]. It could be said that, TCDD-induced decrease in activities of antioxidant enzymes might be caused by the expense of these enzymes in overcoming the oxidative stress conditions. In the present study, pre-treatment of EA followed by TCDD treatment significantly improved the antioxidant status. EA has been shown to offer protection by its free radical scavenging action and improved antioxidant status through Nrf-2 expression [11, 16]. The observed results on increase in antioxidant status and nephroprotective action of EA might be due to the above mentioned properties of EA. In addition, the ability of EA to render protective effect by enhancing the antioxidant mechanism against various oxidative conditions, support the present study findings. Das et al. [55] has shown protection offered by EA through increasing GST activity in liver and lungs of BALB/c mice against oxidative stress induced by benzo[a]pyrene. Protective effect of EA by improving the antioxidant status against selenite induced cataractogenesis [56] and cisplatin induced injuries to sperm [19] has been reported earlier. The membrane-bound enzymes such as Na?, K?-ATPase, Mg2?-ATPase and Ca2?-ATPase are responsible for the transport of sodium/potassium, magnesium and calcium ions across the cell membranes at the expense of ATP hydrolysis [57]. It is important to evaluate membrane bound ATPases enzyme activity during stress conditions, since changes in the activities of these enzymes affect the normal functioning of any organ. It is well documented that TCDD-induced Ca2? signaling plays a major role in TCDD-induced toxic
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events [58–60]. TCDD-dependent increase in intracellular calcium levels activates phospholipase A2, which in turn mobilizes arachidonic acid from cell membrane. The released arachidonic acid acts as a substrate for COX-2 and initiates eicosanoid synthesis. TCDD induced early and sustained increase in intracellular free calcium and subsequent formation of eicosanoids has been reported by Puga et al. [59]. Studies have documented that eicosanoids, especially arachidonic acid metabolites have profound inhibitory action on Na?, K?-ATPase and Mg2?-ATPase activities [61]. The results of the present study reveal a significant decrease in Na?, K?-ATPase and Mg2?-ATPase activities during TCDD treatment when compared to control rats. Decline in Na?, K?-ATPase activity after TCDD treatment has been reported in liver tissues [62] and renal medullary cells [63]. However the Schwartzman et al. [63] has showed that arachidonic acid metabolite formed from cytochrome P450 played a major role in inhibition of in Na?, K?-ATPase activity. But it was interesting to note that there was a significant increase in Ca2?-ATPase activity in the present study on treatment with TCDD. It can be explained that an early and sustained increase in Ca2? levels might have increased the activity of Ca2?-ATPase as a cells adaptive mechanism to overcome Ca2? overloading and prevent subsequent calcium induced toxic events. From the results of the present study and available literature it can be said that TCDD-induced alterations in ATPase enzyme activities may be due the disturbances in Ca2? homeostasis and subsequent formation of eicosanoids. While EA pretreatment significantly restored the ATPase enzyme activities to values close to that of control rats. The ability of EA to maintain Ca2? homeostasis might be attributed to restore ATPase enzyme activities. To this end, Ou et al. [64] have reported that EA maintained Ca2? homeostasis in endothelial cells and thereby prevented oxidized low-density lipoprotein-induced apoptosis. An unpublished data from our laboratory also supports the fact that EA is involved in maintaining Ca2? homeostasis. Our laboratory has been involved in identifying the molecular mechanism of EA in offering cytoprotection against TCDD-induced toxic events by in vitro studies. From the results, we observed that EA significantly maintained the Ca2? homeostasis and prevented TCDD-induced oxidative stress in HepG2 cells. The above results indicate the protective nature of EA against TCDD-induced nephrotoxicity by enhancing antioxidant status and preventing oxidative stress. Since TCDD-induced sustained oxidative stress and other stress response is primarily mediated through TCDD-induced CYP1A1 activation and subsequent imbalance in the redox status [65, 66], we next determined whether EA had any effect on suppressing the activity of CYP1A1. Inhibition of CYP1A1 by natural compounds is an attractive strategy, as these compounds could prevent oxidative stress and subsequent
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induction of various toxic events. The present study demonstrates that EA pre-treatment significantly decreased TCDD-induced CYP1A1 activity. The reduction in CYP1A1 activity might be either due to direct binding of EA to CYP1A1 or it could be due to its AhR antagonism. Further studies are required to understand the exact molecular mechanism of EA mediated CYP1A1 inhibition. In conclusion, the present study demonstrates that TCDDinduced nephrotoxicity is mediated through sustained oxidative stress with altered ATPase enzyme activities. Our data provide clear evidence that over all protective effect offered by EA against TCDD-induced nephrotoxicity is mediated through suppressing CYP1A1 activity and enhancing the antioxidant mechanism. Thus the present study highlights that consumption of ellagic acid helps in protection against environmental toxins which primarily mediate their toxic effects through oxidative stress. Conflict of interest The authors declare that there are no conflict of interest.
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Protective effect of ellagic acid against TCDD-induced renal oxidative stress: Modulation of CYP1A1 activity and antioxidant defense mechanisms Viswanadha Vijaya Padma • Palaniswamy Kalai Selvi Samadi Sravani
•
Received: 16 March 2013 / Accepted: 13 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) belongs to toxicologically important class of poly halogenated aromatic hydrocarbons and produce wide variety of adverse effects in humans. The present study investigated the protective effect of ellagic acid, a natural polyphenolic compound against TCDD-induced nephrotoxicity in Wistar rats. TCDD-induced nephrotoxicity was reflected in marked changes in the histology of kidney, increase in levels of kidney markers (serum urea, serum creatinine) and lipid peroxides. A significant increase in activity of phase I enzyme CYP1A1 with concomitant decline in the activities of phase II enzymes [non-enzymic antioxidant and various enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione-stransferase] was also observed. In addition, TCDD treated rats showed alterations in ATPase enzyme activities such as Na? K?-ATPase, Mg2? ATPase and Ca2? ATPase. Oral pre-treatment with ellagic acid prevented TCDD-induced alterations in levels of kidney markers. Ellagic acid pretreatment significantly counteracted TCDD-induced oxidative stress by decreasing CYP1A1 activity and enhancing the antioxidant status. Furthermore, ellagic acid restored TCDD-induced histopathological changes and alterations in ATPase enzyme activities. The results of the present study show that significant protective effect rendered by ellagic acid against TCDD-induced nephrotoxicity might be attributed to its antioxidant potential.
V. Vijaya Padma (&) P. Kalai Selvi S. Sravani Department of Biotechnology, School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641046, Tamil Nadu, India e-mail: [email protected]
Keywords ATPase CYP1A1 Oxidative stress Reactive oxygen species TCDD Abbreviations AhR Aryl hydrocarbon receptor ARNT Aryl hydrocarbon receptor nuclear translocator CAT Catalase EA Ellagic acid GPx Glutathione peroxidase GSH Glutathione GST Glutathione-s-transferase ROS Reactive oxygen species SOD Superoxide dismutase TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin XRE Xenobiotic responsive elements
Introduction 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic and the representative compound of dioxins. TCDD exposure results in adverse effects such as wasting syndrome, dermal toxicity, immunotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, reproductive disorders, endocrine disruption, neurotoxicity and numerous other biochemical alterations [1–3]. TCDD is unintentionally produced from various manufacturing industries and during incineration of municipal and industrial wastes. The released TCDD and their congeners, are found in the environment including air, food, and soil. The exposure of TCDD among the general population mainly occurs through dietary consumption of meat, dairy products and fish. Since, TCDD is lipophilic in nature it tends to bioaccumulate in the food
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chain. The half-life of TCDD is estimated to be 5.8–11.3 years in humans [4]. Adverse health effects induced by TCDD are mediated through aryl hydrocarbon receptor (AhR) signaling mechanism [5, 6]. The AHR is a ubiquitous cytosolic protein, which gets activated upon binding to TCDD and translocates into nucleus and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR/ARNT heterodimer binds to xenobiotic responsive elements (XREs) and activates transcription of numerous phase I and II xenobiotic-metabolizing genes. One of the most sensitive targets of this heterodimer is CYP1A1 gene [7]. Cytochrome P450 is a super family of microsomal hemoproteins involved in the process of chemically induced carcinogenesis involved in conversion of procarcinogens into ultimate carcinogens [8]. During such metabolic process it generates reactive oxygen species and initiates oxidative stress [9]. ROS mediates toxic responses by creating an atmosphere of oxidative stress by increased lipid peroxidation and decreased antioxidant status [10]. Ellagic acid (EA), a natural polyphenolic antioxidant found in high amounts in the fruits and seeds of raspberries, strawberries, blueberries, pomegranates, walnuts and other plant foods, is being hailed as a potent antioxidant, possessing broad chemo protective properties against a variety of different carcinogens [11]. EA has been found to possess cardioprotecive, hepatoprotective and nephroprotective properties against various conditions of oxidative stress [12– 15]. Recently Hseu et al. [16] have reported that Nrf-2 is believed to play a role in protective effect of EA against UVA induced oxidative stress and apoptosis. Previous reports have documented that EA exhibits better protection than vitamin E against TCDD- induced fetal growth redardation [17]. However TCDD-induced nephrotoxicity is sparsely reported in the literature and protection offered by antioxidants against these effects is not well understood. So the present study, aimed at studying TCDD-induced oxidative stress in kidney tissue and extent of protection offered by EA against these effects. Protection offered by EA was evaluated by determining kidney markers (serum urea and creatinine), antioxidant status (GSH, SOD, CAT, GST and GPx), lipid peroxidation levels, ATPase enzyme activities (Na? K? ATPase, Ca2? ATPase and Mg2? ATPase) and CYP1A1 activity.
Materials and methods Chemicals TCDD was a kind gift from Dr. Howard G. Shertzer, Professor, Director, Environmental Genetics and Molecular Toxicology Division, University of Cincinnati. Ellagic Acid (purity [99 %), 7-ethoxy resorufin, dicumarol, resorufin were purchased from Sigma Aldrich Private
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Limited, Bangalore, India. All other chemicals were purchased from Hi Media Laboratories, Mumbai, India. Animals Male albino wistar rats weighing between 150 and 200 g were procured from Kerala Agricultural University Mannuthy, Kerala, India. The study protocol was approved from the Institutional Animal Ethics Committee constituted in accordance with the rules and guidelines of the Committee for the purpose of control and supervision of experiments and animals (CPCSEA), India. The rats were maintained in a controlled environment with standard temperature (28 ± 2 °C) and humidity with an alternating light and dark cycle. The animals were fed with commercially available pelleted rat chow (Sai Durga Private Limited, Bangalore, India) and water ad libitum. After a week of acclimatization, animals were randomly divided into control and test groups with six rats maintained in each group. Treatment schedule Rats were given intra peritoneal injection of TCDD (15 lg/kg body weight) dissolved in 0.15 ml of corn oil) and EA (10 mg/kg body weight) dissolved in 0.15 ml of 50 % DMSO was administered orally. Experimental procedure The animals were divided into six groups with 6 rats in each group. The dosing protocol of TCDD (15 lg/kg body weight) was chosen from the previous reports of Shertzer et al. [18], which showed that such a dosing schedule intensifies TCDD inducible oxidative stress response. EA dose (10 mg/kg body weight) was fixed based on the reports of Turk et al. [19] and our preliminary studies also confirmed the same. Groups
Concentration and volume of toxin/ antioxidant/vehicle administered
Treatment period
Sacrifice
Group I (control)
–
–
On 21st day
Group II (DMSO— vehicle for EA)
0.15 ml of 50 % DMSO administered/day
From 1st– 10th day
On 21st day
Group III (corn oil—vehicle for TCDD)
0.15 ml of corn oil administered/day
From 11th– 13th day
On 21st day
Group IV (EA)
10 mg of EA/kg body weight dissolved in 0.15 ml of 50 % DMSO per day by oral administration
From 1st– 10th day
On 21st day
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Groups
Concentration and volume of toxin/ antioxidant/vehicle administered
Treatment period
Sacrifice
Group V (TCDD)
15 lg TCDD/kg body weight dissolved in 0.15 ml corn oil per day by intraperitoneal injection
From 11th– 13th day
On 21st day
10 mg of EA/kg body weight dissolved in 0.15 ml of 50 % DMSO per day by oral administration
From 1st– 10th day
15 lg TCDD/kg body weight dissolved in 0.15 ml corn oil per day by intraperitoneal injection
From 11th– 13th day
Group VI (pretreatment with EA followed by TCDD treatment)
On 21st day
After the treatment period the animals were fasted overnight, weighed and anesthetized by exposing to diethyl ether. Blood was collected from jugular vein and serum was separated and used for kidney marker enzyme assays. The kidney tissues were dissected out, washed in chilled physiological saline, patted dry and weighed. A small portion of the kidney tissue was stored in 10 % formalin for histopathological examination. The remaining tissue was stored at -80 °C for further analysis. About 100 mg kidney tissue was weighed and homogenized in chilled 0.1 M Tris–HCl buffer in Potter–Elvehjem Teflon homogenizer. The homogenates were centrifuged at 3,000 rpm for 15 min and supernatant was aliquoted and used for assessing oxidative stress parameters and ATPase enzyme activities. Serum kidney marker analysis Blood samples were allowed to clot at room temperature and then centrifuged at 1,200 g for 15 min. The clear serum was separated and used for biochemical assays. Renal function was assessed by measurement of serum urea [20] and serum creatinine levels [21]. Histopathology A small portion of kidney tissue from the experimental animals was fixed in 10 % neutral buffered formalin and processed by standard procedure for paraffin embedding
and serial sections of about 5 lm sizes were cut and were stained with hematoxylin and eosin (H and E) dyes and studied under light microscope. Assessment of oxidative stress status and ATPases activities Oxidative stress was measured by estimating the enzymic and non-enzymic antioxidants such as catalase (CAT) [22], superoxide dismutase (SOD) [23], glutathione-s-transferase (GST) [24], glutathione peroxidase (GPX) [25], reduced glutathione (GSH) [26] and thio barbituric acid reactive substances (TBARS) [27]. Membrane bound ATPase enzyme activities namely Na? K? ATPase [28], Ca2? ATPase [29] and Mg2? ATPase [30] were assayed in the tissue homogenate. Metabolic activation of TCDD by CYP1A1 Microsomes from kidney tissues were isolated by the method described by Walawalkar and Iyer, [31] and CYP1A1 activity (EROD) was determined by the method of Peters et al. [32]. Statistical analysis The data was analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.
Results Effect of EA on renal biomarkers and histopathological alterations induced by TCDD The biomarkers of renal function, serum urea and creatinine were estimated in the present study. The TCDD induced kidney toxicity was reflected in the significant increase (P \ 0.01) in levels of these two kidney markers when compared to control rats. Pre-treatment with EA followed by TCDD treatment significantly decreased (P \ 0.001) their levels in serum when compared to TCDD alone treated group. Rats treated with corn oil/DMSO/EA alone did not show any change in the serum kidney markers when compared to control rats (Fig. 1). Histological changes on TCDD treatment showed sclerosed glomerulus with lose of shape, shrinkage and increase in interstium (Fig. 2a, b) shows interstitial inflammation with dilated tubules. Histopathological examination of control (Fig. 3a) and rats which received DMSO (Fig. 3b)/corn oil (Fig. 3c)/EA (Fig. 3d) alone showed normal kidney tissue architecture with prominent shape of glomerules and tubules. Rats pre-treated with EA followed by TCDD
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Fig. 1 Effect of ellagic acid and TCDD on renal markers a serum creatinine b serum urea. Group I control, Group II DMSO (vehicle for EA), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDD, Group VI pre-treatment of EA followed by TCDD treatment. Each bar represents, mean ± standard error of mean (SEM) of each
group. **P \ 0.01, when compared to control. ###P \ 0.001, when compared to TCDD treated group. NS non significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison)
Fig. 2 Histopathological alterations induced by TCDD in kidney of wistar rats. a Sclerosed glomeruli with lose of shape, shrinkage and increase in interstium b interstitial inflammation, sclerosed glomeruli and dilated tubules. (H&E, magnification 9400)
Fig. 3 Histopathology of rats treated with ellagic acid and TCDD. a Control rats, b rats treated with DMSO (solvent control for ellagic acid), c rats treated with corn oil (solvent control for TCDD), d rats treated with ellagic acid (10 mg/kg body weight), showing typical
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kidney morphology with normal glomeruli and tubules. e Rats pretreated with ellagic acid (10 mg/kg body weight) followed by TCDD (15 lg/kg body weight) shows normal glomeruli with no signs of sclerosis and inflammation. (H&E, magnification 9400)
treatment (Fig. 3e) showed normal kidney architecture which was comparable to that of control rats. Protective effect of EA on oxidative stress induced by TCDD
##
P \ 0.01,
###
P \ 0.001, when compared to TCDD treated group
The results of the present study showed a significant increase (P \ 0.001) in the levels of TBARS with concomitant decrease (P \ 0.001) in the levels of non- enzymic antioxidant (GSH) in rats treated with TCDD when compared to control. The activities of various antioxidant enzymes like SOD, CAT, GST and GPX were found to be significantly decreased (P \ 0.001) in TCDD treated rats when compared to control. Pre-treatment with EA followed by TCDD treatment significantly increased the antioxidant status with SOD, GPX (P \ 0.001) and GSH, GST, CAT (P \ 0.01) whereas the levels of TBARS significantly decreased (P \ 0.001) when compared to TCDD treated rats. Rats treated with EA/corn oil/DMSO showed nonsignificant levels when compared to control rats (Table 1).
*** P \ 0.001, when compared to control.
2.4 ± 0.01 5.4 ± 0.05 5.4 ± 0.04 5.0 ± 0.05 GST
5.1 ± 0.04
59 ± 1.12 GPX
Group I control, Group II DMSO (vehicle for ellagic acid), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDD, Group VI pre-treatment of EA followed by TCDD treatment. Values are mean ± standard error of mean of each group. NS not significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison). Units are expressed as: LPO in nanomoles of TBA reactants/mg of protein, GSH in nanomoles of GSH/g of tissue, SOD, CAT, GST and GPx activities in Units/mg protein. SOD 1 U the amount of enzyme required to give 50 % inhibition of pyrogallol auto-oxidation. Catalase 1 U the amount of enzyme that consumes 1 nmol of H2O2/min, GST 1 U the amount of enzyme that conjugates 1 lmol CDNB/min, GPx 1 U the amount of enzyme that converts 1 l mole GSH to GSSG in the presence of H2O2/min
4.2 ± 0.02###
49 ± 0.96##
*** NS NS
0.030 ± 0.002## 0.050 ± 0.001 CAT
56 ± 1.28
59 ± 1.09
NS
58 ± 0.90
41 ± 1.65
***
0.010 ± 0.002 0.049 ± 0.002
NS NS
0.041 ± 0.003
98 ± 1.60### 119 ± 2.10 SOD
0.042 ± 0.001
NS
*** NS
32 ± 1.70 120 ± 2.10
45 ± 2.00 GSH
115 ± 1.90
NS
NS NS
111 ± 1.54
41 ± 1.30NS 42 ± 1.90NS
30.65 ± 1.50 32.00 ± 1.11 LPO
30.21 ± 1.17
NS
*** NS
37 ± 1.06## 22 ± 1.24*** 42 ± 1.12NS
39.20 ± 1.37### 31.45 ± 1.22
72.45 ± 1.45
***
Group V Group IV
NS NS
Group III Group II Group I Parameters
Table 1 Effect of ellagic acid on TCDD-induced oxidative stress and antioxidant status in experimental rats
NS
Group VI
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Effect of EA and TCDD on ATPases enzyme activities The rats treated with TCDD showed a significant decrease in both Na?/K? ATPase (P \ 0.001) and Mg2? ATPase (P \ 0.01) activities and significant increase in the Ca2? ATPase (P \ 0.001) activity when compared to control rats. In rats pre-treated with EA followed by TCDD treatment showed a marked increase in Na?/K? ATPase (P \ 0.01) and Mg2? ATPase activities (P \ 0.01) with significant decrease (P \ 0.01) in Ca2? ATPases activity when compared to TCDD alone treated rats. Whereas rats which received EA/corn oil/DMSO alone did not show any significant change in ATPases activities when compared to control rats (Fig. 4). Inhibition of metabolic activation of TCDD by EA EROD activity describes the rate of the CYP1A mediated de ethylation of the substrate 7-ethoxyresorufin (7-ER) to form the product resorufin. The catalytic activity towards this substrate is an indication of the amount of enzyme present and is measured as the concentration of resorufin produced [33]. The results are expressed as percentage fold induction of EROD activity with control values considered as 100 %. In the present study, the activities of CYP1A1 were found to be significantly increased (P \ 0.01) in TCDD treated rats when compared to control rats. In rats pre-treated with EA followed by TCDD treatment there was a significant (P \ 0.001) decrease in activity when compared to TCDD treated rats. Rats which received EA/Corn oil/DMSO alone did not show significant change in the activity when compared to control rats (Fig. 5).
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Mol Biol Rep Fig. 4 Effect of EA on TCDDinduced changes in membrane bound ATPase enzyme activities. Group I control, Group II DMSO (vehicle for DMSO), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDD, Group VI pre-treatment of EA followed by TCDD treatment. Each bar represents mean ± standard error of mean of each group. ***P \ 0.001, **P \ 0.01 when compared to control. ## P \ 0.01, when compared to TCDD treated group. NS non significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison)
Fig. 5 Effect of EA on TCDD-induced CYP1A1 activity. Group I control, Group II DMSO (vehicle for DMSO), Group III corn oil (vehicle for TCDD), Group IV EA, Group V TCDDm, Group VI pretreatment of EA followed by TCDD treatment. Each bar represents mean ± standard error of mean of each group. ***P \ 0.001, when compared to control. ###P \ 0.001, when compared to TCDD treated group. NS non significant when compared to control. (One way ANOVA followed by Tukey’s multiple comparison)
Discussion TCDD is classified as a group I carcinogen by the International Agency for Research on Cancer [34]. TCDD is resistant to degradation which allows it to bioaccumulate in food chain [35]. Since liver is the target organ for detoxification of environmental toxins, most of the studies have focused on TCDD-induced liver toxicity. Recent reports show that TCDD also causes adverse effects in various
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extra hepatic tissues and sparse literature is available on TCDD induced nephrotoxicity and its protection by antioxidants. Lu et al. [36] has first demonstrated combined effect of TCDD and PCB congeners on kidney toxicity in rats. Ciftci et al. [37] have reported protective effect offered by quercetin and chrysin on TCDD induced renal damage by evaluating the antioxidant status. In present study the antioxidant ellagic acid was evaluated for its protective effect by assessing CYP1A1 activity (prime candidate involved in the generation of ROS), ATP ase enzyme activities and serum kidney markers in addition to oxidative stress parameters in TCDD-induced nephrotoxicity in male wistar rats. Phenolic antioxidants play a pivotal role in offering protection during various oxidative stress conditions. EA (gallic acid dimer), a naturally occurring polyphenolic compound possesses lipophilic and free radical scavenging property. EA exhibits antioxidant [11], anti-proliferative [38] and anti-mutagenic properties [39]. Studies on structure–function relationship of EA revealed that phenolic hydroxyl group and lactone are responsible for its protective action against various conditions of oxidative stress [40]. So, the present study evaluated the oxidative stress responses induced by TCDD in kidney of wistar rats and possible protective effect of EA on TCDD induced nephrotoxicity. Histopathological observation and levels of serum kidney markers reveal that TCDD causes severe damage to the kidney tissues. The rats treated with TCDD showed sclerosis in glomeruli with interstitial inflammation and dilation in distal tubules. The observed changes are consistent with earlier reports of Ciftci et al. [37] and Lu et al. [36]. Along with these changes the present study observed that
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TCDD also induced inflammation and damages to the proximal and distal tubules. Pre-treatment with EA rendered significant protection against the histological changes induced by TCDD by maintaining the near normal architecture of glomerulus and tubules. The results of the present study corroborates with the earlier report of Atessahin et al. [15], where EA offered protective effect by reducing the histopathological alterations against cisplatininduced nephrotoxicity. Oxidative stress results when production of reactive oxidative species exceeds the capacity of cellular antioxidant defenses to remove these toxic species. Oxidative stress conditions induced by various xenobiotics result from upstream signaling mechanisms and in the case of TCDD, AhR play an important role in mediating the toxicity. Upon TCDD binding to AhR, it gets translocated to the nucleus where it associates with ARNT. The whole complex then acts as a transcription factor that binds to DNA promoter sequences termed xenobiotic responsive elements, thereby enhancing the transcription of array of genes [41]. Out of which induction of CYP1A1 by AhR is of toxicological relevance. TCDD acts as substrate for CYP1A1 and the formed metabolites potentially initiates toxic responses by increasing oxidative stress status [42, 43]. The ROS generated can directly react with cellular membrane phospholipid moieties due to the presence of their methylene group [44]. Polyunsaturated fatty acid oxidation yields short-lived lipid hydroperoxide molecules known to further perpetuate ROS production [45]. Lipid peroxidation and oxidative stress have been indicated as factors which lead to acute toxicity of TCDD [46, 47]. The cells exhibit various non- enzymic and enzymic antioxidants which act as natural defense mechanisms to counteract the oxidative stress conditions. Of which, reduced glutathione (GSH) is a non protein thiol which is actively present inside the cells and acts as first defense against oxidative attack. It participates in a variety of detoxification reactions, in addition to providing protection against oxidative damage by virtue of its thiol group. Peroxide level and thiol status is indicative of oxidative stress conditions, since increase in lipid peroxidation causes depletion in GSH levels [48]. So, in the present study these two levels were measured to analyze the extent of oxidative stress caused by TCDD. In the present study there was a significant decrease in total GSH and with concomitant increase in lipid peroxide levels in TCDD treated rats. Earlier report by Stohs, [49] also showed decreased thiol levels and increased lipid peroxide levels in mitochondria of TCDD treated rats. In rats pre-treated with EA followed by TCDD treatment there was a significant increase in GSH levels with concomitant decrease in lipid peroxide levels. Thus EA by its potent free radical scavenging property and glutathione sparing action might have
attenuated the oxidative stress status invoked by TCDD. The free radical scavenging property of EA leads to the inhibition of LPO and 8-OGDG formation both in vitro and in vivo [50, 51]. Protection offered by EA by decreasing the levels of lipid peroxides on fetotoxicity induced by TCDD in C57BL/67 mice [17] and increasing the levels of GSH in N-nitrosodiethylamine induced lung tumorigenesis in mice [52] has been reported earlier which supports the present findings. Apart from GSH, various antioxidant enzymes are involved to overcome the conditions of oxidative stress. Optimum levels and proper balance among these enzymes may be critical for the prevention of cellular oxidative stress. In the present study, a significant decrease in the activities of the antioxidant enzymes (SOD, GSH, GPx, and CAT) reflects TCDD-induced oxidative stress in renal tissue. The observed decline in antioxidant status after TCDD treatment is in line with the previous reports of Jin et al. [53] who has demonstrated significant decrease in the activities of antioxidant enzymes in reproductive system of mouse treated with TCDD. In addition, a dose-dependent suppression of SOD activity in cerebral cortex and hippocampus regions following TCDD treatment has been reported earlier by Hassoun et al. [54]. It could be said that, TCDD-induced decrease in activities of antioxidant enzymes might be caused by the expense of these enzymes in overcoming the oxidative stress conditions. In the present study, pre-treatment of EA followed by TCDD treatment significantly improved the antioxidant status. EA has been shown to offer protection by its free radical scavenging action and improved antioxidant status through Nrf-2 expression [11, 16]. The observed results on increase in antioxidant status and nephroprotective action of EA might be due to the above mentioned properties of EA. In addition, the ability of EA to render protective effect by enhancing the antioxidant mechanism against various oxidative conditions, support the present study findings. Das et al. [55] has shown protection offered by EA through increasing GST activity in liver and lungs of BALB/c mice against oxidative stress induced by benzo[a]pyrene. Protective effect of EA by improving the antioxidant status against selenite induced cataractogenesis [56] and cisplatin induced injuries to sperm [19] has been reported earlier. The membrane-bound enzymes such as Na?, K?-ATPase, Mg2?-ATPase and Ca2?-ATPase are responsible for the transport of sodium/potassium, magnesium and calcium ions across the cell membranes at the expense of ATP hydrolysis [57]. It is important to evaluate membrane bound ATPases enzyme activity during stress conditions, since changes in the activities of these enzymes affect the normal functioning of any organ. It is well documented that TCDD-induced Ca2? signaling plays a major role in TCDD-induced toxic
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events [58–60]. TCDD-dependent increase in intracellular calcium levels activates phospholipase A2, which in turn mobilizes arachidonic acid from cell membrane. The released arachidonic acid acts as a substrate for COX-2 and initiates eicosanoid synthesis. TCDD induced early and sustained increase in intracellular free calcium and subsequent formation of eicosanoids has been reported by Puga et al. [59]. Studies have documented that eicosanoids, especially arachidonic acid metabolites have profound inhibitory action on Na?, K?-ATPase and Mg2?-ATPase activities [61]. The results of the present study reveal a significant decrease in Na?, K?-ATPase and Mg2?-ATPase activities during TCDD treatment when compared to control rats. Decline in Na?, K?-ATPase activity after TCDD treatment has been reported in liver tissues [62] and renal medullary cells [63]. However the Schwartzman et al. [63] has showed that arachidonic acid metabolite formed from cytochrome P450 played a major role in inhibition of in Na?, K?-ATPase activity. But it was interesting to note that there was a significant increase in Ca2?-ATPase activity in the present study on treatment with TCDD. It can be explained that an early and sustained increase in Ca2? levels might have increased the activity of Ca2?-ATPase as a cells adaptive mechanism to overcome Ca2? overloading and prevent subsequent calcium induced toxic events. From the results of the present study and available literature it can be said that TCDD-induced alterations in ATPase enzyme activities may be due the disturbances in Ca2? homeostasis and subsequent formation of eicosanoids. While EA pretreatment significantly restored the ATPase enzyme activities to values close to that of control rats. The ability of EA to maintain Ca2? homeostasis might be attributed to restore ATPase enzyme activities. To this end, Ou et al. [64] have reported that EA maintained Ca2? homeostasis in endothelial cells and thereby prevented oxidized low-density lipoprotein-induced apoptosis. An unpublished data from our laboratory also supports the fact that EA is involved in maintaining Ca2? homeostasis. Our laboratory has been involved in identifying the molecular mechanism of EA in offering cytoprotection against TCDD-induced toxic events by in vitro studies. From the results, we observed that EA significantly maintained the Ca2? homeostasis and prevented TCDD-induced oxidative stress in HepG2 cells. The above results indicate the protective nature of EA against TCDD-induced nephrotoxicity by enhancing antioxidant status and preventing oxidative stress. Since TCDD-induced sustained oxidative stress and other stress response is primarily mediated through TCDD-induced CYP1A1 activation and subsequent imbalance in the redox status [65, 66], we next determined whether EA had any effect on suppressing the activity of CYP1A1. Inhibition of CYP1A1 by natural compounds is an attractive strategy, as these compounds could prevent oxidative stress and subsequent
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induction of various toxic events. The present study demonstrates that EA pre-treatment significantly decreased TCDD-induced CYP1A1 activity. The reduction in CYP1A1 activity might be either due to direct binding of EA to CYP1A1 or it could be due to its AhR antagonism. Further studies are required to understand the exact molecular mechanism of EA mediated CYP1A1 inhibition. In conclusion, the present study demonstrates that TCDDinduced nephrotoxicity is mediated through sustained oxidative stress with altered ATPase enzyme activities. Our data provide clear evidence that over all protective effect offered by EA against TCDD-induced nephrotoxicity is mediated through suppressing CYP1A1 activity and enhancing the antioxidant mechanism. Thus the present study highlights that consumption of ellagic acid helps in protection against environmental toxins which primarily mediate their toxic effects through oxidative stress. Conflict of interest The authors declare that there are no conflict of interest.
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