ARTICLE IN PRESS
PATPHY-802; No. of Pages 7
Pathophysiology xxx (2014) xxx–xxx
Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis Vaddi Damodara Reddy a , Pannuru Padmavathi b , Reddyvari Hymavathi a , Paramahamsa Maturu c , N.Ch. Varadacharyulu a,∗ b
a Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515 055, India Department of Medicine, Hematology/Oncology, University of Illinois, Chicago 60612, USA c Department of Genetics, MD Anderson Cancer Center, Houston, TX 77030, USA
Received 10 June 2013; received in revised form 7 December 2013; accepted 15 December 2013
Abstract The protective effect of Emblica officinalis fruit extract (EFE) against alcohol-induced oxidative damage in liver microsomes was investigated in rats. EFE (250 mg/kg b.wt/day) and alcohol (5 g/kg b.wt/day, 20%, w/v) were administered orally to animals for 60 days. Alcohol administration significantly increased lipid peroxidation, protein carbonyls with decreased sulfhydryl groups in microsomes, which were significantly restored to normal levels in EFE and alcohol co-administered rats. Alcohol administration also markedly decreased the levels of reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in the liver microsomes, which were prevented with EFE administration. Further, alcohol administration significantly increased the activities of cytochrome P-450, Na+ /K+ and Mg2+ ATPases and also membrane fluidity. But, administration of EFE along with alcohol restored the all above enzyme activities and membrane fluidity to normal level. Thus, EFE showed protective effects against alcohol-induced oxidative damage by possibly reducing the rate of lipid peroxidation and restoring the various membrane bound and antioxidant enzyme activities to normal levels, and also by protecting the membrane integrity in rat liver microsomes. In conclusion, the polyphenolic compounds including flavonoid and tannoid compounds present in EFE might be playing a major role against alcohol-induced oxidative stress in rats. © 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol; CYP-450; Emblica officinalis; Microsomes; Oxidative stress
1. Introduction Liver is the principle organ responsible for alcohol metabolism and is more susceptible to alcohol-induced toxicity. Oxidative stress has been suggested as key factor capable of both initiating and sustaining the mechanisms of pathogenesis leading to alcohol liver disease (ALD) [1]. As the main source of reactive oxygen species (ROS), hepatic microsomes are susceptible to ROS attack, especially upon cytochrome P450 2E1 (CYP-450) activation by ethanol [2]. ROS generated from ethanol metabolism can directly damage cell membranes by peroxidation of membrane polyunsaturated fatty ∗ Corresponding author at: Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515 055, AP, India. Tel.: +91 8554 255761; fax: +91 8554 255244. E-mail address: vdp [email protected] (N.Ch. Varadacharyulu).
acids. Increased lipid peroxidation changes the activities of various membrane bound enzymes. Especially ATPases are very sensitive to peroxidation reactions and membrane fluidity [3]. Chronic alcohol consumption also alters redox thiol status which might have biological relevance and contribute to the pathologies associated with several disease states. The free radicals generated during ethanol metabolism interact with proteins, lipids and DNA and thereby forming adducts [4]. Adduct formation may lead to interference with protein function, stimulation of fibrogenesis and induction of immune responses, which finally contributes to the progression of alcohol-induced liver diseases [5,6]. Alcohol-induced oxidative stress has been found mainly due to an increased production of oxygen free radicals and lower cellular antioxidant levels [7]. The endogenous antioxidant enzymes are responsible for the detoxification of deleterious oxygen radicals. Antioxidants play an important
0928-4680/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pathophys.2013.12.001
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
2
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
role to protect liver against alcohol-induced damage caused by reactive oxygen species [8]. Dietary phytochemicals are capable of removing free radicals. Among them, phenolic and polyphenolic compounds, such as flavonoids and tannins in edible fruits, exhibit potent antioxidant activities. Therefore, the possibility of preventing the onset of alcohol toxicity using herbal medicines has attracted considerable attention [9]. Literature mapping reveal that the fruit of Emblica officinalis, commonly known as amla, is a potential source with many therapeutic principles and has been used in more than hundred herbal formulations of Ayurveda, an Indian traditional medicine [10]. Studies showed that Emblica contains more than 20 hydrolyzable tannins [11]. The fruit contains flavonoids (e.g. kaempherol-3--O-d-glucoside, quercetin3-O--glucoside), polyphenols (e.g. emblicanin A and B, punigluconin and pedunculagin) and also contains phyllantine and zeatin alkaloids, and a number of benzenoids including amlaic acid, corilagin, ellagic acid, 3-6-di-Ogalloyl-glucose, ethyl gallate, 1,6-di-O-galloyl--d-glucose, 1-di-O-galloyl--d-glucose, putranjivain A, digallic acid, phyllemblic acid, emblicol, and alactaric acid and gallic acid [12–14]. In vitro and in vivo studies showed that EFE had strong antioxidant and radical scavenging activities against DPPH, O2 •– , OH• , and NO radicals [8]. Moreover, studies have shown that Emblica possess antidiabetic [15], hypolipidemic, anticancer [16], antiatherogenic [17], hepatoprotective [18,19] and neuroprotective properties [20]. Most of the reports suggest that these health effects could be attributed to the antioxidant activities of the EFE. Phenolic compounds, especially hydrolyzable tannins and flavonoids in combination with vitamin C, are considered to be the major antioxidants and bioactive components in EFE. Based on these considerations, we hypothesized that EFE may be a promising phytomedicine that could attenuate alcoholinduced oxidative stress in the liver microsomes.
2. Materials and methods
tap water ad libitum. The animals were procured from Sri Venkateswara Enterprises, Bangalore, India, were divided into four groups of eight rats in each group. Group I control rats (C), which received glucose instead of alcohol (i.e. caloric equivalent to alcohol), group II (A), which received 20% (v/v) alcohol in water, group III rats (EFE), which received EFE alone in water, group IV rats (A + EFE), which received 20% (v/v) alcohol and then EFE after 8 h. Alcohol 5 g/kg b.wt/day and EFE 250 mg/kg b.wt/day was administered through stomach tube daily to each rat for 60 days. The dose of Emblica fruit extract was fixed based on earlier reports [8]. Food and water intake of all the animals was recorded daily and weight of rats was followed on alternate days. At the end of the experimental period, the rats in each group were fasted overnight and then sacrificed by cervical dislocation. Tissues were collected and processed immediately for further analysis. Institutional ethical committee approved this study.
2.3. Preparation of rat liver microsomes Rat liver microsomes were prepared according to the method described previously [21]. Microsomes were prepared by centrifuging the post mitochondrial supernatant from the first 7000 × g centrifugation at 12,000 × g for 15 min. The pellet and the floating lipid were discarded, and the supernatant was centrifuged at 144,000 × g for 1 h for sedimentation of microsomes. All operations of preparation and subsequent fractionation were carried out at 4 ◦ C.
2.4. Measurement of TBARS, protein carbonyls and sulfhydryl groups Thiobarbituric acid reactive species (TBARS) were measured by the formation of malondialdehyde [22]. Protein carbonyls were determined as described previously [23]. Protein sulfhydryl groups were measured using Ellman’s reagent [24].
2.1. Chemicals 2.5. Determination of cytochrome P-450 activity The chemicals used in the present study were procured from Sigma Chemical Co. (St. Louis, MO, USA) and SISCO Research Laboratories (Mumbai, India). Ethanol used for administration to rats was obtained by re-distillation. E. officinalis fruit extract dry powder (90.8% water soluble extractives including 49.5% tannins) was obtained from Chemiloids Ltd., Vijayawada, India (Manufacturers and exporters of herbal extracts). 2.2. Animals and experimental design Two-month-old male albino Wistar rats, weighing about 120–140 g, were maintained in animal house with commercial pellet diet (Hindustan Lever Ltd., Bangalore, India) and
The activity of the CYP-450 was assayed as described previously [25]. Briefly, microsomal suspensions (1.0 mg protein/ml) in phosphate buffer treated with a few grains of sodium dithionite and take it in the reference and sample cuvettes (or in the two halves of the split cuvette) and record the baseline from 400 to 510 nm. The contents of the sample cuvette were transferred to new tube and bubble gently with carbon monoxide (CO) for 60 s. Transfer back to the sample cuvette and record the difference spectrum from 400 to 510 nm again. The difference in absorbance between 450 and 490 nm can then be used for the calculation of CYP-450 content using the extinction coefficient difference (E450–490 nm of 91 cm−1 mM−1 ).
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
3
2.6. Determination of antioxidant status Liver microsomal catalase (CAT) was assayed as described [26]. The CAT activity was expressed as nmol H2 O2 decreased/mg protein/min. Superoxide dismutase (SOD) was assayed [27]. A single unit of enzyme was expressed as 50% inhibition of NBT (nitro blue tetrazolium) reduction/min/mg protein. Glutathione peroxidase (GPx) activity was measured as described [28]. GPx activity was expressed as mol GSH oxidized/min/mg protein. Total reduced glutathione (GSH) content was measured as described previously [29]. 2.7. Measurement of Na+ /K+ -ATPase and Mg2+ -ATPase activity The activities of Na+ /K+ and Mg2+ -ATPases were determined [30]. The activities were indirectly measured by estimating the phosphorus liberated after the incubation of erythrocyte membrane in a reaction mixture containing the substrate ATP with the co-substrate elements at 37 ◦ C for 15 min. The Mg2+ ATPase activity was measured in the presence of 1 mM ouabain, a specific inhibitor of Na+ , K+ ATPase. The reactions were arrested by adding 1.0 ml of 10% TCA. The phosphorus content from the TCA supernatants was then determined [31]. 2.8. Measurement of membrane fluidity The quantitative measurement of membrane fluidity was performed by the fluorescence polarization technique described previously with 1,6-diphenyl 1,3,5-hexatriene (DPH) as fluorescence probe as described [32]. Membranes (50 g protein) were suspended in 50 mmol/l DPH solubilized in tetrahydrofuran, and incubated at 37 ◦ C for 30 min. Fluorescence polarization was determined using a Hitachi fluorescence spectrophotometer (Hitachi, Japan) equipped with rotating polarizing filters with samples held at 25 ◦ C. Samples were excited at 360 nm and the emission intensity was read at 435 nm. Polarization (P) and fluorescence anisotropy (γ) were calculated using the equation. P = IVV − IVH·G /IVV + IVH·G . Where IVV and IVH are the intensities measured parallel and perpendicular to the vertical axis of the excitation beam, and G is the correction factor. IVH /IHH·γ is calculated using the formula γ = 2P/(3 − P). Protein concentration was determined as previously described [33].
Fig. 1. Effect of EFE administration on TBARS in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
3. Results 3.1. Effect of EFE on alcohol-induced alterations in TBARS, protein carbonyls and sulfhydryl groups The effect of alcohol administration on lipid peroxidation, protein carbonyls and sulfhydryl groups in microsomes are shown in Figs. 1–3. As an index of lipid peroxidation we measured MDA levels and an index of protein oxidation we measured protein carbonyls and sulfhydryl groups in controls and experimental animals. Alcohol administration resulted a significant (p < 0.05) increase in MDA levels, protein carbonyls with a significant (p < 0.05) decrease in sulfhydryl groups compared to control animals. But, co-administration of alcohol and EFE significantly (p < 0.05) prevented the elevation of MDA levels, protein carbonyls and diminished sulfhydryl groups compared with alcohol alone administered
2.9. Statistical analysis Mean and standard deviation values of all the parameters were determined for each group. ANOVA followed by Student “t” test were performed to determine significant difference among the groups. A p < 0.001 and p < 0.05 was considered statistically significant.
Fig. 2. Effect of EFE administration on protein carbonyls in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
4
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
Fig. 3. Effect of EFE administration on protein sulfhydryl groups in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
animals. EFE treatment alone did not show any significant changes in the above parameters compared to control group. 3.2. Protective role of EFE on alcohol-induced microsomal redox disturbance Non-enzymatic GSH, as well as enzymatic catalase, SOD and GPx are crucial antioxidants for ROS derivatives generated in endoplasmic reticulum during ethanol metabolism, to maintain the redox homeostasis. The data presented in Table 1 shows the effect of EFE on alcohol-induced redox disturbance in microsomes. Chronic alcohol administration significantly (p < 0.05) depleted microsomal catalase, SOD and GPx enzyme activities and non-enzymatic GSH content. All the above enzymes and GSH content were restored to normal levels after co-administration of alcohol and EFE to rats. The compounds present in EFE quench free radicals generated from alcohol metabolism and help cells to maintain redox homeostasis constantly.
Fig. 4. Effect of EFE administration on cytochrome P-450 enzyme activity in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls and “**” indicates significant from control and alcohol administered rats.
3.4. Effect of EFE on ethanol-induced alterations in membrane fluidity The steady state fluorescent anisotropy studies in microsomes using DPH were carried out and the anisotropic (γ) values are presented in Fig. 5. The fluorescent anisotropic values of alcohol administered rats revealed significant (p < 0.01) increase in anisotropic (γ) values in the present study. Upon administration of EFE to alcohol receiving rats significantly (p < 0.01) prevented from these alterations and almost restored to normal levels. No significant change was seen in EFE alone administered rats compared to controls. 3.5. Alterations in membrane bound enzymes Fig. 6 shows the effect of alcohol administration on the activities of Na+ /K+ and Mg2+ ATPases. Na+ /K+ and Mg2+ ATPases activity was increased in alcohol administered rats
3.3. Effect of EFE on alcohol-induced alterations in CYP-450 activity Data presented in Fig. 4 reveals the effect of EFE on alcohol-induced alterations in CYP-450 enzyme activity in microsomes. Results showed that alcohol administration significantly (p < 0.05) increased CYP-450 enzyme activity in microsomes compared to controls. Administration of EFE to alcohol receiving rats significantly (p < 0.05) decreased CYP450 enzyme activity compare to alcohol alone administered rats. Though, administration of EFE to alcohol receiving rats did not brought back the enzyme to normal level, but still it is significantly different from EFE alone administered rats. EFE alone administered rats showed normal enzyme activity to that of controls.
Fig. 5. Effect of EFE administration on anisotropic (γ) value in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.001 is considered as statistically significant between groups. Asterisk “#” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
5
Table 1 Effect of EFE administration on liver microsomal antioxidant status in alcohol administered rats. Parameter
Groups Control
GSH (g/mg protein) Catalase (nmol/min/mg protein) SOD (Units/mg protein) GPX (g of GSH oxidized/min/mg protein)
3.37 38.1 5.38 2.95
± ± ± ±
Alcohol 0.14 3.66 0.24 0.16
1.91 23.3 3.21 0.99
EFE
± ± 2.3* ± 0.13* ± 0.11* 0.17*
3.35 38.6 5.36 2.90
A + EFE ± ± ± ±
0.19 2.4 0.16 0.08
3.12 34.6 4.24 1.65
± ± ± ±
0.10ns 3.2ns 0.19ns 0.1ns
Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
compared to control rats. However, administration of EFE to alcohol administered rats significantly (p < 0.05) offered protection and prevented the rise of the above ATPase activities. EFE alone treated rats had no significant (p < 0.05) effect on Na+ /K+ and Mg2+ ATPases activity comparable to controls.
4. Discussion Reactive oxygen species (ROS) are small, highly reactive, oxygen-containing molecules that can react with and damage complex cellular molecules, particularly in the liver [34]. Ethanol metabolism is directly involved in not only the production of reactive oxygen species, but also related in the formation of an environment favorable to oxidative stress such as hypoxia, endotoxaemia and cytokine release [35]. Previous studies showed that alcohol administration to rats significantly increased lipid peroxidation in the liver and make hepatocytes leaky [8]. Upon hepatic damage or necrosis enzymes will be leaked into the circulation and raise their concentrations. Our previous study also showed that elevated serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in alcohol treated animals indicated cellular breakage and loss of functional integrity of cell membranes in
Fig. 6. Effect of EFE administration on Na+ /K+ -ATPase and Mg2+ -ATPase enzyme activities in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Values are expressed as mol pi/h/mg protein. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
liver [10]. Induction of CYP-450 by ethanol is a central pathway by which ethanol generates oxidative stress in hepatocytes. During ethanol oxidation ROS production increases dramatically due to induction of CYP2E1 and by activation of Kupffer cells in the liver [36]. In the present study, the activity of CYP-450 was increased and consistent with earlier reports [2,37]. Increased activity of CYP-450 results in accelerated production of lipid hydroperoxides and consequently increases oxidative stress [38]. This effect on the redox status of the liver and can cause activation of Kupffer cells and subsequently, hepatic stellate cells, and thus contributing to the generation of alcoholic liver disease [39]. Administration of EFE to alcohol administered rats significantly prevented these alterations. The polyphenolic compounds present in EFE extract scavenge free radicals generated during alcohol metabolism in microsomes and protects liver. The metabolism of ethanol via the CYP-450 pathway results in increased ROS production, including superoxide, hydrogen peroxide (H2 O2 ), and hydroxyl radicals. Microsomes are the known targets for alcohol action due to their high content of polyunsaturated fatty acids and calcium storage [2]. Alcohol administration significantly increased lipid peroxidation and protein carbonyls with decreased sulfhydryl groups. Moreover, various lipid peroxidation products bind to proteins forming stable adducts stimulating fibrogenesis and histological damages in alcoholic liver disease. In addition, hydroxyethyl radical (HER), hydroxynonenal (HNE) and other radicals are involved in modifying cellular proteins [40,41]. Previous studies also showed that greater increase in liver protein carbonyl content, which was attributed to the development of pathogenesis related to alcoholism [42]. Studies using rats administered with alcohol demonstrated that alterations in protein thiols have biological relevance and contribute to the pathologies associated with several diseased states such as alcoholic liver damage [43]. In the present study, increased lipid peroxidation, protein oxidation and diminished protein sulfhydryls may be due to enhanced ROS and lowered antioxidant levels. Administration of EFE to alcohol receiving rats prevented from the augment of lipid peroxidation and protein carbonyls. The protective action of Emblica is usually due to the inhibition of free radical-induced chain reaction and the resultant prevention of peroxidative deterioration of structural lipids in membranes. Antioxidant enzymes play important role in the elimination of ROS in cells. In this study, we found diminished
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
6
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
activities of SOD, catalase and GPx as well as GSH in ethanol receiving rats. The decrease in the SOD activity may be associated with the elevation of the intracellular concentration of H2 O2 as a result of catalase inactivation [44]. Catalase has been reported to be responsible for detoxification of H2 O2 which is an effective inhibitor of SOD [45]. The decrease in catalase activity could be due to low production of the enzyme and/or the inhibition of the enzyme as a result of the increased production of free radicals [46]. GSH is a major non-protein thiol and plays a central role in coordinating the antioxidant defense process. It is involved in the maintenance of normal cell structure and function through its redox and detoxification reactions [47]. GPx with the help of GSH metabolizes hydrogen peroxide to water thereby protecting mammalian cells against oxidative damage. Metabolism of ethanol by CYP2E1 also results in a significant increase in free radical and acetaldehyde production which, in turn, diminish reduced glutathione (GSH) and other defense systems against oxidative stress leading to further hepatocyte damage. Administration of EFE to alcohol-administered rats prevented the down-regulation of antioxidant enzymes. The phytochemical compounds present in EFE boost antioxidant activity of the cells by scavenging free radicals generated during ethanol metabolism. The compounds especially hydrolysable tannoid principles emblicanin A, emblicanin B, punigluconin and pedunculagin compounds present in Emblica might have contributed largely for the above mechanism [48–50]. Biological membranes exerting important biological functions are readily susceptible to free radical attack. A major protective mechanism against oxidative attack is the membrane integrity. Free radicals generated from alcohol metabolism can directly damage cell membrane structure and function, including membrane fluidity, ion permeability and modulation of membrane spanning protein activities [3]. One of the most important membrane proteins are ATPases. Lipid peroxidation is known to disturb structural integrity of the membrane that might in turn affect the activities of Na+ /K+ -ATPase and Mg2+ -ATPase enzymes. Alcohol-induced generation of free radicals also affect membrane-linked enzyme activity through modification of membrane fluidity [51]. Studies also have shown that alcoholinduced generation of free radicals affects membrane-linked enzyme activity through modification of membrane fluidity [52]. In this study, increased Na+ /K+ -ATPase and Mg2+ ATPase activities in ethanol treated rats may be due to alteration in the membrane fluidity. Co-administration of EFE and ethanol to rats showed lowered lipid peroxidation, which in turn might be responsible for normalization of membrane structure and membrane fluidity, consequently restores the activities of membrane bound enzymes. In conclusion, restoration of all these components and activities of microsomal enzymes by EFE administration in alcohol administered rats suggested the therapeutic potential of Emblica principles at molecular level and protecting the cells and organelles from alcohol-induced oxidative stress. In
EFE several phytocompounds work together synergistically to fight against the complications of alcohol consumption. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments The authors thank the Director and staff of the Center for Cellular and Molecular Biology, Hyderabad, India for providing facilities to carry out fluidity studies. This study was supported in part by the University Grants Commission, New Delhi, India (Grant No. F.No.40-203/2011 SR). References [1] D. Wu, X. Wang, R. Zhou, L. Yang, A.I. Cederbaum, Alcohol steatosis and cytotoxicity: the role of cytochrome P4502E1 and autophagy, Free Radic. Biol. Med. 53 (2012) 1346–1357. [2] Y. Tang, C. Gao, Y. Shi, L. Zhu, X. Hu, D. Wang, Y. Lv, X. Yang, L. Liu, P. Yao, Quercetin attenuates ethanol-derived microsomal oxidative stress: implication of haem oxygenase-1 induction, Food Chem. 132 (2012) 1769–1774. [3] V.D. Reddy, P. Padmavathi, M. Paramahamsa, N.C. Varadacharyulu, Modulatory role of Emblica officinalis against alcohol induced biochemical and biophysical changes in rat erythrocyte membranes, Food Chem. Toxicol. 47 (2009) 1958–1963. [4] O. Niemela, Aldehyde–protein adducts in the liver as a result of ethanol induced oxidative stress, Front. Biosci. 4 (1999) D506–D513. [5] J.B. Hoek, J.G. Pastorino, Ethanol, oxidative stress, and cytokine induced liver cell injury, Alcohol 27 (2002) 63–68. [6] P. Abraham, G. Wilfred, B. Ramakrishna, Oxidative damage to the hepatocellular proteins after chronic ethanol intake in the rat, Clin. Chim. Acta 325 (2002) 117–125. [7] A. Dey, A.I. Cederbaum, Alcohol and oxidative liver injury, Hepatology 43 (2006) S63–S74. [8] V.D. Reddy, P. Padmavathi, S. Gopi, M. Paramahamsa, N.C. Varadacharyulu, Protective effect of Emblica officinalis against alcohol induced hepatic injury by ameliorating oxidative stress in rats, Indian J. Clin. Biochem. 4 (2010) 419–424. [9] B.J. Xu, Y.N. Zheng, C.K. Sung, Natural medicines for alcoholism treatment: a review, Drug Alcohol Rev. 24 (2005) 525–536. [10] V.D. Reddy, P. Padmavathi, N.C. Varadacharyulu, Emblica officinalis protects against ethanol-induced liver mitochondrial dysfunction in rats, J. Med. Food 12 (2009) 327–333. [11] B. Yang, M. Kortesniemi, P. Liu, M. Karonen, J.P. Salminen, Analysis of hydrolyzable tannins and other phenolic compounds in emblic leafflower (Phyllanthus emblica L.) fruits by high performance liquid chromatography-electrospray ionization mass spectrometry, J. Agric. Food Chem. 60 (2012) 8672–8683. [12] Y.J. Zhang, T. Tanaka, C. Yang, I. Kouno, New phenolic constituents from the fruit juice of Phyllanthus emblica, Chem. Pharm. Bull. (Tokyo) 49 (2001) 537–540. [13] S. Ghosal, V.K. Tripathi, S. Chauhan, Active constituent of Emblica officinalis. Part 1: The chemistry and antioxidant effects of two new hydrolyzable tannins, emblicanin A and B, Indian J. Chem. 35 (1996) 941–948. [14] M. Majeed, B. Bhat, A. Jadhav, J. Srivastava, K. Nagabhushanam, Ascorbic acid and tannins from Emblica officinalis Gaertn Fruits—a revisit, J. Agric. Food Chem. 57 (2009) 220–225.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
[15] P. Suryanarayana, M. Saraswat, J.M. Petrashm, G.B. Reddy, Emblica officinalis and its enriched tannoids delay streptozotocin induced diabetic cataract in rats, Mol. Vis. 13 (2007) 1291–1297. [16] M.S. Baliga, J.J. Dsouza, Amla (Emblica officinalis Gaertn), a wonder berry in the treatment and prevention of cancer, Eur. J. Cancer Prev. 20 (2011) 225–239. [17] S.V. Nampoothiri, A. Prathapan, O.L. Cherian, K.G. Raghu, V.V. Venugopalan, A. Sundaresan, In vitro antioxidant and inhibitory potential of Terminalia bellerica and Emblica officinalis fruits against LDL oxidation and key enzymes linked to type 2 diabetes, Food Chem. Toxicol. 49 (2011) 125–131. [18] R. Srirama, H.B. Deepak, U. Senthilkumar, G. Ravikanth, B.R. Gurumurthy, M.B. Shivanna, C.V. Chandrasekaran, A. Agarwal, R.U. Shaanker, Hepatoprotective activity of Indian Phyllanthus, Pharm. Biol. 50 (2012) 948–953. [19] B.D. Hiraganahalli, V.C. Chinampudur, S. Dethe, D. Mundkinajeddu, M.K. Pandre, J. Balachandran, A. Agarwal, Hepatoprotective and antioxidant activity of standardized herbal extracts, Pharmacogn. Mag. 8 (2012) 116–123. [20] V.D. Reddy, P. Padmavathi, G. Kavitha, S. Gopi, N.C. Varadacharyulu, Emblica officinalis ameliorates alcohol-induced brain mitochondrial dysfunction in rats, J. Med. Food 14 (2011) 62–68. [21] C.C. Cunningham, S. Filus, R.E. Bottenus, P.I. Spach, Effect of ethanol consumption on the phospholipid composition of rat liver microsomes and mitochondria, Biochim. Biophys. Acta 712 (1982) 225–233. [22] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [23] A.Z. Reznick, L. Packer, Oxidative damage to proteins: spectroscopic method for carbonyl assay, Methods Enzymol. 233 (1994) 357–363. [24] P. Padmavathi, V.D. Reddy, G. Kavitha, P. Maturu, N.C. Varadacharyulu, Chronic cigarette smoking alters erythrocyte membrane lipid composition and properties in male human volunteers, Nitric Oxide 23 (2010) 181–186. [25] T. Omura, R. Sato, The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties, J. Biol. Chem. 239 (1964) 2379–2385. [26] H. Abei, Catalase in vitro, Methods Enzymol. 10 (1988) 121–126. [27] K.R. Reddy, V.D. Reddy, P. Padmavathi, G. Kavitha, B. Saradamma, N.C. Varadacharyulu, Gender differences in alcohol induced oxidative stress and altered membrane properties in erythrocytes of rats, Indian J. Biochem. Biophys. 50 (2013) 32–39. [28] P. Maturu, V.D. Reddy, P. Padmavathi, N.C. Varadacharyulu, Alcohol induced oxidative stress: an adaptive role of erythrocyte antioxidant system in human chronic alcoholics, Exp. Toxicol. Pathol. 64 (2012) 697–703. [29] G.S. Krushna, M.A. Kareem, V.D. Reddy, P. Padmavathi, S.A. Hussain, L. Devi Kodidhela, Aegle marmelos fruit extract attenuates isoproterenol-induced oxidative stress in rats, J. Clin. Biochem. Nutr. 50 (2012) 199–204. [30] B. Ismail, I.S. Edelman, Assay of Na+ , K+ ATPase, Biochem. Pharmacol. 34 (1985) 2685–2689. [31] P. Padmavathi, V.D. Reddy, M. Narendra, N.C. Varadacharyulu, Bidis – hand-rolled, Indian cigarettes: induced biochemical changes in plasma and red cell membranes of human male volunteers, Clin. Biochem. 42 (2009) 1041–1045. [32] P. Padmavathi, V.D. Reddy, P. Maturu, N.C. Varadacharyulu, Smoking-induced alterations in platelet membrane fluidity and Na(+) /K(+) -ATPase activity in chronic cigarette smokers, J. Atheroscler. Thromb. 17 (2010) 619–627. [33] V.D. Reddy, P. Padmavathi, G. Kavitha, B. Saradamma, N.C. Varadacharyulu, Alcohol-induced oxidative/nitrosative stress alters brain mitochondrial membrane properties, Mol. Cell. Biochem. 375 (2013) 39–47. [34] Y. Lu, D. Wu, X. Wang, S.C. Ward, A.I. Cederbaum, Chronic alcohol-induced liver injury and oxidant stress are decreased in
[35] [36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45] [46] [47]
[48]
[49]
[50]
[51]
[52]
7
cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice, Free Radic. Biol. Med. 49 (2010) 1406–1416. D. Wu, A.I. Cederbaum, Oxidative stress and alcoholic liver disease, Semin. Liver Dis. 29 (2009) 141–154. Y. Xu, Y. Feng, H. Li, Z. Gao, Ferric citrate CYP2E1-independently promotes alcohol-induced apoptosis in HepG2 cells via oxidative/nitrative stress which is attenuated by pretreatment with baicalin, Food Chem. Toxicol. 50 (2012) 3264–3272. A. Dey, A.I. Cederbaum, Induction of cytochrome P450 2E1 promotes liver injury in ob/ob mice, Hepatology 45 (2007) 1355–1365. A.I. Cederbaum, L. Yang, X. Wang, D. Wu, CYP2E1 sensitizes the liver to LPS- and TNF ␣-induced toxicity via elevated oxidative and nitrosative stress and activation of ASK-1 and JNK mitogen-activated kinases, Int. J. Hepatol. 2012 (2012) 582790. T. Zima, E. Albano, M. Ingelman-Sunderberg, G.E. Arteel, G.M. Thiele, L.W. Klassen, A.Y. Sun, Modulation of oxidative stress by alcohol, Alcohol. Clin. Exp. Res. 29 (2005) 1060–1068. V.B. Patel, S. Worrall, P.W. Emery, V.P. Reddy, Protein adducts species in muscle and liver of rats following acute ethanol administration, Alcohol Alcohol. 40 (2005) 485–493. M.J. Ronis, A. Butura, B.P. Sampey, K. Shankar, R.L. Prior, S. Korourian, E. Albano, M. Ingelman-Sundberg, D.R. Petersen, T.M. Badger, Effects of N-acetylcysteine on ethanol-induced hepatotoxicity in rats fed via total enteral nutrition, Free Radic. Biol. Med. 39 (2005) 619–630. S.M. Bailey, A review of the role of reactive oxygen and nitrogen species in alcohol-induced mitochondrial dysfunction, Free Radic. Res. 37 (2003) 585–596. A. Venkatraman, S. Shiva, A.J. Davism, S.M. Bailey, P.S. Brookes, V.M. Darley-Usmar, Chronic alcohol consumption increases the sensitivity of rat liver mitochondrial respiration to inhibition by nitric oxide, Hepatology 38 (2003) 141–147. A.D. Antonekov, L.F. Panchenko, Effect of chronic ethanol treatment under partial catalase inhibition on the activity of enzymes related to peroxide metabolism in rat liver and heart, Int. J. Biochem. 20 (1988) 823–828. C. De duve, P. Baudhhuin, Peroxisomes (microbodies and related particles), Physiol. Rev. 46 (1996) 323–357. Y. Kono, I. Fridovich, Superoxide radicals inhibit catalase, J. Biol. Chem. 257 (1982) 5751–5754. C. Gueeri, S. Grisolia, Influence of prolonged ethanol intake on the levels and turnover of alcohol and aldehyde dehydrogenases and glutathione, Adv. Exp. Med. Biol. 126 (1980) 365–384. A. Bhattacharya, A. Chatterjee, S. Ghosal, S.K. Bhattacharya, Antioxidant activity of tannoid principles of Emblica officinalis (amla), Indian J. Exp. Biol. 37 (1999) 676–680. A. Bhattacharya, S. Ghosal, S.K. Bhattacharya, Antioxidant activity of tannoid principles of Emblica officinalis (amla) in chronic stress induced changes in rat brain, Indian J. Exp. Biol. 38 (2000) 877–880. P. Scartezzini, F. Antognoni, M.A. Raggi, F. Poli, C. Sabbioni, Vitamin C content and antioxidant activity of the fruit and of the Ayurvedic preparation Emblica officinalis Gaertn, J. Ethnopharmacol. 104 (2006) 113–118. M. Levental, B. Tabakoff, Sodium–potassium-activated adenosine triphosphatases activity as a measure of neuronal membrane characteristics in ethanol-tolerant mice, J. Pharmacol. Exp. Ther. 212 (1980) 315–319. E. Sutherland, B.S. Dixon, H.L. Leffert, H. Skally, L. Zaccaro, F.R. Simon, Biochemical localization of hepatic surface membrane Na+ , K+ -ATPase activity depends on membrane lipid fluidity, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 8673–8677.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
Pathophysiology xxx (2014) xxx–xxx
Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis Vaddi Damodara Reddy a , Pannuru Padmavathi b , Reddyvari Hymavathi a , Paramahamsa Maturu c , N.Ch. Varadacharyulu a,∗ b
a Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515 055, India Department of Medicine, Hematology/Oncology, University of Illinois, Chicago 60612, USA c Department of Genetics, MD Anderson Cancer Center, Houston, TX 77030, USA
Received 10 June 2013; received in revised form 7 December 2013; accepted 15 December 2013
Abstract The protective effect of Emblica officinalis fruit extract (EFE) against alcohol-induced oxidative damage in liver microsomes was investigated in rats. EFE (250 mg/kg b.wt/day) and alcohol (5 g/kg b.wt/day, 20%, w/v) were administered orally to animals for 60 days. Alcohol administration significantly increased lipid peroxidation, protein carbonyls with decreased sulfhydryl groups in microsomes, which were significantly restored to normal levels in EFE and alcohol co-administered rats. Alcohol administration also markedly decreased the levels of reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in the liver microsomes, which were prevented with EFE administration. Further, alcohol administration significantly increased the activities of cytochrome P-450, Na+ /K+ and Mg2+ ATPases and also membrane fluidity. But, administration of EFE along with alcohol restored the all above enzyme activities and membrane fluidity to normal level. Thus, EFE showed protective effects against alcohol-induced oxidative damage by possibly reducing the rate of lipid peroxidation and restoring the various membrane bound and antioxidant enzyme activities to normal levels, and also by protecting the membrane integrity in rat liver microsomes. In conclusion, the polyphenolic compounds including flavonoid and tannoid compounds present in EFE might be playing a major role against alcohol-induced oxidative stress in rats. © 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol; CYP-450; Emblica officinalis; Microsomes; Oxidative stress
1. Introduction Liver is the principle organ responsible for alcohol metabolism and is more susceptible to alcohol-induced toxicity. Oxidative stress has been suggested as key factor capable of both initiating and sustaining the mechanisms of pathogenesis leading to alcohol liver disease (ALD) [1]. As the main source of reactive oxygen species (ROS), hepatic microsomes are susceptible to ROS attack, especially upon cytochrome P450 2E1 (CYP-450) activation by ethanol [2]. ROS generated from ethanol metabolism can directly damage cell membranes by peroxidation of membrane polyunsaturated fatty ∗ Corresponding author at: Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515 055, AP, India. Tel.: +91 8554 255761; fax: +91 8554 255244. E-mail address: vdp [email protected] (N.Ch. Varadacharyulu).
acids. Increased lipid peroxidation changes the activities of various membrane bound enzymes. Especially ATPases are very sensitive to peroxidation reactions and membrane fluidity [3]. Chronic alcohol consumption also alters redox thiol status which might have biological relevance and contribute to the pathologies associated with several disease states. The free radicals generated during ethanol metabolism interact with proteins, lipids and DNA and thereby forming adducts [4]. Adduct formation may lead to interference with protein function, stimulation of fibrogenesis and induction of immune responses, which finally contributes to the progression of alcohol-induced liver diseases [5,6]. Alcohol-induced oxidative stress has been found mainly due to an increased production of oxygen free radicals and lower cellular antioxidant levels [7]. The endogenous antioxidant enzymes are responsible for the detoxification of deleterious oxygen radicals. Antioxidants play an important
0928-4680/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pathophys.2013.12.001
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
2
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
role to protect liver against alcohol-induced damage caused by reactive oxygen species [8]. Dietary phytochemicals are capable of removing free radicals. Among them, phenolic and polyphenolic compounds, such as flavonoids and tannins in edible fruits, exhibit potent antioxidant activities. Therefore, the possibility of preventing the onset of alcohol toxicity using herbal medicines has attracted considerable attention [9]. Literature mapping reveal that the fruit of Emblica officinalis, commonly known as amla, is a potential source with many therapeutic principles and has been used in more than hundred herbal formulations of Ayurveda, an Indian traditional medicine [10]. Studies showed that Emblica contains more than 20 hydrolyzable tannins [11]. The fruit contains flavonoids (e.g. kaempherol-3--O-d-glucoside, quercetin3-O--glucoside), polyphenols (e.g. emblicanin A and B, punigluconin and pedunculagin) and also contains phyllantine and zeatin alkaloids, and a number of benzenoids including amlaic acid, corilagin, ellagic acid, 3-6-di-Ogalloyl-glucose, ethyl gallate, 1,6-di-O-galloyl--d-glucose, 1-di-O-galloyl--d-glucose, putranjivain A, digallic acid, phyllemblic acid, emblicol, and alactaric acid and gallic acid [12–14]. In vitro and in vivo studies showed that EFE had strong antioxidant and radical scavenging activities against DPPH, O2 •– , OH• , and NO radicals [8]. Moreover, studies have shown that Emblica possess antidiabetic [15], hypolipidemic, anticancer [16], antiatherogenic [17], hepatoprotective [18,19] and neuroprotective properties [20]. Most of the reports suggest that these health effects could be attributed to the antioxidant activities of the EFE. Phenolic compounds, especially hydrolyzable tannins and flavonoids in combination with vitamin C, are considered to be the major antioxidants and bioactive components in EFE. Based on these considerations, we hypothesized that EFE may be a promising phytomedicine that could attenuate alcoholinduced oxidative stress in the liver microsomes.
2. Materials and methods
tap water ad libitum. The animals were procured from Sri Venkateswara Enterprises, Bangalore, India, were divided into four groups of eight rats in each group. Group I control rats (C), which received glucose instead of alcohol (i.e. caloric equivalent to alcohol), group II (A), which received 20% (v/v) alcohol in water, group III rats (EFE), which received EFE alone in water, group IV rats (A + EFE), which received 20% (v/v) alcohol and then EFE after 8 h. Alcohol 5 g/kg b.wt/day and EFE 250 mg/kg b.wt/day was administered through stomach tube daily to each rat for 60 days. The dose of Emblica fruit extract was fixed based on earlier reports [8]. Food and water intake of all the animals was recorded daily and weight of rats was followed on alternate days. At the end of the experimental period, the rats in each group were fasted overnight and then sacrificed by cervical dislocation. Tissues were collected and processed immediately for further analysis. Institutional ethical committee approved this study.
2.3. Preparation of rat liver microsomes Rat liver microsomes were prepared according to the method described previously [21]. Microsomes were prepared by centrifuging the post mitochondrial supernatant from the first 7000 × g centrifugation at 12,000 × g for 15 min. The pellet and the floating lipid were discarded, and the supernatant was centrifuged at 144,000 × g for 1 h for sedimentation of microsomes. All operations of preparation and subsequent fractionation were carried out at 4 ◦ C.
2.4. Measurement of TBARS, protein carbonyls and sulfhydryl groups Thiobarbituric acid reactive species (TBARS) were measured by the formation of malondialdehyde [22]. Protein carbonyls were determined as described previously [23]. Protein sulfhydryl groups were measured using Ellman’s reagent [24].
2.1. Chemicals 2.5. Determination of cytochrome P-450 activity The chemicals used in the present study were procured from Sigma Chemical Co. (St. Louis, MO, USA) and SISCO Research Laboratories (Mumbai, India). Ethanol used for administration to rats was obtained by re-distillation. E. officinalis fruit extract dry powder (90.8% water soluble extractives including 49.5% tannins) was obtained from Chemiloids Ltd., Vijayawada, India (Manufacturers and exporters of herbal extracts). 2.2. Animals and experimental design Two-month-old male albino Wistar rats, weighing about 120–140 g, were maintained in animal house with commercial pellet diet (Hindustan Lever Ltd., Bangalore, India) and
The activity of the CYP-450 was assayed as described previously [25]. Briefly, microsomal suspensions (1.0 mg protein/ml) in phosphate buffer treated with a few grains of sodium dithionite and take it in the reference and sample cuvettes (or in the two halves of the split cuvette) and record the baseline from 400 to 510 nm. The contents of the sample cuvette were transferred to new tube and bubble gently with carbon monoxide (CO) for 60 s. Transfer back to the sample cuvette and record the difference spectrum from 400 to 510 nm again. The difference in absorbance between 450 and 490 nm can then be used for the calculation of CYP-450 content using the extinction coefficient difference (E450–490 nm of 91 cm−1 mM−1 ).
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
3
2.6. Determination of antioxidant status Liver microsomal catalase (CAT) was assayed as described [26]. The CAT activity was expressed as nmol H2 O2 decreased/mg protein/min. Superoxide dismutase (SOD) was assayed [27]. A single unit of enzyme was expressed as 50% inhibition of NBT (nitro blue tetrazolium) reduction/min/mg protein. Glutathione peroxidase (GPx) activity was measured as described [28]. GPx activity was expressed as mol GSH oxidized/min/mg protein. Total reduced glutathione (GSH) content was measured as described previously [29]. 2.7. Measurement of Na+ /K+ -ATPase and Mg2+ -ATPase activity The activities of Na+ /K+ and Mg2+ -ATPases were determined [30]. The activities were indirectly measured by estimating the phosphorus liberated after the incubation of erythrocyte membrane in a reaction mixture containing the substrate ATP with the co-substrate elements at 37 ◦ C for 15 min. The Mg2+ ATPase activity was measured in the presence of 1 mM ouabain, a specific inhibitor of Na+ , K+ ATPase. The reactions were arrested by adding 1.0 ml of 10% TCA. The phosphorus content from the TCA supernatants was then determined [31]. 2.8. Measurement of membrane fluidity The quantitative measurement of membrane fluidity was performed by the fluorescence polarization technique described previously with 1,6-diphenyl 1,3,5-hexatriene (DPH) as fluorescence probe as described [32]. Membranes (50 g protein) were suspended in 50 mmol/l DPH solubilized in tetrahydrofuran, and incubated at 37 ◦ C for 30 min. Fluorescence polarization was determined using a Hitachi fluorescence spectrophotometer (Hitachi, Japan) equipped with rotating polarizing filters with samples held at 25 ◦ C. Samples were excited at 360 nm and the emission intensity was read at 435 nm. Polarization (P) and fluorescence anisotropy (γ) were calculated using the equation. P = IVV − IVH·G /IVV + IVH·G . Where IVV and IVH are the intensities measured parallel and perpendicular to the vertical axis of the excitation beam, and G is the correction factor. IVH /IHH·γ is calculated using the formula γ = 2P/(3 − P). Protein concentration was determined as previously described [33].
Fig. 1. Effect of EFE administration on TBARS in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
3. Results 3.1. Effect of EFE on alcohol-induced alterations in TBARS, protein carbonyls and sulfhydryl groups The effect of alcohol administration on lipid peroxidation, protein carbonyls and sulfhydryl groups in microsomes are shown in Figs. 1–3. As an index of lipid peroxidation we measured MDA levels and an index of protein oxidation we measured protein carbonyls and sulfhydryl groups in controls and experimental animals. Alcohol administration resulted a significant (p < 0.05) increase in MDA levels, protein carbonyls with a significant (p < 0.05) decrease in sulfhydryl groups compared to control animals. But, co-administration of alcohol and EFE significantly (p < 0.05) prevented the elevation of MDA levels, protein carbonyls and diminished sulfhydryl groups compared with alcohol alone administered
2.9. Statistical analysis Mean and standard deviation values of all the parameters were determined for each group. ANOVA followed by Student “t” test were performed to determine significant difference among the groups. A p < 0.001 and p < 0.05 was considered statistically significant.
Fig. 2. Effect of EFE administration on protein carbonyls in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
4
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
Fig. 3. Effect of EFE administration on protein sulfhydryl groups in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
animals. EFE treatment alone did not show any significant changes in the above parameters compared to control group. 3.2. Protective role of EFE on alcohol-induced microsomal redox disturbance Non-enzymatic GSH, as well as enzymatic catalase, SOD and GPx are crucial antioxidants for ROS derivatives generated in endoplasmic reticulum during ethanol metabolism, to maintain the redox homeostasis. The data presented in Table 1 shows the effect of EFE on alcohol-induced redox disturbance in microsomes. Chronic alcohol administration significantly (p < 0.05) depleted microsomal catalase, SOD and GPx enzyme activities and non-enzymatic GSH content. All the above enzymes and GSH content were restored to normal levels after co-administration of alcohol and EFE to rats. The compounds present in EFE quench free radicals generated from alcohol metabolism and help cells to maintain redox homeostasis constantly.
Fig. 4. Effect of EFE administration on cytochrome P-450 enzyme activity in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Asterisk “*” indicates significant from controls and “**” indicates significant from control and alcohol administered rats.
3.4. Effect of EFE on ethanol-induced alterations in membrane fluidity The steady state fluorescent anisotropy studies in microsomes using DPH were carried out and the anisotropic (γ) values are presented in Fig. 5. The fluorescent anisotropic values of alcohol administered rats revealed significant (p < 0.01) increase in anisotropic (γ) values in the present study. Upon administration of EFE to alcohol receiving rats significantly (p < 0.01) prevented from these alterations and almost restored to normal levels. No significant change was seen in EFE alone administered rats compared to controls. 3.5. Alterations in membrane bound enzymes Fig. 6 shows the effect of alcohol administration on the activities of Na+ /K+ and Mg2+ ATPases. Na+ /K+ and Mg2+ ATPases activity was increased in alcohol administered rats
3.3. Effect of EFE on alcohol-induced alterations in CYP-450 activity Data presented in Fig. 4 reveals the effect of EFE on alcohol-induced alterations in CYP-450 enzyme activity in microsomes. Results showed that alcohol administration significantly (p < 0.05) increased CYP-450 enzyme activity in microsomes compared to controls. Administration of EFE to alcohol receiving rats significantly (p < 0.05) decreased CYP450 enzyme activity compare to alcohol alone administered rats. Though, administration of EFE to alcohol receiving rats did not brought back the enzyme to normal level, but still it is significantly different from EFE alone administered rats. EFE alone administered rats showed normal enzyme activity to that of controls.
Fig. 5. Effect of EFE administration on anisotropic (γ) value in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.001 is considered as statistically significant between groups. Asterisk “#” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
5
Table 1 Effect of EFE administration on liver microsomal antioxidant status in alcohol administered rats. Parameter
Groups Control
GSH (g/mg protein) Catalase (nmol/min/mg protein) SOD (Units/mg protein) GPX (g of GSH oxidized/min/mg protein)
3.37 38.1 5.38 2.95
± ± ± ±
Alcohol 0.14 3.66 0.24 0.16
1.91 23.3 3.21 0.99
EFE
± ± 2.3* ± 0.13* ± 0.11* 0.17*
3.35 38.6 5.36 2.90
A + EFE ± ± ± ±
0.19 2.4 0.16 0.08
3.12 34.6 4.24 1.65
± ± ± ±
0.10ns 3.2ns 0.19ns 0.1ns
Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
compared to control rats. However, administration of EFE to alcohol administered rats significantly (p < 0.05) offered protection and prevented the rise of the above ATPase activities. EFE alone treated rats had no significant (p < 0.05) effect on Na+ /K+ and Mg2+ ATPases activity comparable to controls.
4. Discussion Reactive oxygen species (ROS) are small, highly reactive, oxygen-containing molecules that can react with and damage complex cellular molecules, particularly in the liver [34]. Ethanol metabolism is directly involved in not only the production of reactive oxygen species, but also related in the formation of an environment favorable to oxidative stress such as hypoxia, endotoxaemia and cytokine release [35]. Previous studies showed that alcohol administration to rats significantly increased lipid peroxidation in the liver and make hepatocytes leaky [8]. Upon hepatic damage or necrosis enzymes will be leaked into the circulation and raise their concentrations. Our previous study also showed that elevated serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in alcohol treated animals indicated cellular breakage and loss of functional integrity of cell membranes in
Fig. 6. Effect of EFE administration on Na+ /K+ -ATPase and Mg2+ -ATPase enzyme activities in control and experimental rats. Values are mean ± SD of eight rats in each group. A p < 0.05 is considered as statistically significant between groups. Values are expressed as mol pi/h/mg protein. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and EFE alone administered rats.
liver [10]. Induction of CYP-450 by ethanol is a central pathway by which ethanol generates oxidative stress in hepatocytes. During ethanol oxidation ROS production increases dramatically due to induction of CYP2E1 and by activation of Kupffer cells in the liver [36]. In the present study, the activity of CYP-450 was increased and consistent with earlier reports [2,37]. Increased activity of CYP-450 results in accelerated production of lipid hydroperoxides and consequently increases oxidative stress [38]. This effect on the redox status of the liver and can cause activation of Kupffer cells and subsequently, hepatic stellate cells, and thus contributing to the generation of alcoholic liver disease [39]. Administration of EFE to alcohol administered rats significantly prevented these alterations. The polyphenolic compounds present in EFE extract scavenge free radicals generated during alcohol metabolism in microsomes and protects liver. The metabolism of ethanol via the CYP-450 pathway results in increased ROS production, including superoxide, hydrogen peroxide (H2 O2 ), and hydroxyl radicals. Microsomes are the known targets for alcohol action due to their high content of polyunsaturated fatty acids and calcium storage [2]. Alcohol administration significantly increased lipid peroxidation and protein carbonyls with decreased sulfhydryl groups. Moreover, various lipid peroxidation products bind to proteins forming stable adducts stimulating fibrogenesis and histological damages in alcoholic liver disease. In addition, hydroxyethyl radical (HER), hydroxynonenal (HNE) and other radicals are involved in modifying cellular proteins [40,41]. Previous studies also showed that greater increase in liver protein carbonyl content, which was attributed to the development of pathogenesis related to alcoholism [42]. Studies using rats administered with alcohol demonstrated that alterations in protein thiols have biological relevance and contribute to the pathologies associated with several diseased states such as alcoholic liver damage [43]. In the present study, increased lipid peroxidation, protein oxidation and diminished protein sulfhydryls may be due to enhanced ROS and lowered antioxidant levels. Administration of EFE to alcohol receiving rats prevented from the augment of lipid peroxidation and protein carbonyls. The protective action of Emblica is usually due to the inhibition of free radical-induced chain reaction and the resultant prevention of peroxidative deterioration of structural lipids in membranes. Antioxidant enzymes play important role in the elimination of ROS in cells. In this study, we found diminished
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
6
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
activities of SOD, catalase and GPx as well as GSH in ethanol receiving rats. The decrease in the SOD activity may be associated with the elevation of the intracellular concentration of H2 O2 as a result of catalase inactivation [44]. Catalase has been reported to be responsible for detoxification of H2 O2 which is an effective inhibitor of SOD [45]. The decrease in catalase activity could be due to low production of the enzyme and/or the inhibition of the enzyme as a result of the increased production of free radicals [46]. GSH is a major non-protein thiol and plays a central role in coordinating the antioxidant defense process. It is involved in the maintenance of normal cell structure and function through its redox and detoxification reactions [47]. GPx with the help of GSH metabolizes hydrogen peroxide to water thereby protecting mammalian cells against oxidative damage. Metabolism of ethanol by CYP2E1 also results in a significant increase in free radical and acetaldehyde production which, in turn, diminish reduced glutathione (GSH) and other defense systems against oxidative stress leading to further hepatocyte damage. Administration of EFE to alcohol-administered rats prevented the down-regulation of antioxidant enzymes. The phytochemical compounds present in EFE boost antioxidant activity of the cells by scavenging free radicals generated during ethanol metabolism. The compounds especially hydrolysable tannoid principles emblicanin A, emblicanin B, punigluconin and pedunculagin compounds present in Emblica might have contributed largely for the above mechanism [48–50]. Biological membranes exerting important biological functions are readily susceptible to free radical attack. A major protective mechanism against oxidative attack is the membrane integrity. Free radicals generated from alcohol metabolism can directly damage cell membrane structure and function, including membrane fluidity, ion permeability and modulation of membrane spanning protein activities [3]. One of the most important membrane proteins are ATPases. Lipid peroxidation is known to disturb structural integrity of the membrane that might in turn affect the activities of Na+ /K+ -ATPase and Mg2+ -ATPase enzymes. Alcohol-induced generation of free radicals also affect membrane-linked enzyme activity through modification of membrane fluidity [51]. Studies also have shown that alcoholinduced generation of free radicals affects membrane-linked enzyme activity through modification of membrane fluidity [52]. In this study, increased Na+ /K+ -ATPase and Mg2+ ATPase activities in ethanol treated rats may be due to alteration in the membrane fluidity. Co-administration of EFE and ethanol to rats showed lowered lipid peroxidation, which in turn might be responsible for normalization of membrane structure and membrane fluidity, consequently restores the activities of membrane bound enzymes. In conclusion, restoration of all these components and activities of microsomal enzymes by EFE administration in alcohol administered rats suggested the therapeutic potential of Emblica principles at molecular level and protecting the cells and organelles from alcohol-induced oxidative stress. In
EFE several phytocompounds work together synergistically to fight against the complications of alcohol consumption. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments The authors thank the Director and staff of the Center for Cellular and Molecular Biology, Hyderabad, India for providing facilities to carry out fluidity studies. This study was supported in part by the University Grants Commission, New Delhi, India (Grant No. F.No.40-203/2011 SR). References [1] D. Wu, X. Wang, R. Zhou, L. Yang, A.I. Cederbaum, Alcohol steatosis and cytotoxicity: the role of cytochrome P4502E1 and autophagy, Free Radic. Biol. Med. 53 (2012) 1346–1357. [2] Y. Tang, C. Gao, Y. Shi, L. Zhu, X. Hu, D. Wang, Y. Lv, X. Yang, L. Liu, P. Yao, Quercetin attenuates ethanol-derived microsomal oxidative stress: implication of haem oxygenase-1 induction, Food Chem. 132 (2012) 1769–1774. [3] V.D. Reddy, P. Padmavathi, M. Paramahamsa, N.C. Varadacharyulu, Modulatory role of Emblica officinalis against alcohol induced biochemical and biophysical changes in rat erythrocyte membranes, Food Chem. Toxicol. 47 (2009) 1958–1963. [4] O. Niemela, Aldehyde–protein adducts in the liver as a result of ethanol induced oxidative stress, Front. Biosci. 4 (1999) D506–D513. [5] J.B. Hoek, J.G. Pastorino, Ethanol, oxidative stress, and cytokine induced liver cell injury, Alcohol 27 (2002) 63–68. [6] P. Abraham, G. Wilfred, B. Ramakrishna, Oxidative damage to the hepatocellular proteins after chronic ethanol intake in the rat, Clin. Chim. Acta 325 (2002) 117–125. [7] A. Dey, A.I. Cederbaum, Alcohol and oxidative liver injury, Hepatology 43 (2006) S63–S74. [8] V.D. Reddy, P. Padmavathi, S. Gopi, M. Paramahamsa, N.C. Varadacharyulu, Protective effect of Emblica officinalis against alcohol induced hepatic injury by ameliorating oxidative stress in rats, Indian J. Clin. Biochem. 4 (2010) 419–424. [9] B.J. Xu, Y.N. Zheng, C.K. Sung, Natural medicines for alcoholism treatment: a review, Drug Alcohol Rev. 24 (2005) 525–536. [10] V.D. Reddy, P. Padmavathi, N.C. Varadacharyulu, Emblica officinalis protects against ethanol-induced liver mitochondrial dysfunction in rats, J. Med. Food 12 (2009) 327–333. [11] B. Yang, M. Kortesniemi, P. Liu, M. Karonen, J.P. Salminen, Analysis of hydrolyzable tannins and other phenolic compounds in emblic leafflower (Phyllanthus emblica L.) fruits by high performance liquid chromatography-electrospray ionization mass spectrometry, J. Agric. Food Chem. 60 (2012) 8672–8683. [12] Y.J. Zhang, T. Tanaka, C. Yang, I. Kouno, New phenolic constituents from the fruit juice of Phyllanthus emblica, Chem. Pharm. Bull. (Tokyo) 49 (2001) 537–540. [13] S. Ghosal, V.K. Tripathi, S. Chauhan, Active constituent of Emblica officinalis. Part 1: The chemistry and antioxidant effects of two new hydrolyzable tannins, emblicanin A and B, Indian J. Chem. 35 (1996) 941–948. [14] M. Majeed, B. Bhat, A. Jadhav, J. Srivastava, K. Nagabhushanam, Ascorbic acid and tannins from Emblica officinalis Gaertn Fruits—a revisit, J. Agric. Food Chem. 57 (2009) 220–225.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
PATPHY-802; No. of Pages 7
ARTICLE IN PRESS V.D. Reddy et al. / Pathophysiology xxx (2014) xxx–xxx
[15] P. Suryanarayana, M. Saraswat, J.M. Petrashm, G.B. Reddy, Emblica officinalis and its enriched tannoids delay streptozotocin induced diabetic cataract in rats, Mol. Vis. 13 (2007) 1291–1297. [16] M.S. Baliga, J.J. Dsouza, Amla (Emblica officinalis Gaertn), a wonder berry in the treatment and prevention of cancer, Eur. J. Cancer Prev. 20 (2011) 225–239. [17] S.V. Nampoothiri, A. Prathapan, O.L. Cherian, K.G. Raghu, V.V. Venugopalan, A. Sundaresan, In vitro antioxidant and inhibitory potential of Terminalia bellerica and Emblica officinalis fruits against LDL oxidation and key enzymes linked to type 2 diabetes, Food Chem. Toxicol. 49 (2011) 125–131. [18] R. Srirama, H.B. Deepak, U. Senthilkumar, G. Ravikanth, B.R. Gurumurthy, M.B. Shivanna, C.V. Chandrasekaran, A. Agarwal, R.U. Shaanker, Hepatoprotective activity of Indian Phyllanthus, Pharm. Biol. 50 (2012) 948–953. [19] B.D. Hiraganahalli, V.C. Chinampudur, S. Dethe, D. Mundkinajeddu, M.K. Pandre, J. Balachandran, A. Agarwal, Hepatoprotective and antioxidant activity of standardized herbal extracts, Pharmacogn. Mag. 8 (2012) 116–123. [20] V.D. Reddy, P. Padmavathi, G. Kavitha, S. Gopi, N.C. Varadacharyulu, Emblica officinalis ameliorates alcohol-induced brain mitochondrial dysfunction in rats, J. Med. Food 14 (2011) 62–68. [21] C.C. Cunningham, S. Filus, R.E. Bottenus, P.I. Spach, Effect of ethanol consumption on the phospholipid composition of rat liver microsomes and mitochondria, Biochim. Biophys. Acta 712 (1982) 225–233. [22] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [23] A.Z. Reznick, L. Packer, Oxidative damage to proteins: spectroscopic method for carbonyl assay, Methods Enzymol. 233 (1994) 357–363. [24] P. Padmavathi, V.D. Reddy, G. Kavitha, P. Maturu, N.C. Varadacharyulu, Chronic cigarette smoking alters erythrocyte membrane lipid composition and properties in male human volunteers, Nitric Oxide 23 (2010) 181–186. [25] T. Omura, R. Sato, The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties, J. Biol. Chem. 239 (1964) 2379–2385. [26] H. Abei, Catalase in vitro, Methods Enzymol. 10 (1988) 121–126. [27] K.R. Reddy, V.D. Reddy, P. Padmavathi, G. Kavitha, B. Saradamma, N.C. Varadacharyulu, Gender differences in alcohol induced oxidative stress and altered membrane properties in erythrocytes of rats, Indian J. Biochem. Biophys. 50 (2013) 32–39. [28] P. Maturu, V.D. Reddy, P. Padmavathi, N.C. Varadacharyulu, Alcohol induced oxidative stress: an adaptive role of erythrocyte antioxidant system in human chronic alcoholics, Exp. Toxicol. Pathol. 64 (2012) 697–703. [29] G.S. Krushna, M.A. Kareem, V.D. Reddy, P. Padmavathi, S.A. Hussain, L. Devi Kodidhela, Aegle marmelos fruit extract attenuates isoproterenol-induced oxidative stress in rats, J. Clin. Biochem. Nutr. 50 (2012) 199–204. [30] B. Ismail, I.S. Edelman, Assay of Na+ , K+ ATPase, Biochem. Pharmacol. 34 (1985) 2685–2689. [31] P. Padmavathi, V.D. Reddy, M. Narendra, N.C. Varadacharyulu, Bidis – hand-rolled, Indian cigarettes: induced biochemical changes in plasma and red cell membranes of human male volunteers, Clin. Biochem. 42 (2009) 1041–1045. [32] P. Padmavathi, V.D. Reddy, P. Maturu, N.C. Varadacharyulu, Smoking-induced alterations in platelet membrane fluidity and Na(+) /K(+) -ATPase activity in chronic cigarette smokers, J. Atheroscler. Thromb. 17 (2010) 619–627. [33] V.D. Reddy, P. Padmavathi, G. Kavitha, B. Saradamma, N.C. Varadacharyulu, Alcohol-induced oxidative/nitrosative stress alters brain mitochondrial membrane properties, Mol. Cell. Biochem. 375 (2013) 39–47. [34] Y. Lu, D. Wu, X. Wang, S.C. Ward, A.I. Cederbaum, Chronic alcohol-induced liver injury and oxidant stress are decreased in
[35] [36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45] [46] [47]
[48]
[49]
[50]
[51]
[52]
7
cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice, Free Radic. Biol. Med. 49 (2010) 1406–1416. D. Wu, A.I. Cederbaum, Oxidative stress and alcoholic liver disease, Semin. Liver Dis. 29 (2009) 141–154. Y. Xu, Y. Feng, H. Li, Z. Gao, Ferric citrate CYP2E1-independently promotes alcohol-induced apoptosis in HepG2 cells via oxidative/nitrative stress which is attenuated by pretreatment with baicalin, Food Chem. Toxicol. 50 (2012) 3264–3272. A. Dey, A.I. Cederbaum, Induction of cytochrome P450 2E1 promotes liver injury in ob/ob mice, Hepatology 45 (2007) 1355–1365. A.I. Cederbaum, L. Yang, X. Wang, D. Wu, CYP2E1 sensitizes the liver to LPS- and TNF ␣-induced toxicity via elevated oxidative and nitrosative stress and activation of ASK-1 and JNK mitogen-activated kinases, Int. J. Hepatol. 2012 (2012) 582790. T. Zima, E. Albano, M. Ingelman-Sunderberg, G.E. Arteel, G.M. Thiele, L.W. Klassen, A.Y. Sun, Modulation of oxidative stress by alcohol, Alcohol. Clin. Exp. Res. 29 (2005) 1060–1068. V.B. Patel, S. Worrall, P.W. Emery, V.P. Reddy, Protein adducts species in muscle and liver of rats following acute ethanol administration, Alcohol Alcohol. 40 (2005) 485–493. M.J. Ronis, A. Butura, B.P. Sampey, K. Shankar, R.L. Prior, S. Korourian, E. Albano, M. Ingelman-Sundberg, D.R. Petersen, T.M. Badger, Effects of N-acetylcysteine on ethanol-induced hepatotoxicity in rats fed via total enteral nutrition, Free Radic. Biol. Med. 39 (2005) 619–630. S.M. Bailey, A review of the role of reactive oxygen and nitrogen species in alcohol-induced mitochondrial dysfunction, Free Radic. Res. 37 (2003) 585–596. A. Venkatraman, S. Shiva, A.J. Davism, S.M. Bailey, P.S. Brookes, V.M. Darley-Usmar, Chronic alcohol consumption increases the sensitivity of rat liver mitochondrial respiration to inhibition by nitric oxide, Hepatology 38 (2003) 141–147. A.D. Antonekov, L.F. Panchenko, Effect of chronic ethanol treatment under partial catalase inhibition on the activity of enzymes related to peroxide metabolism in rat liver and heart, Int. J. Biochem. 20 (1988) 823–828. C. De duve, P. Baudhhuin, Peroxisomes (microbodies and related particles), Physiol. Rev. 46 (1996) 323–357. Y. Kono, I. Fridovich, Superoxide radicals inhibit catalase, J. Biol. Chem. 257 (1982) 5751–5754. C. Gueeri, S. Grisolia, Influence of prolonged ethanol intake on the levels and turnover of alcohol and aldehyde dehydrogenases and glutathione, Adv. Exp. Med. Biol. 126 (1980) 365–384. A. Bhattacharya, A. Chatterjee, S. Ghosal, S.K. Bhattacharya, Antioxidant activity of tannoid principles of Emblica officinalis (amla), Indian J. Exp. Biol. 37 (1999) 676–680. A. Bhattacharya, S. Ghosal, S.K. Bhattacharya, Antioxidant activity of tannoid principles of Emblica officinalis (amla) in chronic stress induced changes in rat brain, Indian J. Exp. Biol. 38 (2000) 877–880. P. Scartezzini, F. Antognoni, M.A. Raggi, F. Poli, C. Sabbioni, Vitamin C content and antioxidant activity of the fruit and of the Ayurvedic preparation Emblica officinalis Gaertn, J. Ethnopharmacol. 104 (2006) 113–118. M. Levental, B. Tabakoff, Sodium–potassium-activated adenosine triphosphatases activity as a measure of neuronal membrane characteristics in ethanol-tolerant mice, J. Pharmacol. Exp. Ther. 212 (1980) 315–319. E. Sutherland, B.S. Dixon, H.L. Leffert, H. Skally, L. Zaccaro, F.R. Simon, Biochemical localization of hepatic surface membrane Na+ , K+ -ATPase activity depends on membrane lipid fluidity, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 8673–8677.
Please cite this article in press as: V.D. Reddy, et al., Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis, Pathophysiology (2014), http://dx.doi.org/10.1016/j.pathophys.2013.12.001
