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Antioxidant effect of Sargassum polycystum (Phaeophyceae) against acetaminophen induced changes in hepatic mitochondrial enzymes during toxic hepatitis
Chemosphere 61 (2005) 276–281 www.elsevier.com/locate/chemosphere
Antioxidant effect of Sargassum polycystum (Phaeophyceae) against acetaminophen induced changes in hepatic mitochondrial enzymes during toxic hepatitis Hanumantha Rao Balaji Raghavendran, Arumugam Sathivel, Thiruvengadum Devaki * Department of Biochemistry, University of Madras, Guindy Campus, Patel Road, Chennai 600 025, India Received 14 June 2004; received in revised form 13 January 2005; accepted 18 January 2005 Available online 25 April 2005
Abstract The present study was aimed to examine the protective effects of Sargassum polycystum (Phaeophyceae) alcoholic extract on changes in liver mitochondrial enzymes against acetaminophen induced toxic hepatitis in experimental rats. The levels of protein, lipid peroxide, glutathione (GSH) in mitochondrial fraction, superoxide dismutase (SOD) and catalase (CAT) were also determined. The activities of tricarboxylic acid enzymes such as isocitrate dehydrogenase (ICD), a-ketoglutarate dehydrogenase (a-KGD), succinate dehydrogenase (SD), malate dehydrogenase (MD), NADH dehydrogenase, and cytochrome-c-oxidase were determined in mitochondrial fraction. The rats intoxicated with acetaminophen showed significant elevation in the levels of lipid peroxides with decreased levels of protein, GSH, SOD, CAT and impaired tricarboxylic acid cycle enzyme activities. The prior oral administration of S. polycystum alcoholic extract showed significant diminution in the severity of toxic hepatitis in acetaminophen-induced rats by maintaining the activities of tricarboxylic acid enzymes with concomitant improvement in the hepatic mitochondrial antiperoxidative status when compared with intoxicated animals. The results obtained in the present study indicate that the protective effects of S. polycystum extract may be due to the presence of some active compounds that are inhibitory against the free radicals generated during lipid peroxidation in acetaminophen induced toxic hepatitis. 2005 Published by Elsevier Ltd. Keywords: Sargassum polycystum; Acetaminophen; Antiperoxidative; Toxic hepatitis; Lipid peroxidation
1. Introduction Acetaminophen (AP) is one of the most commonly used analgesics/antipyretics worldwide. The toxic doses
*
Corresponding author. Tel.: +91 2235 1269 (O), 2441 2292 (R); fax: +91 244235 2494. E-mail addresses: searag13@rediffmail.com (H.R. Balaji Raghavendran), [email protected] (T. Devaki). 0045-6535/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.chemosphere.2005.01.049
of AP can cause hepatocellular necrosis (Albano et al., 1985). In particular, the mechanism of cell damage is mediated by the metabolic activation of AP to a highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is able to deplete hepatocellular glutathione (GSH) and to bind covalently with cell macromolecules (Black, 1984). The concentration of intracellular GSH is therefore a vital determinant in the extent of AP induced hepatic necrosis. The GSH depletion has been suggested to markedly enhance the
H.R. Balaji Raghavendran et al. / Chemosphere 61 (2005) 276–281
susceptibility of mitochondrial structural dysfunction from oxidative stress and to induce mitochondrial structural degeneration (Martensson and Meister, 1989). The levels of mitochondrial GSH and alterations in mitochondrial energy metabolism, which are most likely due to the free radical accumulation, observed during liver regeneration (Vendemiale et al., 1995). The food values of marine algae are currently being reconsidered in the hope of coping with future food shortages. Recently the marine algae have been shown to produce a variety of compounds and some of them are found to have key biological activity of potential medicinal value (Faulkner, 1993). Several studies (in vitro) have investigated to show the antioxidant activity of Sargassum spp. (Matsukawa, 1997; Yan et al., 1998). Initially, Sargassum polycystum alcoholic extract was screened for its protective nature against acetaminophen induced hepatotoxicity in experimental rats (Balaji Raghavendran et al., 2004) The present study was designed to examine the protective effect of S. polycystum alcoholic extract on changes in liver mitochondrial enzymes against acetaminophen induced toxic hepatitis in experimental rats.
2. Materials and methods 2.1. Seaweed material Seaweed S. polycystum was collected from low tide area of mandapam (Gulf of Mannar) coasts. The species authentication was done by Prof. N. Kaliaperumal (CMFRI, Rameswaram, India). Alga was thoroughly washed in running tap water and deionized water to remove epiphytes and other contamination. 2.2. Seaweed extraction The contamination free seaweed sample was air-dried under shade and then coarsely powdered. The powdered seaweed material was extracted with ethanol in cold for a period of 72 h. The crude extract was filtered, concentrated on a water bath, and then dried in vaccum. The extract was subjected to TLC and phytochemical analysis, which showed positive result for the presence of terpenoids and flavonoids (Harbone, 1973). 2.3. 1,1-Diphenyl-2-picrylhydrazyl assay (in vitro) The seaweed extract was subjected to 1, 1-diphenyl2-picrylhydrazyl (DPPH) assay to determine the free radical scavenging property of seaweed extract. DPPH solution was prepared at the concentration of 3 · 105 mol/l; the solvent was dimethylsulfoxide (DMSO). During the test, an organic solvent (alcohol) extract
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(50, 100, 200, 300, 500 and 750 mg) of 2 ml was mixed with 2 ml of DPPH solution. The test tube was capped and kept for 60 min; absorbance was read at 517 nm by UV-visible spectrophotometer. DMSO was used as the blank. Another measure was done after 30 min. Meanwhile, the absorbance of the organic solvent adding 2 ml DMSO was also determined. Every sample was done in triplet and mean was obtained to calculate the free radical scavenging activity (Brad-Williams, 1995). ½1 ðAi Aj Þ=Ac 100 For DPPH in the above equation, Ai = the absorbance of organic solvent extract mixed with DPPH solution; Aj = the absorbance of the same organic extract mixed with 2 ml DMSO; Ac = absorbance of DPPH solution upon adding 2 ml of DMSO. 2.4. Histochemical analysis of S. polycystum The sectional and macerated seaweed material was dehydrated by passing through ascending and descending alcoholic (100–50%) series and kept in water. The section was then stained with diluted 5% aluminum sulphate solution of toluidine blue (TB) pH at 4.4. The sectional seaweed showed deep purple color indicating the presence of sulphated polysaccharide. Alcian blue (5% 8GX of 100 ml acid water at pH 5.0) a specific stain for sulphated polysaccharide was also used for the further confirmation (Krishnamurthy, 1988). 2.5. Experimental animals Male Wistar strain albino rats, weighing about 120– 150 g was obtained from the Fredrick institute for plant protection and toxicology, Padappai, Chennai, India. The animals were maintained in a 12-h light and dark, at 22 ± 3 C cycle and fed with a commercial pelleted diet (M/s. Hindustan Foods Ltd., Bangalore, India) and had free access of water. The rats were randomized into four groups, each group comprising six animals. Group I rats were served as normal control, group II rats were given acetaminophen (Sigma, chemical company, St. Louis, MO, USA: 800 mg/kg b.wt. intraperitoneally), group III rats were pre-treated with seaweed extract alone (200 mg/kg b.wt. daily for a period of 15 days, orally) group IV rats were pre-treated orally with seaweed extract (200 mg/kg b.wt. daily for a period of 15 days, orally) prior to acetaminophen induction (800 mg/kg b.wt. intraperito-neally). At the end of the experiment, the rats were anesthetized with sodium pentobarbitone (35 mg/kg b.wt. intraperitoneally) and sacrificed by cervical decapitation. The liver was excised immediately and washed in ice-cold saline. Liver mitochondria was isolated by homogenization and differential centrifugation in a buffer containing 200 mmol/l mannitol, 50 mmol/l sucrose, 10 mmol/l
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KCl, 1 mmol/l EDTA, 10 mmol/l Hepes-KOH (pH 7.4). Liver was homogenized in 5 · volume of mannitol-sucrose buffer and centrifuged at 1000g for 5 m. The supernatant was then centrifuged at 8000g for 10 m. Then the resulting mitochondrial pellets were washed 3 times in the same buffer (Graham, 1993). The mitochondrial pellets suspended in Tris-HCl buffer (0.1 M, pH 7.4) were used for estimation of enzyme activities and protein (Lowry et al., 1951). 2.6. Biochemical assays The activities of TCA cycle enzymes, viz isocitrate dehydrogenase (Bell and Baron, 1960), a-ketoglutarate dehydrogenase (Reed and Mukherjee, 1969), succinate dehydrogenase (Slater and Bonner, 1952), malate dehydrogenase (Meheler et al., 1948), NADH dehydrogenase (Minakami et al., 1962) and cytochrome-c-oxidase (Pearl et al., 1963) were assayed. The levels of glutathione (Moron et al., 1979), superoxide dismutase (Misra and Fridovich, 1972) catalase (Beers and Sizer, 1952) and lipid peroxides (Ohkawa et al., 1979) were determined.
2.7. Statistical analysis Results are expressed as mean ± SD, and studentÕs t-test was used to assess statistical significance.
3. Results Table 1 shows the TCA cycle enzyme activities in experimental animals and Table 2 depicts the antioxidant profile of control and experimental animals. Fig. 2 shows the appearance of deep blue color indicating the presence of seaweed polysaccharide. Fig. 1 shows the DPPH scavenging activity of seaweed alcoholic extract. The minimum concentration of the extract that gave maximum scavenging activity was chosen for the animal experimental study. The TCA cycle enzyme activities and antiperoxidative enzymes such as SOD, CAT and non-enzymic antioxidant GSH were significantly decreased with increased levels of LPO in acetaminophen induced group II rats when compared with group I normal control rats. The S. polycystum extract pre-treated group IV rats prior
Table 1 Effect of S. polycystum extract on liver mitochondrial protein and tricarboxylic acid cycle enzymes in control and experimental animals Parameters
Group I Control
Group II Acetaminophen intoxicated
Group III S. polycystum extract
Group IV S. polycystum prior to acetaminophen induction
Protein Isocitrate dehydrogenase a-Ketoglutarate dehydrogenase Succinate dehydrogenase Malate dehydrogenase NADH dehydrogenase Cytochrome-c-oxidase
33.42 ± 3.17 810.73 ± 70.4 196.00 ± 18.3 36.30 ± 3.1 349.71 ± 25.64 31.07 ± 1.94 6.35 ± 0.58
22.17 ± 1.72*** 657.20 ± 65.2*** 125.00 ± 12.7*** 16.11 ± 2.1*** 247.15 ± 21.74*** 24.80 ± 2.1*** 4.32 ± 0.36***
34.41 ± 3.19 811.61 ± 68.9 185.00 ± 18.1 32.1 ± 2.6 348.31 ± 29.08 30.40 ± 2.8 6.37 ± 0.51
30.1 ± 3.06*** 798.21 ± 71.3** 192.00 ± 18.9*** 35.60 ± 3.3*** 307.53 ± 25.14** 31.70 ± 3.6** 6.31 ± 0.62***
Values are mean ± SD (n = 6). **P < 0.01, ***P < 0.001; Group II vs. Group I, Group IV vs. Group II. Unit: Protein expressed as microgram/g; nmol of a-ketoglutarate formed/h/mg for isocitrate dehydrogenase; nmol of ferrocynaide formed/h/mg protein for aketoglutarate dehydrogenase; nmol of succinate oxidized/min/mg protein for Succinate dehydrogenase; nmol of NADH oxidized/min/ mg protein for malate dehydrogenase and NADH dehydrogenase and change in OD/min/mg protein for cytochrome-c-oxidase.
Table 2 Effect of S. polycystum extract on lipid peroxides (LPO), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), in control experimental group of rats Parameters
Group I Control
Group II Acetaminophen induced
Group III S. polycystum extract
Group IV S. polycystum prior to acetaminophen induction
LPO GSH SOD CAT
168 ± 15.43 3.20 ± 0.28 42.14 ± 3.28 128.71 ± 11.31
285.78 ± 25.16*** 2.21 ± 0.17*** 28.8 ± 2.11*** 68.81 ± 5.99***
164.33 ± 13.19 3.29 ± 0.31 43.60 ± 3.74 125.67 ± 10.97
188.94 ± 17.18*** 3.16 ± 0.29*** 38.6 ± 1.91*** 101.46 ± 7.94***
Values are mean ± SD, n = 6; **P < 0.01, ***P < 0.001; Group II vs. Group I, Group IV vs. Group II; StudentÕs t-test. Unit: nmol of malondialdehyde formed/mg protein for LPO and nmol/mg protein for GSH. One unit of SOD activity is the amount of enzymes required to give 50% inhibition of epinephrine auto-oxidation. nmol H2O2 decomposed/min/mg protein.
DPPH Radical scavenging activity (%)
H.R. Balaji Raghavendran et al. / Chemosphere 61 (2005) 276–281 80 60 40 20 0 50
100
150
200
300
500
750
Concentration of seaweed extract (mg/ml)
Fig. 1. Effects of S. polycystum extract on DPPH.
Fig. 2. Arrow shows the appearance of deep blue color indicating the presence of sulphated polysaccharide.
to acetaminophen intoxication showed considerable reduction in impairment of TCA cycle enzyme activities and improved antiperoxidative enzymes with reduced level of lipid peroxide when compared to group II intoxicated rats. The group III rats did not show any severe alterations in the activities of liver mitochondrial enzymes when compared with that of group I control rats.
4. Discussion The mitochondria are of particular importance to cell viability due to the nature of their functions (Carafoli, 1987). The lipid peroxidation is recognized as an important deteriorative reaction in biological membrane. The depletion in the levels of mitochondrial GSH seems to be a major mechanism in inducing imbalance of mitochondrial function (Anderson et al., 1990). The depletion of GSH in acetaminophen intoxicated animals sensitizes mitochondria to oxidative modifications by drugs or xenobiotics (Fernandez-Checa et al., 1991). The S. polycystum pre-treated rats showed an improved level of mitochondrial GSH, and prevented the excessive depletion of SOD and CAT with concomitant reduction in the levels of lipid peroxides when compared with acetaminophen induced animals. This indicates that the seaweed extract may able to reduce the acetaminophen induced hepatic toxicity.
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Succinate dehydrogenase is an important enzyme of TCA cycle and is also associated with the electron transport chain due to its ability to transfer electrons to respiratory chain (Singh et al., 1990). Succinate dehydrogenase is known to contain a number of cysteine rich sulfur clusters and can be inhibited by a number of agents that modify sulfhydryl groups. The reactive metabolite NAPQI directly interacts with sulfhydryl groups on succinate dehydrogenase, causing the loss of its activity (Burcham and Harman, 1991). The pretreatment with S. polycystum extract prevented the severe impairment in the activities of tricarboxylic acid cycle enzymes probably by promoting the elimination of NAPQI and improving the mitochondrial antioxidant defense system thereby protecting the critical nucleophilic sites on the enzymes against toxic electrophilic metabolite NAPQI. The respiratory enzyme NADH dehydrogenase is located in the mitochondrial membrane (Nicolay et al., 1985). NAPQI, the toxic metabolite of acetaminophen arylates and oxidizes essential protein sulfhydryls in the mitochondrial respiratory chain thereby limiting the ability of the mitochondria to meet the energy demand of the cell and affecting cellular energy homeostasis (Streeter et al., 1984). The rats pretreated with S. polycystum extract showed substantial prevention in the excessive impairment of NADH dehydrogenase activity further suggests that the extract may able to restore energy status of the mitochondria, thereby maintaining membrane integrity. Acetaminophen causes lipid peroxidation in membrane, which initiates the formation of free radicals (Jaeschke, 1990). Biological membranes and subcellular organelles rich in polyunsaturated fatty acids are the major sites for the free radical mediated damage. The increased lipid peroxidation alters the lipid environment of the membrane thus affecting the activity of some enzymes like cytochrome-c-oxidase (Padama and Setty, 1999). The cytochrome-c-oxidase, the terminal enzyme of the respiratory chain requires phospholipid for its optimal activity. Any change in the lipid composition of the mitochondrial membrane may decrease the activity of cytochrome-c-oxidase as in acetaminophenintoxicated rats (Aria et al., 1984). The pretreatment with S. polycystum extract may able to reduce the excessive generation of free radicals thereby prevented the severe impairment in the cytochrome-c-oxidase activity. Reports show that S. polycystum contains fucoidan, alginates and triterpenes, which are found to have broad spectrum of biological properties (Jothi Saraswathi et al., 2003; Chotigeat et al., 2004). Although the sulphated polysaccharide are found to have antioxidant properties, the relationship between the structure of sulphated polysaccharide and free radical quenching mechanism have not been elucidated. According to Xue et al. (1998) the antioxidant abilities of alginates
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might be related to sulfate content and their molecular weight. Moreover anionic groups in alginate would display a significant antioxidant effect. The presence of the sulphated compound was confirmed by seaweed histochemical analysis. Thus the results obtained in the present study indicate that the S. polycystum alcoholic extract may able to improve the hepatic mitochondrial antioxidant defense system against free radicals generated, which might be attributed to the sulphated compounds present in the extract. The extract also showed considerable prevention in the severe impairment of TCA enzyme activities thereby restoring the mitochondrial functional status against acetaminophen induced toxic hepatitis. Thus the study suggests that selective prevention of mitochondrial oxidative damage and maintenance of citric acid cycle enzyme activities by natural antioxidants may be an effective approach for a wide range of human diseases. The characterization of S. polycystum extracts and its mode of action on microsomal system is underway.
Acknowledgments We thank Dr. N. Kaliaperumal (CMFRI, Rameswaram India) and Prof. V. Krishnamurthy (Krishnamurthy Institute of Algology, Chennai, India) for their help during the period of seaweed collection and species identification. We are grateful to CSIR India for providing financial support for the work. References Albano, E., Rundgren, M., Harvison, P.J., Nelson, S.D., Moldeus, P., 1985. Mechanism of N-acetyl-p-benzoquninone imine cytotoxicity. Mol. Pharmacol. 28, 306–311. Anderson, B.S., Rundgren, M., Nelson, S.D., Harder, S., 1990. N-acetyl-p-benzoquinone imine-induced changes in the energy metabolism in hepatocytes. Chem. Biol. Interact. 75, 201–211. Aria, M., Gord, E.R., Leiber, C.S., 1984. Increased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids. Biochim. Biophys. Acta 797, 320–327. Balaji Raghavendran, H., Sathivel, A., Devaki, T., 2004. Hepatoprotective nature of seaweed alcoholic extract in acetaminophen induced hepatic oxidative stress. J. Health Sci. 50 (1), 42. Beers, R.F., Sizer, I.W., 1952. A spectroscopic method for measuring the break down of the hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Bell, J.L., Baron, D.N., 1960. A colorimetric method for determination of isocitrate dehydrogenase. Clin. Chem. Acta 5, 740. Black, M., 1984. Acetaminophen hepatotoxicity. Annu. Rev. Med. 35, 577–593.
Brad-Williams, W., 1995. Use of a free radical method to evaluate antioxidant activity. Food. Sci. Technol. (London) 28, 25–30. Burcham, P.C., Harman, A.W., 1991. Acetaminophen toxicity results in site-specific mitochondrial damage in isolated hepatocytes. J. Biol. Chem. 226, 5049–5054. Carafoli, E., 1987. Intracellular calcium homeostasis. Annu. Rev. Biochem. 56, 395–433. Chotigeat, W., Tongsupa, S., Supamataya, K., Phongdara, A., 2004. Effect of fucoidan on disease resistance of black tiger shrimp. Aquaculture 233, 23–30. Faulkner, D.J., 1993. Marine natural products chemistry: introduction. Chem. Rev. 93, 1671–1673. Fernandez-Checa, J.C., Garcia-Ruiz, C., Ookhetns, M., Kapowitz, N., 1991. Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress. J. Clin. Invest. 87, 397–405. Graham, J.M., 1993. Isolation of mitochondria, mitochondrial membrane, lysosomes, peroxisomes and golgi membrane from rat liver. Meth. Mol. Biol. 19, 29–40. Harbone, J.B., 1973. Phytochemical Methods. Chapman and Hall, London. Jaeschke, H.R., 1990. Glutathione disulfide formation and oxidant stress during acetaminophen induced hepatotoxicity in mice in vivo. The protective effect of allopurinol. J. Pharmacol. Exp. Ther. 255, 935–941. Jothi Saraswathi, S., Babu, B., Rengaswamy, R., 2003. Seasonal studies on the alginate and its biochemical composition. I: Sargassum polycystum (Fucales), Phaeophyceae. Phycolological Res. 51 (4), 240. Krishnamurthy, K.V., 1988. Methods in Plant Histochemistry. S. Viswanathan Pvt. Ltd., Madras, pp. 14–15. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265–275. Martensson, J., Meister, A., 1989. Mitochondrial damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoester. Proc. Natl. Acad. Sci., USA 86, 471–475. Matsukawa, R., 1997. A comparison of screening methods for antioxidant activity in seaweeds. J. Appl. Phycol. 9 (1), 29– 35. Meheler, A.H., Kornberg, A., Crisolia, S., Ochoa, S., 1948. The enzymatic mechanism of oxidation reductions between malate, isocitrate and pyruvate. J. Biol. Chem. 174, 961– 977. Minakami, S., Ringler, R.L., Singer, T.P., 1962. Studies on respiratory chain linked dihydrodiphosphopyridine nucleotide dehydrogenase I. Assay of the enzyme in particulate and in soluble preparation. J. Biol. Chem. 237, 569– 576. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the auto-oxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione-s-transferase activities in rat lung and Liver. Biochim. Biophys. Acta 5820, 62–68. Nicolay, K., Van der Neut, R., Fok, J.J., De Kruijff, B., 1985. Effects of adriamycin on lipid polymorphism in
H.R. Balaji Raghavendran et al. / Chemosphere 61 (2005) 276–281 cardiolipin-containing model and mitochondrial membranes. Biochim. Biophys. Acta 819, 55–65. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay of lipidperoxides in animal tissues by thiobarbituric acid reaction. Ann. Biochem. 95, 351–358. Padama, P., Setty, O.H., 1999. Studies on cytochrome oxidase in carbon tetrachloride treated rats. Ind. J. Exp. Res. 37, 1139–1141. Pearl, W.C., Cancercao, J., Zweifach, B.W., 1963. Microdetermination of cytochrome oxidase in rat tissues by the oxidation of N-phenyl-p-phenylene diamine of ascorbic acid. J. Histochem. Cytochem. 11, 102. Reed, L.J., Mukherjee, R.B., 1969. Keto glutarate dehydrogenase complex from Escherichia coli. Meth. Enzymol. 13, 53– 61. Singh, U.K., Agarwal, K.N., Shankar, R., 1990. Effect of under nutrition on succinate dehydrogenase and acetylcholine esterase in developing rat brain. Ind. J. Exp. Biol. 28, 868– 870.
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Slater, E.C., Bonner, W.D., 1952. The effect of fluoride on succinic oxidase system. Biochem. J. 52, 185. Streeter, A.J., Dahlin, D.C., Nelson, S.D., Baillie, T.A., 1984. The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro. Chem. Biol. Interact. 48, 349–366. Vendemiale, G., Guerrieri, F., Grattangliano, I., Didonna, D., Mwla, L., Altomare, E., 1995. Mitochondrial oxidative stress phosphorylation and intracellular glutathione compartmentation during rat liver regeneration. Hepatology 21, 1450–1454. Xue, C., Yu, G., Hirata, T., Terao, J., Lin, H., 1998. Antioxidative activities of several marine polysaccharides evaluated in a phosphatidylcholine–liposomal suspension and organic solvents. Biosci. Biotechnol. Biochem. 62, 206– 209. Yan, X., Nagata, T., Fan, X., 1998. Antioxidant activities in some common seaweeds. Plant Foods Hum. Nutr. 52, 253– 262.
Antioxidant effect of Sargassum polycystum (Phaeophyceae) against acetaminophen induced changes in hepatic mitochondrial enzymes during toxic hepatitis Hanumantha Rao Balaji Raghavendran, Arumugam Sathivel, Thiruvengadum Devaki * Department of Biochemistry, University of Madras, Guindy Campus, Patel Road, Chennai 600 025, India Received 14 June 2004; received in revised form 13 January 2005; accepted 18 January 2005 Available online 25 April 2005
Abstract The present study was aimed to examine the protective effects of Sargassum polycystum (Phaeophyceae) alcoholic extract on changes in liver mitochondrial enzymes against acetaminophen induced toxic hepatitis in experimental rats. The levels of protein, lipid peroxide, glutathione (GSH) in mitochondrial fraction, superoxide dismutase (SOD) and catalase (CAT) were also determined. The activities of tricarboxylic acid enzymes such as isocitrate dehydrogenase (ICD), a-ketoglutarate dehydrogenase (a-KGD), succinate dehydrogenase (SD), malate dehydrogenase (MD), NADH dehydrogenase, and cytochrome-c-oxidase were determined in mitochondrial fraction. The rats intoxicated with acetaminophen showed significant elevation in the levels of lipid peroxides with decreased levels of protein, GSH, SOD, CAT and impaired tricarboxylic acid cycle enzyme activities. The prior oral administration of S. polycystum alcoholic extract showed significant diminution in the severity of toxic hepatitis in acetaminophen-induced rats by maintaining the activities of tricarboxylic acid enzymes with concomitant improvement in the hepatic mitochondrial antiperoxidative status when compared with intoxicated animals. The results obtained in the present study indicate that the protective effects of S. polycystum extract may be due to the presence of some active compounds that are inhibitory against the free radicals generated during lipid peroxidation in acetaminophen induced toxic hepatitis. 2005 Published by Elsevier Ltd. Keywords: Sargassum polycystum; Acetaminophen; Antiperoxidative; Toxic hepatitis; Lipid peroxidation
1. Introduction Acetaminophen (AP) is one of the most commonly used analgesics/antipyretics worldwide. The toxic doses
*
Corresponding author. Tel.: +91 2235 1269 (O), 2441 2292 (R); fax: +91 244235 2494. E-mail addresses: searag13@rediffmail.com (H.R. Balaji Raghavendran), [email protected] (T. Devaki). 0045-6535/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.chemosphere.2005.01.049
of AP can cause hepatocellular necrosis (Albano et al., 1985). In particular, the mechanism of cell damage is mediated by the metabolic activation of AP to a highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is able to deplete hepatocellular glutathione (GSH) and to bind covalently with cell macromolecules (Black, 1984). The concentration of intracellular GSH is therefore a vital determinant in the extent of AP induced hepatic necrosis. The GSH depletion has been suggested to markedly enhance the
H.R. Balaji Raghavendran et al. / Chemosphere 61 (2005) 276–281
susceptibility of mitochondrial structural dysfunction from oxidative stress and to induce mitochondrial structural degeneration (Martensson and Meister, 1989). The levels of mitochondrial GSH and alterations in mitochondrial energy metabolism, which are most likely due to the free radical accumulation, observed during liver regeneration (Vendemiale et al., 1995). The food values of marine algae are currently being reconsidered in the hope of coping with future food shortages. Recently the marine algae have been shown to produce a variety of compounds and some of them are found to have key biological activity of potential medicinal value (Faulkner, 1993). Several studies (in vitro) have investigated to show the antioxidant activity of Sargassum spp. (Matsukawa, 1997; Yan et al., 1998). Initially, Sargassum polycystum alcoholic extract was screened for its protective nature against acetaminophen induced hepatotoxicity in experimental rats (Balaji Raghavendran et al., 2004) The present study was designed to examine the protective effect of S. polycystum alcoholic extract on changes in liver mitochondrial enzymes against acetaminophen induced toxic hepatitis in experimental rats.
2. Materials and methods 2.1. Seaweed material Seaweed S. polycystum was collected from low tide area of mandapam (Gulf of Mannar) coasts. The species authentication was done by Prof. N. Kaliaperumal (CMFRI, Rameswaram, India). Alga was thoroughly washed in running tap water and deionized water to remove epiphytes and other contamination. 2.2. Seaweed extraction The contamination free seaweed sample was air-dried under shade and then coarsely powdered. The powdered seaweed material was extracted with ethanol in cold for a period of 72 h. The crude extract was filtered, concentrated on a water bath, and then dried in vaccum. The extract was subjected to TLC and phytochemical analysis, which showed positive result for the presence of terpenoids and flavonoids (Harbone, 1973). 2.3. 1,1-Diphenyl-2-picrylhydrazyl assay (in vitro) The seaweed extract was subjected to 1, 1-diphenyl2-picrylhydrazyl (DPPH) assay to determine the free radical scavenging property of seaweed extract. DPPH solution was prepared at the concentration of 3 · 105 mol/l; the solvent was dimethylsulfoxide (DMSO). During the test, an organic solvent (alcohol) extract
277
(50, 100, 200, 300, 500 and 750 mg) of 2 ml was mixed with 2 ml of DPPH solution. The test tube was capped and kept for 60 min; absorbance was read at 517 nm by UV-visible spectrophotometer. DMSO was used as the blank. Another measure was done after 30 min. Meanwhile, the absorbance of the organic solvent adding 2 ml DMSO was also determined. Every sample was done in triplet and mean was obtained to calculate the free radical scavenging activity (Brad-Williams, 1995). ½1 ðAi Aj Þ=Ac 100 For DPPH in the above equation, Ai = the absorbance of organic solvent extract mixed with DPPH solution; Aj = the absorbance of the same organic extract mixed with 2 ml DMSO; Ac = absorbance of DPPH solution upon adding 2 ml of DMSO. 2.4. Histochemical analysis of S. polycystum The sectional and macerated seaweed material was dehydrated by passing through ascending and descending alcoholic (100–50%) series and kept in water. The section was then stained with diluted 5% aluminum sulphate solution of toluidine blue (TB) pH at 4.4. The sectional seaweed showed deep purple color indicating the presence of sulphated polysaccharide. Alcian blue (5% 8GX of 100 ml acid water at pH 5.0) a specific stain for sulphated polysaccharide was also used for the further confirmation (Krishnamurthy, 1988). 2.5. Experimental animals Male Wistar strain albino rats, weighing about 120– 150 g was obtained from the Fredrick institute for plant protection and toxicology, Padappai, Chennai, India. The animals were maintained in a 12-h light and dark, at 22 ± 3 C cycle and fed with a commercial pelleted diet (M/s. Hindustan Foods Ltd., Bangalore, India) and had free access of water. The rats were randomized into four groups, each group comprising six animals. Group I rats were served as normal control, group II rats were given acetaminophen (Sigma, chemical company, St. Louis, MO, USA: 800 mg/kg b.wt. intraperitoneally), group III rats were pre-treated with seaweed extract alone (200 mg/kg b.wt. daily for a period of 15 days, orally) group IV rats were pre-treated orally with seaweed extract (200 mg/kg b.wt. daily for a period of 15 days, orally) prior to acetaminophen induction (800 mg/kg b.wt. intraperito-neally). At the end of the experiment, the rats were anesthetized with sodium pentobarbitone (35 mg/kg b.wt. intraperitoneally) and sacrificed by cervical decapitation. The liver was excised immediately and washed in ice-cold saline. Liver mitochondria was isolated by homogenization and differential centrifugation in a buffer containing 200 mmol/l mannitol, 50 mmol/l sucrose, 10 mmol/l
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KCl, 1 mmol/l EDTA, 10 mmol/l Hepes-KOH (pH 7.4). Liver was homogenized in 5 · volume of mannitol-sucrose buffer and centrifuged at 1000g for 5 m. The supernatant was then centrifuged at 8000g for 10 m. Then the resulting mitochondrial pellets were washed 3 times in the same buffer (Graham, 1993). The mitochondrial pellets suspended in Tris-HCl buffer (0.1 M, pH 7.4) were used for estimation of enzyme activities and protein (Lowry et al., 1951). 2.6. Biochemical assays The activities of TCA cycle enzymes, viz isocitrate dehydrogenase (Bell and Baron, 1960), a-ketoglutarate dehydrogenase (Reed and Mukherjee, 1969), succinate dehydrogenase (Slater and Bonner, 1952), malate dehydrogenase (Meheler et al., 1948), NADH dehydrogenase (Minakami et al., 1962) and cytochrome-c-oxidase (Pearl et al., 1963) were assayed. The levels of glutathione (Moron et al., 1979), superoxide dismutase (Misra and Fridovich, 1972) catalase (Beers and Sizer, 1952) and lipid peroxides (Ohkawa et al., 1979) were determined.
2.7. Statistical analysis Results are expressed as mean ± SD, and studentÕs t-test was used to assess statistical significance.
3. Results Table 1 shows the TCA cycle enzyme activities in experimental animals and Table 2 depicts the antioxidant profile of control and experimental animals. Fig. 2 shows the appearance of deep blue color indicating the presence of seaweed polysaccharide. Fig. 1 shows the DPPH scavenging activity of seaweed alcoholic extract. The minimum concentration of the extract that gave maximum scavenging activity was chosen for the animal experimental study. The TCA cycle enzyme activities and antiperoxidative enzymes such as SOD, CAT and non-enzymic antioxidant GSH were significantly decreased with increased levels of LPO in acetaminophen induced group II rats when compared with group I normal control rats. The S. polycystum extract pre-treated group IV rats prior
Table 1 Effect of S. polycystum extract on liver mitochondrial protein and tricarboxylic acid cycle enzymes in control and experimental animals Parameters
Group I Control
Group II Acetaminophen intoxicated
Group III S. polycystum extract
Group IV S. polycystum prior to acetaminophen induction
Protein Isocitrate dehydrogenase a-Ketoglutarate dehydrogenase Succinate dehydrogenase Malate dehydrogenase NADH dehydrogenase Cytochrome-c-oxidase
33.42 ± 3.17 810.73 ± 70.4 196.00 ± 18.3 36.30 ± 3.1 349.71 ± 25.64 31.07 ± 1.94 6.35 ± 0.58
22.17 ± 1.72*** 657.20 ± 65.2*** 125.00 ± 12.7*** 16.11 ± 2.1*** 247.15 ± 21.74*** 24.80 ± 2.1*** 4.32 ± 0.36***
34.41 ± 3.19 811.61 ± 68.9 185.00 ± 18.1 32.1 ± 2.6 348.31 ± 29.08 30.40 ± 2.8 6.37 ± 0.51
30.1 ± 3.06*** 798.21 ± 71.3** 192.00 ± 18.9*** 35.60 ± 3.3*** 307.53 ± 25.14** 31.70 ± 3.6** 6.31 ± 0.62***
Values are mean ± SD (n = 6). **P < 0.01, ***P < 0.001; Group II vs. Group I, Group IV vs. Group II. Unit: Protein expressed as microgram/g; nmol of a-ketoglutarate formed/h/mg for isocitrate dehydrogenase; nmol of ferrocynaide formed/h/mg protein for aketoglutarate dehydrogenase; nmol of succinate oxidized/min/mg protein for Succinate dehydrogenase; nmol of NADH oxidized/min/ mg protein for malate dehydrogenase and NADH dehydrogenase and change in OD/min/mg protein for cytochrome-c-oxidase.
Table 2 Effect of S. polycystum extract on lipid peroxides (LPO), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), in control experimental group of rats Parameters
Group I Control
Group II Acetaminophen induced
Group III S. polycystum extract
Group IV S. polycystum prior to acetaminophen induction
LPO GSH SOD CAT
168 ± 15.43 3.20 ± 0.28 42.14 ± 3.28 128.71 ± 11.31
285.78 ± 25.16*** 2.21 ± 0.17*** 28.8 ± 2.11*** 68.81 ± 5.99***
164.33 ± 13.19 3.29 ± 0.31 43.60 ± 3.74 125.67 ± 10.97
188.94 ± 17.18*** 3.16 ± 0.29*** 38.6 ± 1.91*** 101.46 ± 7.94***
Values are mean ± SD, n = 6; **P < 0.01, ***P < 0.001; Group II vs. Group I, Group IV vs. Group II; StudentÕs t-test. Unit: nmol of malondialdehyde formed/mg protein for LPO and nmol/mg protein for GSH. One unit of SOD activity is the amount of enzymes required to give 50% inhibition of epinephrine auto-oxidation. nmol H2O2 decomposed/min/mg protein.
DPPH Radical scavenging activity (%)
H.R. Balaji Raghavendran et al. / Chemosphere 61 (2005) 276–281 80 60 40 20 0 50
100
150
200
300
500
750
Concentration of seaweed extract (mg/ml)
Fig. 1. Effects of S. polycystum extract on DPPH.
Fig. 2. Arrow shows the appearance of deep blue color indicating the presence of sulphated polysaccharide.
to acetaminophen intoxication showed considerable reduction in impairment of TCA cycle enzyme activities and improved antiperoxidative enzymes with reduced level of lipid peroxide when compared to group II intoxicated rats. The group III rats did not show any severe alterations in the activities of liver mitochondrial enzymes when compared with that of group I control rats.
4. Discussion The mitochondria are of particular importance to cell viability due to the nature of their functions (Carafoli, 1987). The lipid peroxidation is recognized as an important deteriorative reaction in biological membrane. The depletion in the levels of mitochondrial GSH seems to be a major mechanism in inducing imbalance of mitochondrial function (Anderson et al., 1990). The depletion of GSH in acetaminophen intoxicated animals sensitizes mitochondria to oxidative modifications by drugs or xenobiotics (Fernandez-Checa et al., 1991). The S. polycystum pre-treated rats showed an improved level of mitochondrial GSH, and prevented the excessive depletion of SOD and CAT with concomitant reduction in the levels of lipid peroxides when compared with acetaminophen induced animals. This indicates that the seaweed extract may able to reduce the acetaminophen induced hepatic toxicity.
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Succinate dehydrogenase is an important enzyme of TCA cycle and is also associated with the electron transport chain due to its ability to transfer electrons to respiratory chain (Singh et al., 1990). Succinate dehydrogenase is known to contain a number of cysteine rich sulfur clusters and can be inhibited by a number of agents that modify sulfhydryl groups. The reactive metabolite NAPQI directly interacts with sulfhydryl groups on succinate dehydrogenase, causing the loss of its activity (Burcham and Harman, 1991). The pretreatment with S. polycystum extract prevented the severe impairment in the activities of tricarboxylic acid cycle enzymes probably by promoting the elimination of NAPQI and improving the mitochondrial antioxidant defense system thereby protecting the critical nucleophilic sites on the enzymes against toxic electrophilic metabolite NAPQI. The respiratory enzyme NADH dehydrogenase is located in the mitochondrial membrane (Nicolay et al., 1985). NAPQI, the toxic metabolite of acetaminophen arylates and oxidizes essential protein sulfhydryls in the mitochondrial respiratory chain thereby limiting the ability of the mitochondria to meet the energy demand of the cell and affecting cellular energy homeostasis (Streeter et al., 1984). The rats pretreated with S. polycystum extract showed substantial prevention in the excessive impairment of NADH dehydrogenase activity further suggests that the extract may able to restore energy status of the mitochondria, thereby maintaining membrane integrity. Acetaminophen causes lipid peroxidation in membrane, which initiates the formation of free radicals (Jaeschke, 1990). Biological membranes and subcellular organelles rich in polyunsaturated fatty acids are the major sites for the free radical mediated damage. The increased lipid peroxidation alters the lipid environment of the membrane thus affecting the activity of some enzymes like cytochrome-c-oxidase (Padama and Setty, 1999). The cytochrome-c-oxidase, the terminal enzyme of the respiratory chain requires phospholipid for its optimal activity. Any change in the lipid composition of the mitochondrial membrane may decrease the activity of cytochrome-c-oxidase as in acetaminophenintoxicated rats (Aria et al., 1984). The pretreatment with S. polycystum extract may able to reduce the excessive generation of free radicals thereby prevented the severe impairment in the cytochrome-c-oxidase activity. Reports show that S. polycystum contains fucoidan, alginates and triterpenes, which are found to have broad spectrum of biological properties (Jothi Saraswathi et al., 2003; Chotigeat et al., 2004). Although the sulphated polysaccharide are found to have antioxidant properties, the relationship between the structure of sulphated polysaccharide and free radical quenching mechanism have not been elucidated. According to Xue et al. (1998) the antioxidant abilities of alginates
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might be related to sulfate content and their molecular weight. Moreover anionic groups in alginate would display a significant antioxidant effect. The presence of the sulphated compound was confirmed by seaweed histochemical analysis. Thus the results obtained in the present study indicate that the S. polycystum alcoholic extract may able to improve the hepatic mitochondrial antioxidant defense system against free radicals generated, which might be attributed to the sulphated compounds present in the extract. The extract also showed considerable prevention in the severe impairment of TCA enzyme activities thereby restoring the mitochondrial functional status against acetaminophen induced toxic hepatitis. Thus the study suggests that selective prevention of mitochondrial oxidative damage and maintenance of citric acid cycle enzyme activities by natural antioxidants may be an effective approach for a wide range of human diseases. The characterization of S. polycystum extracts and its mode of action on microsomal system is underway.
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