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Effect of L-cysteine on lipid peroxidation in experimental urolithiatic rats
Pharmacological Research, Vol. 32, No. 3, 1995
165
EFFECT OF L-CYSTEINE ON LIPID PEROXIDATION IN EXPERIMENTAL UROLITHIATIC RATS N. SARAVANAN, D. SENTHIL and P. VARALAKSHMI Department of Medical Biochemistry, Dr A. L. Mudaliar Post Graduate Institute of Basic Medical Sciences, Universi O, of Madras, Taramani Campus, Madras 600 113, India Accepted 5 September 1995 Oxalate, the major stone-forming constituent induces lipid peroxidation during lithogenesis. In experimental condition oxalate formation was induced by the administration of its precursor glycollate. Glycollate-fed rats showed increased susceptibility to lipid peroxidation in the presence of promoters. In addition, antioxidant enzymes--catalase, superoxide dismutase and glutathione peroxidase also showed decreased activity. Reduced glutathione, total thiols and ascorbic acid were also significantly decreased. On the other hand, an increased xanthine oxidase and decreased glucose-6-phosphate dehydrogenase activity was also observed upon glycollate administration. Cysteine, a sulphydryl compound, is known to inhibit free radical toxicity in various pathologies. Cysteine administration to glycollate-fed rats brought about a significant decrease in the peroxidative level, with an increase in the antioxidant status. © 1995 The Italian Pharmacological Society KEYWORDS:glycollate, cysteine, liver, kidney, lipid peroxidation.
INTRODUCTION Lipid peroxidation of unsaturated fatty acids has been shown to be involved in the pathogenesis of cellular damage [1]. Oxalate, the major stone-forming constituent, is known to induce lipid peroxidation which causes disruption of the structural integrity of the membranes [2]. This alters the membrane permeability and thereby impairs the transport of ions and electrons across the cellular organelles. Formation of peroxides in in vivo conditions may produce serious consequences in the tissues [3]. Oxygen and/or hydroxyl radical reaction mechanism can injure renal tubular cells and promote calcium oxalate crystallization within the subcellular fractions of the kidney [4]. Selvam and Sridevi [5] have also reported the effect of lipid peroxidation in rat kidney mitochondria, which may be an important factor for the aetiology of stone formation. The products of lipid peroxidation in cells, is controlled by various cellular defence mechanisms consisting of enzymatic and non-enzymatic scavenger systems [6]. The oxidative damage may be attributed to the destruction of thiol groups of the amino acids and proteins [3], since thiol compounds are well known for their free radical scavenging property [7]. Dietary supplementation of sulphur-containing amino acids
C o r r e s p o n d e n c e to: D r ( M r s ) P. V a r a l a k s h m i .
1043-6618/95/090165-05/$12.00/0
have been reported to improve the tissue antioxidant status in rats [8]. L-cysteine, a sulphur-containing amino acid is known to offer protection to the living system against certain toxicants through its ability to increase the thiol status of the tissues. Cysteine has been reported to exhibit inhibitory effect on endrin induced hepatic and renal lipid peroxidation in rats [9]. Cysteine has also been reported to inhibit oxalate synthesis, thus leading to decreased oxalate excretion [10]. This unique antioxidant property of cysteine motivated us to investigate whether this amino acid could possibly prevent the oxalate-induced peroxidation of lipids in experimental hyperoxaluria.
MATERIALS AND METHODS Male albino Wistar rats weighing between 150 and 180 g were divided into four groups of six rats. Group 1 used as control, were maintained on the commercial feed [1 I]. Group 2 animals were given the commercial diet mixed with 3% w/w sodium glyeollate for 30 days according to the method of Chow et al. [12]. Group 3 animals received L-cysteine intraperitoneatl3/ at a dose of 500 mg (kg body weight) -l day"~ ft~r 30 days. Group 4 rats received glycollate :supplemented diet and L-cysteine intraperitoneallyfor 30-days: • : ?: . ,!-: Water was given ad libitum. At the end of the experimelatal peri0d,.the; a~aimals were killed by decapitation. Liver and ~.'dnvys,~were dissected out and washed in cold 0.15 M KCI:rT,hefi~the © 1995The ItalianPharmacologicalSociety
166
Pharmacological Research, Vol. 32, No. 3, 1995
tissues were homogenized in Tris-HC1 buffer (0.01 M, pH 7.4) using a Potter-Elvehjem homogenizer to give a 10% homogenate. Lipid peroxidation was determined by the method of Devasagayam [13]. The NADPH-induced system in a total volume of 2 ml contained 0.2 ml of tissue homogenate, 50/.tM FeCI3, 5 mr~ ADP, 1 mM KH2POa and 0.4mM NADPH in 0.15M Tris-HC1 buffer, pH 7.4. The ascorbate-induced system contained 0.2 ml of tissue homogenate, 50/IM FeSO4, I mM KH2PO4, 0.4 mM ascorbic acid in 0.15 M Tris-HCl buffer, pH 7.4. Antioxidant enzyme catalase was assayed by the method of Sinha [14]. Superoxide dismutase (SOD) was estimated according to the method of Marklund and Marklund [ 15]. Glutathione peroxidase (GPx) was assayed by the method of Rotruck et al. [ 16]. Antioxidants such as total reduced glutathione [17], ascorbic acid [18] and total thiol groups [19] were also determined. Xanthine oxidase activity was determined by
the method of Fried and Fried [20] using xanthine as substrate and glucose-6-phosphate dehydrogenase activity was assayed by the method of Balinsky and Bernstein [21], using glucose-6-phosphate as substrate. Tissue protein content was estimated by the method of Lowry et al. [22]. The data were analysed using one way analysis of variance followed by Student's Newman-Keuls test. The values are expressed as mean+sD.
RESULTS Table I presents the enzymatic, non-enzymatic and ferrous sulphate induced lipid peroxidation of liver and kidney homogenates of control and experimental rats. Basal, NADPH (enzymatic), ascorbate (nonenzymatic) and ferrous sulphate induced lipid peroxidation were significantly high in glycollate fed rats (P
Table I Effect of glycollate and cysteine on lipid peroxidation in liver and kidneys of control and experimental rats. (Values are m e a n ± s o for six animals in each group) Particulars
Group I Rat chow for 30 days
Liver Basal NADPH system Ascorbate system 10 mM ferrous sulphate Kidney Basal NADPH system Ascorbate system 10 rnM ferrous sulphate
Group 2 Glycollate for 30 days
Group 3 Rat chow+cysteine for 30 days
Group 4 Glycollate+cysteine for 30 days
1.70+0.20 3.53+0.45 9.98+ 1.34 11.84+ 1.49
2.70_+0.15a* ** 5.60+_0.42a*** 12.53+ 1.27a** 14.84+ 1.17a**
1.74+0.22 3.48+0.20 10.05+ 1.13 11.97+ 1.29
1.80+0.14b*** 3.82+0.26b*** 11.08+ 1.14 12.77+ 1.19b*
1.58+0.36 3.71_+0.32 10.62+1.06 12.01+1.02
2.30_+0.23a*** 5.69+0.39a*** 12.84+ 1.08a** 15.01+1.13a***
1.65+0.25 3.68+0.34 10.57+1.08 12.10_+1.21
1.70+0.2 lb** 4.01+0,27b*** 11.25+1.02b** 13.14+1.10b**
Units: LPO-nanomoles of MDA formed per milligram of protein Comparisons were made between: agroup 1 and 2, group 1 and 3, group 1 and 4, bgroup 2 and 4, ~group 3 and 4. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
Table II Activities of antioxidant e n z y m e s in liver and kidneys of control and experimental rats. (Values are m e a n ± s o for six animals in each group) Particulars
Liver Catalase Superoxide dismutase Glutathione peroxidase Kidney Catalase Superoxide dismutase Glutathione peroxidase
Group 1 Rat chow for 30 days 168.84+10.65 7.10+0.63 9.44+1.02 52.75+3.09. 5.40+0.39 5.37:k-0.28
Group 2 Glycollate for 30 days 152.85+ 10.49a* 6.22+0.56a* 8.03+ 1.03 46.90+3.17a** 4.10+0.30a*** 4.50+0.40a***
Group 3 Rat chow+cysteine for 30 days
Group 4 Glycollate+cysteine for 30 days
173.30+10.99 7.18+0.59 9.90+ 1.00
162.46+11 .l 1 7.02+0.64b* 9.01 + 1.01
56.12+2.80 5.33+0.34 5.70+0.30
Enzyme activities are expressed as: Catalase: ltmol of H202 consumed min-t (mg protein)-L SOD: units (rag protein) -~ (1 unit=the amount of enzyme that inhibits the autoxidation reaction by 50%). GPx:/.tg of reduced glutathione utilized min-~ (mg protein)-L Comparison between groups are as in Table I. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
50.01+2.70c*** 5.02~.0.30b*** 5.12+0.33b**c*
Pharmacological Research, Vol. 32. No. 3. 1995
167
<0.001, P<0.01 ). The increased production of malondialdehyde was controlled by the administration of cysteine when compared to calculogenic rats. Changes in the ant±oxidant enzymes are presented in Table II. A significant decrease in the catalase activity was observed in liver and kidney (P<0.05, P<0.01) of glycollate-fed rats. During cysteine supplementation, an increase in the enzyme activity was observed. A similar decrease in the superoxide dismutase activity was observed in the tissue homogenates (P<0.05 in liver, P<0.001 in kidney) of calculogenic rats when compared to control rats. Cysteine administration to glycollate fed rats (group 4) restored the enzyme activity to that of control. Glutathione peroxidase activity was decreased in liver and more significantly in kidney (P<0.001) of glycollate fed rats. Cysteine treatment to lithogenic rats elevated the enzyme level to that of controls in
the kidney, whereas no significant alteration was observed in the liver. The changes observed with respect to the ant±oxidant status are depicted in Table III. Glutathione, inhibitor of free radical toxicity, was significantly decreased (P<0.001) in calculogenic rats (group 2). Cysteine administration to glycollate fed rats showed elevation in the tissue concentration of glutathione. Total thiol concentration showed a significant decrease in liver and kidney of lithogenic rats (P< 0.01) when compared to controls. Cysteine supplementation significantly increased the total thiol level in group 4 animals to the extent of controls. Ascorbic acid level was significantly decreased in the liver and kidney of group 2 animals (P<0.01, P<0.05, respectively). No significant changes were observed during cysteine treatment to glycollate fed rats. The activities of superoxide generating enzyme, xanthine oxidase and NADPH producing enzyme, glu-
Table III Concentrations of ant±oxidants in liver and kidneys of control and experimental rats. (Values are mean--sD for six animals in each group)
Particulars [llg (mg protein) -~]
Group 1 Rat chow for 30 days
Liver Reduced glutathione Total thiols Ascorbic acid
4.92±0.56 12.01± 1.02 2.52±0.34
Kidney Reduced glutathione Total thiols Ascorbic acid
2.39±0.22 6.03±0.58 1.38±0.24
Group 2 Glycollate for 30 clays
Group 3 Rat chow+cysteine for 30 days
3.40_+0.42a*** 10.02± 1.01 a** 2.01±0.23a** 1.40±0.16a*** 4.90±0.61a** 1.01±0.21a*
Group 4 Glycollate+cysteine for 30 days
5.30±0.50 12.30± 1.00 2.60±0.22
4.73±0.39b*** 11.32±1.01b* 2.30±0.22
2.51±0.20 6.20±0.50 1.48±0.16
2.34±0.18b*** 5.98±0.63b** 1.20±0.23
Comparison between groups are as in Table I. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
Table IV Activities of xanthine oxidase and glucose-6-phosphate dehydrogenase in liver and kidneys of control and experimental rats. (Values are mean--sD for six animals in each group)
Particulars
Group 1 Rat chow for 30 days
Group 2 Glycollate for 30 days
Group 3 Rat chow+cysteine for 30 days
Group 4 Glycollate+cysteine for 30 days
Liver Xanthine oxidase Glucose-6-phosphate dehydrogenase
1.78±0.13 2.25±0.20
2.22±0.10a*** 1.98±0.18
1.84_+0.11 2.29±0.16
2.18±0.!0a, c*** 2.15±0.21
Kidney Xanthine oxidase G luc ose- 6-p hos pha te dehydrogenase
0.75+0.04 1.11 +0.12
1.03+0.05a*** 0.90+0.13 a *
0.78+0.03 1.19+ 0.12
0.9 l_+0.05a,b,c*** 0.98 ±0. I 0e*
Enzyme activities are expressed as: units (mg protein) -t (1 unit=the amount of enzyme that brings about a change in O,D, of 0.01 min-t). Comparison between groups are as in Table I. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
168 cose-6-phosphate dehydrogenase are given in Table IV. Xanthine oxidase activity was significantly high (P<0.001) in the liver and kidney of group 2 animals. Cysteine administration lowered the enzyme activity in kidney, but not to that of control level. A slight reduction in glucose-6-phosphate dehydrogenase activity (P<0.05) in kidney was observed in group 2 rats. However, the enzyme activity was not altered during cysteine treatment.
DISCUSSION The increased membrane susceptibility to lipid peroxidation by enzymatic and non-enzymatic systems in glycollate-fed rat tissues may be due to increased promoters or decreased antioxidant levels. Ascorbic acid mediated lipid peroxidation is physiologically important because of its role in being an immediate precursor for oxalate synthesis. The malondialdehyde released in the presence of ferrous ion in both the tissues were high in glycollate-fedrats than controls. It has been suggested that malondialdehyde production was increased during oxalate precipitation. Both in vivo and in vitro studies have revealed that the mechanism of induction of lipid peroxidation by oxalate may be involved through the inhibition of catalase activity [23]. Cysteine administration produced significant decrease in endrin-induced hepatic and renal lipid peroxidation in rats [9] and lanthanum chloride induced lipid peroxidation in the liver of chicks [24]. The sulphydryl group of cysteine is involved in the oxidation-reduction and conjugation reactions, thus accounting for its physiological activity [25]. In our present study, cysteine administration significantly reduced the lipid peroxide level. Catalase present in the peroxisomes, probably serves to destroy H202 generated by oxidase enzymes [26]. Rister and Bachner [27] have speculated that, during oxidative stress catalase activity decreases and H202 accumulates thereby favouring more peroxidation of lipids. Superoxide dismutase is widely distributed in cells and has been proposed to protect them against the deleterious effect of superoxide anion [28]. This enzyme catalytically scavenges the superoxide radicals and thus renders cytoprotection against free radical damage. Decreased superoxide dismutase activity has also been reported in rats fed with glycollate [29]. Glutathione peroxidase is considered to be a most important H202 removing enzyme [30]. Glutathione peroxidase utilizes glutathione as a major source of reducing power for the removal of H202 and hydroperoxides. The increased activity of these enzymes during cysteine treatment in hyperoxaluric condition lowered the levels of malondialdehyde an end product of lipid peroxidation. Glutathione, an ubiquitous tripeptide plays a vital
Pharmacological Research. Vol. 32. No. 3. 1995
role in a number of cellular functions including translocation of amino acids across the cell membrane, retention of protein sulphydryl groups, detoxification of electrophilic compounds and catabolism of hydrogenperoxide [31]. Glutathione depletion in lithogenic rats have been reported [32]. Cysteine is a precursor for glutathione biosynthesis and it has been proposed that rat liver glutathione production is dependent upon cysteine consumption [33]. Supplementation of cysteine in diet to cysteine deficient chicks brought about an increase in the liver glutathione content [34]. A direct link between the thiol status of the membrane and cellular glutathione has been reported [35]. The function of glutathione is to serve as a reductant of membrane protein disulphide and to avert membrane thiol oxidation. Moreover a noted reduction in the level of ascorbic acid might also contribute for the elevated lipid peroxidation. The results observed in the present study highlight the fact that cysteine may increase the intracellular glutathione content and regulate lipid peroxidation in hyperoxaluric rats. However, cysteine administration did not show any influence on the ascorbic acid level. Xanthine oxidase is considered to be one of the enzymes responsible for the production of oxalate from glyoxylate [36]. However, the contribution of xanthine oxidase in oxalate synthesis is thought to be minor. Production of superoxide anion has been reported to increase due to the enhanced activity of xanthine oxidase [37]. Cysteine administration has very little influence on the enzyme activity. The reduction in glucose-6-phosphate dehydrogenase activity observed in the glycollate fed rats may be due to the inhibition of the enzyme activity by glycollic and oxalic acid [38]. The observed decrease in the enzyme activity in lithogenic rats may decrease the regeneration of glutathione by lowered NADPH production. To conclude, cysteine ameliorates the lipid peroxidative damage observed during hyperoxaluria and this can be attributed to its efficiency in reviving the antioxidant status. It is tempting to speculate that cysteine treatment may prove useful as a therapeutic regimen in calcium oxalate urolithiasis.
REFERENCES 1. Eichenberger K, Bohni P, Winterhalter KH, Kawato S, Richter C. Microsomal lipid peroxidation causes an increase in the order of membrane lipid domain. FEBS Lett 1982; 142: 59-62. 2. Ernster L, Nordenbrand K. Microsomal lipid peroxidation. Methods Enzymol 1967; 10: 574-80. 3. Lewis SE, Wills ED. Destruction of sulphydryl groups of proteins and amino acids by peroxides of unsaturated fatty acids. Biochem Pharmacol 1962; 11: 901-12. 4. Ravichandran V, Selvam R. Lipid peroxidation in subcellular fractions of liver and kidney of vitamin B6 deficient rats. Med Sci Res 1990; 18:369-71.
Pharmacological Research, Vol. 32, No. 3, 1995 5. Selvam R, Sridevi D. Induction of oxalate binding by lipid peroxidation in rat kidney mitochondria. Biochena lnt 1991; 24: 857-66. 6. Halliwell B, Gutteridge JMC. Lipid peroxidation, oxygen radicals, cell damage and antioxidant therapy. Lancet 1984; i: 1396-8. 7. Nicotera P, Orrenius S. Role of thiols in protection against biological reactive intermediates. Adv Exp Med Biol 1986; 197: 41-51. 8. Chow CK. Nutritional influence on cellular antioxidant defence system. Am J Clin Nutr 1979; 32:1066-81. 9. Numan IT, Hassan MQ, Stohs SJ. Protective effects of antioxidants against endrin-induced lipid peroxdiation, glutathione depletion, and lethality in rats. Arch Environ Contain Toxicol 1990; 19: 302-6. 10. Bais R, Role AM, Conyers RAJ. The inhibition of metabolic oxalate production by sulfhydryl compounds. J Urol 1991; 145: 1302-5. I I. Selvam GS, Subha K, Varalakshmi P. Effect of L(+)tartrate on some biochemical and enzymatic parameters in normal and glycollate treated rats. Pharmacol Res 1992; 26: 385-94. 12. Chow FHC, Dysart MI, Hamar DW, Udall RH. Control of oxalate urolithiasis by DL-alanine. hn,est Urol 1975; 13:113-16. 13. Devasagayam TPA. Lipid peroxidation in rat uterus. Biochim Biophys Acta 1986; 876: 507-14. 14. Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972; 47: 389-94. 15. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469-74. 16. Rotruck JT, Pope AL, Ganther HE. Selenium: Biochemical role as a component of glutathione peroxidase purification and assay. Science 1973; 179: 588-90. 17. Moron MS, Defierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione-S-transferase activities in rat lung and liver. Biochim Biophys Acta 1979; 582: 67-78. 18. Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Methods Enzymol 1971; 62: 1-I 1. 19. Sedlak J, Lindsay RH. Estimation of total, protein bound and non-protein sulfhydryl groups in tissue with EIIman's reagent. Anal Biochem 1968; 25:192-205. 20. Fried R, Fried LW. Xanthine oxidase. In: Bergmeyer HU, ed. Methods of enzymatic analysis. Vol 2. Academic Press, New York, 1966; 644. 21. Balinsky D, Bernstein RE. The purification and properties of glucose-6-phosphate dehydrogenase from human erythrocytes. Biochim Biophys Acta 1963; 67:313-15. 22. Lowry OH, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem 1951; 193: 265-75. 23. Selvam R, Bijikurien T. Induction of lipid peroxidation
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by oxalate in experimental rat urolithiasis. J Bio sci 1987; 12: 367-73. Basu A, Haldar S, Chakrabarty K, Santra M, Chatterjee GC. Effect of cysteine supplementation on lanthanum chloride induced alterations in the antioxidant defence system of chick liver. IndJ Exp Biol 1984; 22: 432-34. Hawk PB. In: Oser BL, ed. Hawk's physiological chemistry. The Blakiston Division, McGraw-Hill, New York, 1965; 147. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979; 59: 527-605. Rister M, Bachner R. The alteration of superoxide dismutase, catalase, glutathione peroxidase and NAD(P)H cytochrome-C-reductase in guinea pig polymorphonuclear leukocytes and alveolar macrophages during hypoxia. J Clin hlvest 1976; 58: I 174-84. Fridovich I. Superoxide dismutases. Ann Rev Biochem 1975; 44: 147-59. Selvam R, Bijikurien T. Effect of citrate feeding on free radical induced changes in experimental urolithiasis. hid J Exp Biol 1992; 30:705-10. Gaetani GF, Galiano S, Canepa L, Ferraris AM, Kirkman HN. Catalase and glutathione peroxidase are equally active in detoxification of hydrogenperoxide in human erythrocytes. Blood 1989; 73: 334-9. Beutler E. Nutritional and metabolic aspects of glutathione. Ann Rev Nutr 1989; 9: 287-302. Sumathi R, Jayanthi S, Kalpana Devi V, Varalakshmi P. Effect of DL a-lipoic acid on tissue lipid peroxidation and antioxidant systems in normal and glycollate treated rats. Pharmacol Res 1993; 27" 309-18. Tateishi N, Higashi T, Naruse A, Hikita K, Sakamoto Y. Relative contributions of sulfur atoms of dietary cysteine and methionine to rat liver glutathione and proteins. JBiochem 1981 ; 90: 1603-10. Boebel KP, Baker DH. Blood and liver concentrations of glutathione and plasma concentrations of sulfur containing amino acids in chicks fed deficient, adequate or excess levels of dietary cysteine. Proc Soc Exp Biol Med 1983; 172: 498-501. Kosower NS, Kosower EM. Glutathione and cell membrane thiol status. In: Larsson A, Orrenius S, Holmgren A, Mannervik B, eds. Functions of glutathione-biochemical, physiological, toxicological and clinical aspects. Raven Press, New York 1983; 307-15. Hodgkinson A. Oxalate metabolism in animals and man. In: Oxalic acid in biology and medicine. Academic Press, New York, 1977; 159-212. Gutteridge JMC, Westermark T, Halliwell B. In: Johnson JE, Walford R, Harman D, Miquel J, eds. Free radicals, aging and degenerative diseases. Alan R. Liss, New York, 1985; 97. Ribaya JD, Gershoff SN. Interrelationships in rats among dietary vitamin B6, glycine and hydroxyproline. Effects of oxalate, glyoxylate, glycolate and glycine on liver enzymes. J Nutr 1979; 109: 171-83.
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EFFECT OF L-CYSTEINE ON LIPID PEROXIDATION IN EXPERIMENTAL UROLITHIATIC RATS N. SARAVANAN, D. SENTHIL and P. VARALAKSHMI Department of Medical Biochemistry, Dr A. L. Mudaliar Post Graduate Institute of Basic Medical Sciences, Universi O, of Madras, Taramani Campus, Madras 600 113, India Accepted 5 September 1995 Oxalate, the major stone-forming constituent induces lipid peroxidation during lithogenesis. In experimental condition oxalate formation was induced by the administration of its precursor glycollate. Glycollate-fed rats showed increased susceptibility to lipid peroxidation in the presence of promoters. In addition, antioxidant enzymes--catalase, superoxide dismutase and glutathione peroxidase also showed decreased activity. Reduced glutathione, total thiols and ascorbic acid were also significantly decreased. On the other hand, an increased xanthine oxidase and decreased glucose-6-phosphate dehydrogenase activity was also observed upon glycollate administration. Cysteine, a sulphydryl compound, is known to inhibit free radical toxicity in various pathologies. Cysteine administration to glycollate-fed rats brought about a significant decrease in the peroxidative level, with an increase in the antioxidant status. © 1995 The Italian Pharmacological Society KEYWORDS:glycollate, cysteine, liver, kidney, lipid peroxidation.
INTRODUCTION Lipid peroxidation of unsaturated fatty acids has been shown to be involved in the pathogenesis of cellular damage [1]. Oxalate, the major stone-forming constituent, is known to induce lipid peroxidation which causes disruption of the structural integrity of the membranes [2]. This alters the membrane permeability and thereby impairs the transport of ions and electrons across the cellular organelles. Formation of peroxides in in vivo conditions may produce serious consequences in the tissues [3]. Oxygen and/or hydroxyl radical reaction mechanism can injure renal tubular cells and promote calcium oxalate crystallization within the subcellular fractions of the kidney [4]. Selvam and Sridevi [5] have also reported the effect of lipid peroxidation in rat kidney mitochondria, which may be an important factor for the aetiology of stone formation. The products of lipid peroxidation in cells, is controlled by various cellular defence mechanisms consisting of enzymatic and non-enzymatic scavenger systems [6]. The oxidative damage may be attributed to the destruction of thiol groups of the amino acids and proteins [3], since thiol compounds are well known for their free radical scavenging property [7]. Dietary supplementation of sulphur-containing amino acids
C o r r e s p o n d e n c e to: D r ( M r s ) P. V a r a l a k s h m i .
1043-6618/95/090165-05/$12.00/0
have been reported to improve the tissue antioxidant status in rats [8]. L-cysteine, a sulphur-containing amino acid is known to offer protection to the living system against certain toxicants through its ability to increase the thiol status of the tissues. Cysteine has been reported to exhibit inhibitory effect on endrin induced hepatic and renal lipid peroxidation in rats [9]. Cysteine has also been reported to inhibit oxalate synthesis, thus leading to decreased oxalate excretion [10]. This unique antioxidant property of cysteine motivated us to investigate whether this amino acid could possibly prevent the oxalate-induced peroxidation of lipids in experimental hyperoxaluria.
MATERIALS AND METHODS Male albino Wistar rats weighing between 150 and 180 g were divided into four groups of six rats. Group 1 used as control, were maintained on the commercial feed [1 I]. Group 2 animals were given the commercial diet mixed with 3% w/w sodium glyeollate for 30 days according to the method of Chow et al. [12]. Group 3 animals received L-cysteine intraperitoneatl3/ at a dose of 500 mg (kg body weight) -l day"~ ft~r 30 days. Group 4 rats received glycollate :supplemented diet and L-cysteine intraperitoneallyfor 30-days: • : ?: . ,!-: Water was given ad libitum. At the end of the experimelatal peri0d,.the; a~aimals were killed by decapitation. Liver and ~.'dnvys,~were dissected out and washed in cold 0.15 M KCI:rT,hefi~the © 1995The ItalianPharmacologicalSociety
166
Pharmacological Research, Vol. 32, No. 3, 1995
tissues were homogenized in Tris-HC1 buffer (0.01 M, pH 7.4) using a Potter-Elvehjem homogenizer to give a 10% homogenate. Lipid peroxidation was determined by the method of Devasagayam [13]. The NADPH-induced system in a total volume of 2 ml contained 0.2 ml of tissue homogenate, 50/.tM FeCI3, 5 mr~ ADP, 1 mM KH2POa and 0.4mM NADPH in 0.15M Tris-HC1 buffer, pH 7.4. The ascorbate-induced system contained 0.2 ml of tissue homogenate, 50/IM FeSO4, I mM KH2PO4, 0.4 mM ascorbic acid in 0.15 M Tris-HCl buffer, pH 7.4. Antioxidant enzyme catalase was assayed by the method of Sinha [14]. Superoxide dismutase (SOD) was estimated according to the method of Marklund and Marklund [ 15]. Glutathione peroxidase (GPx) was assayed by the method of Rotruck et al. [ 16]. Antioxidants such as total reduced glutathione [17], ascorbic acid [18] and total thiol groups [19] were also determined. Xanthine oxidase activity was determined by
the method of Fried and Fried [20] using xanthine as substrate and glucose-6-phosphate dehydrogenase activity was assayed by the method of Balinsky and Bernstein [21], using glucose-6-phosphate as substrate. Tissue protein content was estimated by the method of Lowry et al. [22]. The data were analysed using one way analysis of variance followed by Student's Newman-Keuls test. The values are expressed as mean+sD.
RESULTS Table I presents the enzymatic, non-enzymatic and ferrous sulphate induced lipid peroxidation of liver and kidney homogenates of control and experimental rats. Basal, NADPH (enzymatic), ascorbate (nonenzymatic) and ferrous sulphate induced lipid peroxidation were significantly high in glycollate fed rats (P
Table I Effect of glycollate and cysteine on lipid peroxidation in liver and kidneys of control and experimental rats. (Values are m e a n ± s o for six animals in each group) Particulars
Group I Rat chow for 30 days
Liver Basal NADPH system Ascorbate system 10 mM ferrous sulphate Kidney Basal NADPH system Ascorbate system 10 rnM ferrous sulphate
Group 2 Glycollate for 30 days
Group 3 Rat chow+cysteine for 30 days
Group 4 Glycollate+cysteine for 30 days
1.70+0.20 3.53+0.45 9.98+ 1.34 11.84+ 1.49
2.70_+0.15a* ** 5.60+_0.42a*** 12.53+ 1.27a** 14.84+ 1.17a**
1.74+0.22 3.48+0.20 10.05+ 1.13 11.97+ 1.29
1.80+0.14b*** 3.82+0.26b*** 11.08+ 1.14 12.77+ 1.19b*
1.58+0.36 3.71_+0.32 10.62+1.06 12.01+1.02
2.30_+0.23a*** 5.69+0.39a*** 12.84+ 1.08a** 15.01+1.13a***
1.65+0.25 3.68+0.34 10.57+1.08 12.10_+1.21
1.70+0.2 lb** 4.01+0,27b*** 11.25+1.02b** 13.14+1.10b**
Units: LPO-nanomoles of MDA formed per milligram of protein Comparisons were made between: agroup 1 and 2, group 1 and 3, group 1 and 4, bgroup 2 and 4, ~group 3 and 4. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
Table II Activities of antioxidant e n z y m e s in liver and kidneys of control and experimental rats. (Values are m e a n ± s o for six animals in each group) Particulars
Liver Catalase Superoxide dismutase Glutathione peroxidase Kidney Catalase Superoxide dismutase Glutathione peroxidase
Group 1 Rat chow for 30 days 168.84+10.65 7.10+0.63 9.44+1.02 52.75+3.09. 5.40+0.39 5.37:k-0.28
Group 2 Glycollate for 30 days 152.85+ 10.49a* 6.22+0.56a* 8.03+ 1.03 46.90+3.17a** 4.10+0.30a*** 4.50+0.40a***
Group 3 Rat chow+cysteine for 30 days
Group 4 Glycollate+cysteine for 30 days
173.30+10.99 7.18+0.59 9.90+ 1.00
162.46+11 .l 1 7.02+0.64b* 9.01 + 1.01
56.12+2.80 5.33+0.34 5.70+0.30
Enzyme activities are expressed as: Catalase: ltmol of H202 consumed min-t (mg protein)-L SOD: units (rag protein) -~ (1 unit=the amount of enzyme that inhibits the autoxidation reaction by 50%). GPx:/.tg of reduced glutathione utilized min-~ (mg protein)-L Comparison between groups are as in Table I. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
50.01+2.70c*** 5.02~.0.30b*** 5.12+0.33b**c*
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167
<0.001, P<0.01 ). The increased production of malondialdehyde was controlled by the administration of cysteine when compared to calculogenic rats. Changes in the ant±oxidant enzymes are presented in Table II. A significant decrease in the catalase activity was observed in liver and kidney (P<0.05, P<0.01) of glycollate-fed rats. During cysteine supplementation, an increase in the enzyme activity was observed. A similar decrease in the superoxide dismutase activity was observed in the tissue homogenates (P<0.05 in liver, P<0.001 in kidney) of calculogenic rats when compared to control rats. Cysteine administration to glycollate fed rats (group 4) restored the enzyme activity to that of control. Glutathione peroxidase activity was decreased in liver and more significantly in kidney (P<0.001) of glycollate fed rats. Cysteine treatment to lithogenic rats elevated the enzyme level to that of controls in
the kidney, whereas no significant alteration was observed in the liver. The changes observed with respect to the ant±oxidant status are depicted in Table III. Glutathione, inhibitor of free radical toxicity, was significantly decreased (P<0.001) in calculogenic rats (group 2). Cysteine administration to glycollate fed rats showed elevation in the tissue concentration of glutathione. Total thiol concentration showed a significant decrease in liver and kidney of lithogenic rats (P< 0.01) when compared to controls. Cysteine supplementation significantly increased the total thiol level in group 4 animals to the extent of controls. Ascorbic acid level was significantly decreased in the liver and kidney of group 2 animals (P<0.01, P<0.05, respectively). No significant changes were observed during cysteine treatment to glycollate fed rats. The activities of superoxide generating enzyme, xanthine oxidase and NADPH producing enzyme, glu-
Table III Concentrations of ant±oxidants in liver and kidneys of control and experimental rats. (Values are mean--sD for six animals in each group)
Particulars [llg (mg protein) -~]
Group 1 Rat chow for 30 days
Liver Reduced glutathione Total thiols Ascorbic acid
4.92±0.56 12.01± 1.02 2.52±0.34
Kidney Reduced glutathione Total thiols Ascorbic acid
2.39±0.22 6.03±0.58 1.38±0.24
Group 2 Glycollate for 30 clays
Group 3 Rat chow+cysteine for 30 days
3.40_+0.42a*** 10.02± 1.01 a** 2.01±0.23a** 1.40±0.16a*** 4.90±0.61a** 1.01±0.21a*
Group 4 Glycollate+cysteine for 30 days
5.30±0.50 12.30± 1.00 2.60±0.22
4.73±0.39b*** 11.32±1.01b* 2.30±0.22
2.51±0.20 6.20±0.50 1.48±0.16
2.34±0.18b*** 5.98±0.63b** 1.20±0.23
Comparison between groups are as in Table I. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
Table IV Activities of xanthine oxidase and glucose-6-phosphate dehydrogenase in liver and kidneys of control and experimental rats. (Values are mean--sD for six animals in each group)
Particulars
Group 1 Rat chow for 30 days
Group 2 Glycollate for 30 days
Group 3 Rat chow+cysteine for 30 days
Group 4 Glycollate+cysteine for 30 days
Liver Xanthine oxidase Glucose-6-phosphate dehydrogenase
1.78±0.13 2.25±0.20
2.22±0.10a*** 1.98±0.18
1.84_+0.11 2.29±0.16
2.18±0.!0a, c*** 2.15±0.21
Kidney Xanthine oxidase G luc ose- 6-p hos pha te dehydrogenase
0.75+0.04 1.11 +0.12
1.03+0.05a*** 0.90+0.13 a *
0.78+0.03 1.19+ 0.12
0.9 l_+0.05a,b,c*** 0.98 ±0. I 0e*
Enzyme activities are expressed as: units (mg protein) -t (1 unit=the amount of enzyme that brings about a change in O,D, of 0.01 min-t). Comparison between groups are as in Table I. The symbols represent statistical significance: *=P<0.05; **=P<0.01; ***=P<0.001.
168 cose-6-phosphate dehydrogenase are given in Table IV. Xanthine oxidase activity was significantly high (P<0.001) in the liver and kidney of group 2 animals. Cysteine administration lowered the enzyme activity in kidney, but not to that of control level. A slight reduction in glucose-6-phosphate dehydrogenase activity (P<0.05) in kidney was observed in group 2 rats. However, the enzyme activity was not altered during cysteine treatment.
DISCUSSION The increased membrane susceptibility to lipid peroxidation by enzymatic and non-enzymatic systems in glycollate-fed rat tissues may be due to increased promoters or decreased antioxidant levels. Ascorbic acid mediated lipid peroxidation is physiologically important because of its role in being an immediate precursor for oxalate synthesis. The malondialdehyde released in the presence of ferrous ion in both the tissues were high in glycollate-fedrats than controls. It has been suggested that malondialdehyde production was increased during oxalate precipitation. Both in vivo and in vitro studies have revealed that the mechanism of induction of lipid peroxidation by oxalate may be involved through the inhibition of catalase activity [23]. Cysteine administration produced significant decrease in endrin-induced hepatic and renal lipid peroxidation in rats [9] and lanthanum chloride induced lipid peroxidation in the liver of chicks [24]. The sulphydryl group of cysteine is involved in the oxidation-reduction and conjugation reactions, thus accounting for its physiological activity [25]. In our present study, cysteine administration significantly reduced the lipid peroxide level. Catalase present in the peroxisomes, probably serves to destroy H202 generated by oxidase enzymes [26]. Rister and Bachner [27] have speculated that, during oxidative stress catalase activity decreases and H202 accumulates thereby favouring more peroxidation of lipids. Superoxide dismutase is widely distributed in cells and has been proposed to protect them against the deleterious effect of superoxide anion [28]. This enzyme catalytically scavenges the superoxide radicals and thus renders cytoprotection against free radical damage. Decreased superoxide dismutase activity has also been reported in rats fed with glycollate [29]. Glutathione peroxidase is considered to be a most important H202 removing enzyme [30]. Glutathione peroxidase utilizes glutathione as a major source of reducing power for the removal of H202 and hydroperoxides. The increased activity of these enzymes during cysteine treatment in hyperoxaluric condition lowered the levels of malondialdehyde an end product of lipid peroxidation. Glutathione, an ubiquitous tripeptide plays a vital
Pharmacological Research. Vol. 32. No. 3. 1995
role in a number of cellular functions including translocation of amino acids across the cell membrane, retention of protein sulphydryl groups, detoxification of electrophilic compounds and catabolism of hydrogenperoxide [31]. Glutathione depletion in lithogenic rats have been reported [32]. Cysteine is a precursor for glutathione biosynthesis and it has been proposed that rat liver glutathione production is dependent upon cysteine consumption [33]. Supplementation of cysteine in diet to cysteine deficient chicks brought about an increase in the liver glutathione content [34]. A direct link between the thiol status of the membrane and cellular glutathione has been reported [35]. The function of glutathione is to serve as a reductant of membrane protein disulphide and to avert membrane thiol oxidation. Moreover a noted reduction in the level of ascorbic acid might also contribute for the elevated lipid peroxidation. The results observed in the present study highlight the fact that cysteine may increase the intracellular glutathione content and regulate lipid peroxidation in hyperoxaluric rats. However, cysteine administration did not show any influence on the ascorbic acid level. Xanthine oxidase is considered to be one of the enzymes responsible for the production of oxalate from glyoxylate [36]. However, the contribution of xanthine oxidase in oxalate synthesis is thought to be minor. Production of superoxide anion has been reported to increase due to the enhanced activity of xanthine oxidase [37]. Cysteine administration has very little influence on the enzyme activity. The reduction in glucose-6-phosphate dehydrogenase activity observed in the glycollate fed rats may be due to the inhibition of the enzyme activity by glycollic and oxalic acid [38]. The observed decrease in the enzyme activity in lithogenic rats may decrease the regeneration of glutathione by lowered NADPH production. To conclude, cysteine ameliorates the lipid peroxidative damage observed during hyperoxaluria and this can be attributed to its efficiency in reviving the antioxidant status. It is tempting to speculate that cysteine treatment may prove useful as a therapeutic regimen in calcium oxalate urolithiasis.
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