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Time-dependent protective effect of selenium against cadmium-induced nephrotoxicity and hepatotoxicity
Chem.-Biol. Interactions, 42 (1982) 345--351 Elsevier Scientific Publishers Ireland Ltd.
345
TIME-DEPENDENT PROTECTIVE EFFECT OF SELENIUM AGAINST CADMIUM-INDUCED NEPHROTOXICITY AND HEPATOTOXICITY
S.J.S. FLORA, JAI RAJ BEHARI, M. ASHQUIN and S.K. TANDON*
Industrial Toxicology Research Centre, Post Box-80, Lucknow-226001 (India) (Received March 16th, 1982) (Revision received June l l t h , 1982) (Accepted June 15th, 1982)
SUMMARY
The role of selenium in protection against nephrotoxicity and hepatotoxicity of cadmium in rats was investigated. The administration of Cd (3 mg/kg, s.c.} for 3 days enhanced the urinary excretion of lactic dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) and total proteins, decreased the renal activity of GOT and alkaline phosphatase (ALP) and increased the renal level of Cd, Cu and Zn. Cadmium also increased the serum GOT and glutamic pyruvic transaminase (GPT), decreased the hepatic activity of GOT and GPT and increased the hepatic level of Cd and Zn. The concomitantly administered Se (2 mg/kg, i.p.) initially reduced most of these Cd-induced alterations. The results show protection by Se against nephrotoxicity and hepatotoxicity of Cd on the 4th day of the c o m m e n c e m e n t of Cd administration, but the signs of Cd intoxication were observed on the 8th day.
Key words: Cadmium nephrotoxicity -- hepatotoxicity -- Selenium protection -- Metal-metal interaction -- Kidney-liver-serum-urinary-enzymesRat
INTRODUCTION
Cadmium, an occupational and environmental pollutant causes liver, kidney and pancreas dysfunction [1--3]. Copper, iron, zinc and particularly selenium protect against Cd toxicity [4--7]. Selenium counteracts Cdinduced lethal effects [8], testicular damage [9,10] and several metabolic *To whom reprint requests be addressed. Abbreviations: ALP, alkaline phosphatase; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; LDH, lactic dehydrogenase. 0009--2797/82/0000---0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
346 and functional alterations [6]. R e d d y et al. [11] demonstrated that Cd causes greater enzymatic and biochemical changes in Se-deficient rats than in Se-supplemented rats. Although antagonism of Se to Cd toxicity is not well understood, the ability of Se to influence the organ distribution of Cd [ 1 0 ] , its strong tendency to complex with metals [12] and its antioxidant property [13] may play important roles in the protective mechanism. The reduced uptake and retention of Cd in b o d y by Cu, Fe or Zn have been responsible for their protective effect [4,14]. The present study was undertaken to investigate the time-dependent protective effect of Se in Cd nephrotoxicity and hepatotoxicity. The hepatic injury was evaluated by the activities of diagnostic enzymes in serum and liver and the renal injury by the urinary excretion of enzymes and total proteins and the enzymatic activities of kidney. The levels of Cd, Cu and Zn in these tissues were also determined to investigate the influence of Se on, the uptake and retention of Cd in relation to the trace metals. MATERIALS AND METHODS Forty-eight female albino rats weighing 170 + 10 g of ITRC Colony maintained on standard pellet diet (Hindustan Lever Ltd., India} were divided equally into 4 groups and treated daily for 3 days as follows: Group I Group II Group III
Group IV
4 ml/kg, Normal saline, s.c. 3 mg/kg, Cd as CdC12 • H20 (E. Merck}, s.c. 3 mg/kg, Cd as CdC12 • H20, s.c. + 2 mg/kg, Se as Na~SeO3 (E. Merck}, i.p. 2 mg/kg, Se as Na2SeO3, i.p.
The animals were kept in metabolic cages for 24 h, urine was collected consecutively for 4 days. Half of the animals from each group were killed by decapitation on the 4th day and remainder on the 8th day after the c o m m e n c e m e n t of treatment. The liver and kidneys were removed, washed free of extraneous material and homogenised in 0.25 M sucrose {10%). The blood was collected from the heart for serum separation. Standard procedures were used for the determination of the activities of GOT [15] in urine, serum, kidney and liver GPT [15] in serum and liver LDH [16] and total proteins [17] in urine and ALP [18] in kidney. The protein content of liver and kidney was estimated by the m e t h o d of Lowry et al. [19]. Cadmium, Cu and Zn were estimated in liver and kidney following wet acid digestion using atomic absorption spectrophotometer, PerkinElmer model 303 [ 2 0 ] . RESULTS The administration of Cd significantly enhanced the urinary excretion of
347
LDH (NADH Oxid,zed )
% Z
GOT (Hydrozone formed)
PROTEIN
3,
Cd
c)
Cd*%e " ~, ~
E
%e
Cd
--
--
~ ~
~d -a i
I1
S,
C T,
E c~
DAY
llllllllllH
.:
1234
Fig. 1. The effect of Cd, Cd + Se or Se administration o n the urinary excretions o f LDH, GOT and proteins in rats ( m e a n ± S.E. o f 5 values), ap < 0.001, b p < 0.01, c p < 0.05 w h e n compared to control ( m e a n +- S.E. o f 8 - - 1 0 values, s h o w n b y horizontal line), * * * P < 0.001, * * P < 0.01, *P < 0.05 w h e n compared to Cd-treated group as evaluated by the S t u d e n t ' s t-test.
LDH, GOT and total proteins indicating kidney damage. The simultaneous administration of Se with Cd reduces this effect significantly. The treatment with selenium alone caused slight proteinurea {Fig. 1). Cadmium caused significant decreases in the activities of renal GOT and ALP on the 4th day but, on the 8th day after the commencement of treatment, the levels of these enzymes were the same as those in the kidneys of control rats and thus indicative of recovery from the initial tissue damage {Table I). The levels of Cd, Cu and Zn however, increased significantly on the 4th and the 8th day of exposure. The simultaneous administration of Se reduced the Cd-induced inhibition of these enzymes and the increased levels of Cu on the 4th and the 8th day and of Zn only at the 4th day. The renal Cd, on the other hand, increased from the 4th day to the 8th of the experiment and this increase
GOT
o
GPT C_d Q
u
E
7-1p
Fsl i °
1
=
DAY &
C d+S_e S_ee
.... ft...
II-HI
Fig. 2. The effect of Cd, C d + Se or Se administration on the serum activitiesof G O T and G P T in rats (mean ± S.E. of 5---6 values), ap < 0.001, bp < 0.01, cp < 0.05 w h e n compared to control (mean + S.E. of 9 values shown by horizontal line) * * * P < 0.001, * * P < 0.01, *P < 0.05 w h e n compared to Cd-treated group as evaluated by the Student's t-test.
13.7 0.34 28.1 12.9 26.9
+ ± ± ± ±
0.67 a ( 4 ) 0.03 a ( 6 ) 3.06a(4) 0.675(5) 0.59 a ( 6 )
21.1 0.54 41.1 22.1 25.4
± ± + + ÷
1.01 (5) 0.02 (5) 6.51a(5) 1.26 a ( 5 ) 0.825 (6)
21.6± 0.42 ± 28.6 ± 8.2 ± 19.6 ±
0 . 7 0 * * * (6) 0.02b,* (6) 2.22a(6) 0 . 5 7 * * (6) 1 . 4 1 " * (6)
Day 4
Day 4
Day 8
Cd + Se
Cd
0.52 (6) 0.02 c (6) 0.14 (4) 1.54 (5) 2.23 c (5)
GOTt GPT ~ Cd~: Cu$ Zn-J:
19.1 20.2 3.1 5.7 36.7
+ 1.81 + 1.09 + 0.56 * 0.37 ÷ 2.31
Control
(9) (8) (7) (7) (8)
5.9 14.1 90.7 6.3 73.8
+ 0.45 a (5) ÷ 0.975 (4) +- 6.31 a (6) + 0.48 (6) ÷ 6.425 (6)
13.9 13.5 63.5 7.0 91.0
+ 0.78 a (5) + 1.22 c (5) ± 11.52 a (4) ~ 0.74 (5) ÷ 7.03 a (6)
8.3 19.3 65.5 6.4 46.0
± ± ± ± *
0.59 a,* (5) 0.52* (5) 2.54 a,* (5) 0.57 (6) 3.92* (5)
Day 4
Day 4
Day 8
Cd + Se
Cd
+ ± + ± +
20.0 25.5 1.6 6.4 33.2
0.90 a (5) 0.46 a ( 5 ) 2.54 a (5) 0.175 (5) 5.45 a (5)
14.7 13.8 99.6 8.0 97.2
± ± + + ±
Day 4
Day 8
Se
0.93 (5) 2.06 c (5) 0.20 (5) 1.08 (5) 3.05 (5)
17.4 19.2 1.7 4.9 38.5
-+ 1.26 + 1.21 ± 0.19 + 0.28 ± 2.20
Day 8 (6) (6) (6) (6) (5)
t n m o l h y d r a z o n e f o r m e d l m i n / m g p r o t e i n ; $ug/g fresh tissue. Each figure is m e a n ± S.E. o f n u m b e r o f rats given in parenthesis~ ap < 0.001, 5 p < 0.01, cp < 0.05 c o m p a r e d t o c o n t r o l , * P < 0.05 c o m p a r e d t o Cd g r o u p as evaluated b y S t u d e n t ' s t-test.
+- 1 . 5 6 ( 6 ) ± 0.03 (6) + 0.15(6) ± 0.68 (6) ± 0.80a(6)
19.1 0.54 1.4 8.9 27.9 + ± ± ± ±
21.2 0.47 2.0 8.9 27.8
0.52 (5) 0.23 (5) 0.50 a ( 4 ) 0 . 5 8 * * * (5) 2.40 c (5)
20.8 0.64 53.7 9.6 28.8
± ± ± + +
Day 8
Day 4
Day 8
Se
E F F E C T OF S E L E N I U M ON C A D M I U M - I N D U C E D C H A N G E S IN H E P A T I C E N Z Y M E S A N D M E T A L C O N T E N T S
T A B L E II
GOT t / 2 2 . 8 ± 0 . 3 2 ( 9 ) ALP $ ~ 0.56 ± 0.02 (9) Cd § 1.2 + 0 . 2 7 ( 9 ) Cu § 7.9 ÷ 0.43 (8) Zn § 21.7 ± 0.72 (6)
Control
t n m o l h y d r a z o n e f o r m e d / r a i n / r a g p r o t e i n , S u m o l p - n i t r o p h e n o l l i b . ] m i n / m g p r o t e i n ; § ug]g fresh tissue. E a c h figure is m e a n ± S.E. o f n u m b e r o f rats given in p a r e n t h e s i s ; a p < 0.001, 5 p < 0.01, c p < 0.05 c o m p a r e d to c o n t r o l ; * * * P < 0.001, * * P < 0.01, *P < 0.05 c o m p a r e d t o Cd g r o u p as evaluated by t h e S t u d e n t ' s t-test.
E F F E C T OF S E L E N I U M ON C A D M I U M - I N D U C E D C H A N G E S IN R E N A L E N Z Y M E S A N D M E T A L C O N T E N T S
TABLE I
O0
349 was unaffected by the treatment with Se. The treatment with Se alone however, increased the renal level of Zn significantly (Table I). The activities of serum GOT and GPT increased significantly on exposure to Cd suggesting liver damage. However, hepatotoxicity of Se cannot be suggested with any certainty on the basis of an increase in the activity of only serum GOT on the 8th day. Cadmium-induced increases were less marked in animals treated with Se simultaneously (Fig. 2). The activities of GOT and GPT decreased and the levels of Cd and Zn increased in liver significantly after the administration of Cd (Table II). The concomitant administration of Se prevented these alterations only at the 4th day. However, at the 8th day the activities of both the enzymes remained inhibited and the levels of Cd, Cu and Zn remained significantly higher in animals supplemented with Se indicative of no such protection against the toxic effects of Cd (Table II). DISCUSSION Enzymuria and proteinuria observed in Cd administered rats are the early signs of Cd nephrotoxicity and precede functional disturbances [21,22] (Fig. 1}. Concomitantly, the activities of the renal GOT and ALP decreased initially after Cd administration. The kidney damage being closely related to the induction of metallothionein synthesis by Cd, the uptake and/or retention of Cu and Zn also increased as expected with the progressive accummulation of Cd in the renal tissue [23] (Table I). The increase in the activities of GOT and GPT in serum and decrease in liver on Cd administration are the indices of Cd hepatotoxicity [24] (Fig. 2, Table II). Enzymatic and metabolic alterations in liver and kidney have been observed in rats exposed to Cd [3,6]. The inital uptake of Cd by liver has been far more than that by kidney. However, it decreased subsequently in liver and increased in kidney, the latter being the target organ. The increase in renal and hepatic Zn in Cd administered animals might be related to the intervening effect of Cd in absorption and disposition rate of Zn [4,25] or the induction of metallothionein by Cd [23,26]. The hepatic level of Cu did not increase significantly on exposure to Cd in this study (Table II). Some investigators reported a decrease [4,14], while others observed an increase in the hepatic Cu on Cd administration [27,28]. However, an excessive increase of Zn in liver and of Cu in kidney on exposure to Cd has been well established in the rat [27--29]. In contrast to Cd, the administration of Se had no effect on most of these parameters, except slight proteinuria and an increase in renal content of Zn indicative of some renotoxicity of Se. However, administration of Se concurrently with Cd significantly reduced the Cd induced enzymuria, proteinuria and elevation of serum enzymes. Selenium also ameliorated the inhibition in the activities of renal and hepatic enzymes observed on the 4th day of Cd administration. The increase in the uptake of renal Cu at the 4th a n d the 8th day and of renal and hepatic Zn on the 4th day of Cd
350 administration was also prevented by Se. Interestingly, the accummulation of Cd in kidney and the increase of Zn in liver and kidney observed on the 8th day of Cd administration could not be checked by Se given simultaneously. Furthermore, hepatic uptake of Cd was successfully prevented by Se on the 4th day. However, the level of hepatic Cd and Cu increased from the 4th to the 8th day on simultaneous administration of Se. These observations clearly suggest an initial protection against Cd toxicity by Se, possibly when the tissue levels of Se are high. Selenium may compete with Cd for binding with the functional biological ligands or the target tissue sites [10,30]. Alternatively, Cd and Se might interact with the reversible formation of cadmium selenide (CdSe), thus reducing the 'free' concentration of toxic Cd 2+ ions in the body [30,31]. Subsequently, CdSe is dissociated and the toxic effects of Cd are observed. The present results thus indicate that nephrotoxicity and hepatotoxicity of Cd could be reduced immediately following Se administration, however, later when the body or tissue level of Se decline, the toxic effects of Cd appear, particularly the hepatotoxicity. ACKNOWLEDGEMENTS
S.J.S. Flora is grateful to Indian Council of Medical Research, New Delhi for a Junior Research fellowship. Thanks are due to Mr. Surendra Singh for the technical assistance. REFERENCES 1 I. Murata, T. Hirono, Y. Saeki and W.S. Nakaga, Cadmium enteropathy, renal osteomalacia (Itai Itai disease in Japan), Bull. Soc. Int. Chir., 1 (1970) 34. 2 H.D. Stowe, M. Wilson and R.A. Goyer, Clinical and morphological effects of oral cadium toxicity in rabbits, Arch. Pathol., 94 (1972) 389. 3 R.L. Singhai, Z. Merali, S. Kacew and D.J.B. Sutherland, Persistence of Cadmiuminduced metabolic changes in liver and kidney, Science (Washington), 183 (1974.) 1094. 4 C.R. Bunn and G. Matrone, In vivo interactions of cadmium, copper, zinc and iron in mouse and rat, J. Nutr., 90 (1966) 395. 5 J. Parizek, I. Ostadalova, A. Kalonskova, A. Babicky and J. Benes, The detoxifying effects of selenium inter-relations between Compounds of selenium and certain metals, in: W. Mertz and W.E. Cornatzer (Eds.), Newer Trace Elements in Nutrition, Marcel Dekker, New York, 1971, pp. 85--122. 6 Z. Merali and R.L. Singhal, Protective effect of selenium on certain hepatotoxic and pancreotoxic manifestations of subacute cadmium administration,J. Pharmacol. Exp. Ther., 195 (1975) 58. 7 Z. Merali and R.L. Singhal, Prevention by zinc of cadmium induced alterations in pancreatic and hepatic functions, Br. J. Pharmacol., 57 (1976) 573. 8 S.A. Gunn, T.C. Gould and W.A.D. Anderson, Specificity in protection against lethality and testicular toxicity from cadium, Proc. Soc. Exp. Biol. Med., 128 (1968) 591. 9 K.E. Mason and J.O. Young, Effectiveness of selenium and zinc in protecting against cadmium induced injury of the rat testis, in: O.H. Muth (Ed.), Selenium in Biomedicine, AVI, Westport, CN, 1967, pp. 383--394. 10 S.A. Gunn, T.C. Gould and W.A.D. Anderson, Selectivity of organ response to cadmium injury and various protective measures, J. Pathol. Bacteriol., 96 (1968) 89.
351 11 K.A. Reddy, S.T. Omaye, G.K. Hasegawa and C.E. Cross, Enhanced lung toxicity of intratracheally instilled cadmium chloride in selenium deficient rats, Toxicol. Appl. Pharmacol., 43 (1978) 249. 12 H.E. Ganther, P. Wagner, M.L, Sunde and W.G. Hoekstra, Protective effects of selenium against heavy metal toxicities, in: D.D. Hemphill (Ed.), Trace Substances in Environmental Health VI, Univ. of Missouri Press, Columbia, MO, 1972, pp. 247 - 2 5 2 . 13 J.T. Rotruck, A.L. Pope. H.E. Ganther, A.B. Swanson, D.G. Hafeman and W.G. Hoekstra, Selenium: Biochemical role as a component of glutathione peroxidase, Science, 179 (1973) 588. 14 R.J. Banis, W.G. Pond, E.F. Walker Jr. and J.R. O'Connor, Dietary cadmium, iron and zinc interactions in the growing rat, Proc. Soc. Exp. Biol. Med., 130 (1969) 802. 15 S. Reitman and S. Frankel, A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases, A m . J. Clin. Pathol., 28 (1957) 56. 16 I.D.P. Wooton, Lactate dehydrogenase, spectrophotometric method, in" E.J. King (Ed.), Microanalysis in Medical Biochemistry, 4th edn, 1964, pp. 115--116. 17 M. Piscator, Proteinurea in chronic cadmium poisoning, Arch. Environ. Health, 5 (1962) 325. 18 P.J. Wright, P.D. Leathwood and D.T. Plummer, Enzymes in rat urine: Alkaline phosphatase, Enzymologia, 42 (1972) 317. 19 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 20 M.N. Parker, F.L. Humoller and D.J. Mahler, Determination of copper and zinc in biological materials, Clin. Chem., 13 (1967) 40. 21 K. Nomiyama, C. Sato and A. Yamamoto, Early signs of cadmium intoxication in rabbits, Toxicol. Appl. Pharmacol., 24 (1973) 625. 22 F.W. Bonner, L.J. King and D.V. Parke, The urinary excretion of enzymes following repeated parenteral administration of cadmium to rats, Environ. Res., 22 (1980) 237. 23 M. Nordberg, Studies on metallothionein and cadmium, Environ. Res., 15 (1978) 381. 24 S. Zelman and C.C. Wang, Transaminases in serum and liver correlated with liver cell necrosis in needle aspiration biopsies, Am. J. Med. Sci., 237 (1959) 323. 25 K. Nakamura, E. Suzuki, Y. Sugiura and T. Takata, Effects of simultaneous administration of cadmium and zinc on the accumulation and the toxicity of cadmium, Ind. Health, 17 (1979) 1. 26 K.S. Squibb and R.J. Cousins, Control of cadmium binding protein synthesis in rat liver, Environ. Physiol. Biochem., 4 (1974) 24. 27 Y. Suzuki and H. Yoshikawa, Effects of cadmium injection on intraceUular distribution of essential metal in rat liver, Ind. Health, 10 (1972) 93. 28 M.D. Stonard and M. Webb, Influence of dietary cadmium on the distribution of the essential metals copper, zinc and iron in tissues of the rat, Chem.-Biol. Interact., 15 (1976) 349. 29 M. Webb, Cadmium, Br. Med. Bull., 31 (1975) 246. 30 T.W. Clarkson, Factors involved in heavy metal poisoning, Fed. Proc., 36 (1977) 1634. 31 T.A. Gasiewicz and T.C. Smith, Interactions of cadmium and selenium in rat plasma in vivo and in vitro, Biochim. Biophys. Acta, 428 (1976) 113.
345
TIME-DEPENDENT PROTECTIVE EFFECT OF SELENIUM AGAINST CADMIUM-INDUCED NEPHROTOXICITY AND HEPATOTOXICITY
S.J.S. FLORA, JAI RAJ BEHARI, M. ASHQUIN and S.K. TANDON*
Industrial Toxicology Research Centre, Post Box-80, Lucknow-226001 (India) (Received March 16th, 1982) (Revision received June l l t h , 1982) (Accepted June 15th, 1982)
SUMMARY
The role of selenium in protection against nephrotoxicity and hepatotoxicity of cadmium in rats was investigated. The administration of Cd (3 mg/kg, s.c.} for 3 days enhanced the urinary excretion of lactic dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) and total proteins, decreased the renal activity of GOT and alkaline phosphatase (ALP) and increased the renal level of Cd, Cu and Zn. Cadmium also increased the serum GOT and glutamic pyruvic transaminase (GPT), decreased the hepatic activity of GOT and GPT and increased the hepatic level of Cd and Zn. The concomitantly administered Se (2 mg/kg, i.p.) initially reduced most of these Cd-induced alterations. The results show protection by Se against nephrotoxicity and hepatotoxicity of Cd on the 4th day of the c o m m e n c e m e n t of Cd administration, but the signs of Cd intoxication were observed on the 8th day.
Key words: Cadmium nephrotoxicity -- hepatotoxicity -- Selenium protection -- Metal-metal interaction -- Kidney-liver-serum-urinary-enzymesRat
INTRODUCTION
Cadmium, an occupational and environmental pollutant causes liver, kidney and pancreas dysfunction [1--3]. Copper, iron, zinc and particularly selenium protect against Cd toxicity [4--7]. Selenium counteracts Cdinduced lethal effects [8], testicular damage [9,10] and several metabolic *To whom reprint requests be addressed. Abbreviations: ALP, alkaline phosphatase; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; LDH, lactic dehydrogenase. 0009--2797/82/0000---0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
346 and functional alterations [6]. R e d d y et al. [11] demonstrated that Cd causes greater enzymatic and biochemical changes in Se-deficient rats than in Se-supplemented rats. Although antagonism of Se to Cd toxicity is not well understood, the ability of Se to influence the organ distribution of Cd [ 1 0 ] , its strong tendency to complex with metals [12] and its antioxidant property [13] may play important roles in the protective mechanism. The reduced uptake and retention of Cd in b o d y by Cu, Fe or Zn have been responsible for their protective effect [4,14]. The present study was undertaken to investigate the time-dependent protective effect of Se in Cd nephrotoxicity and hepatotoxicity. The hepatic injury was evaluated by the activities of diagnostic enzymes in serum and liver and the renal injury by the urinary excretion of enzymes and total proteins and the enzymatic activities of kidney. The levels of Cd, Cu and Zn in these tissues were also determined to investigate the influence of Se on, the uptake and retention of Cd in relation to the trace metals. MATERIALS AND METHODS Forty-eight female albino rats weighing 170 + 10 g of ITRC Colony maintained on standard pellet diet (Hindustan Lever Ltd., India} were divided equally into 4 groups and treated daily for 3 days as follows: Group I Group II Group III
Group IV
4 ml/kg, Normal saline, s.c. 3 mg/kg, Cd as CdC12 • H20 (E. Merck}, s.c. 3 mg/kg, Cd as CdC12 • H20, s.c. + 2 mg/kg, Se as Na~SeO3 (E. Merck}, i.p. 2 mg/kg, Se as Na2SeO3, i.p.
The animals were kept in metabolic cages for 24 h, urine was collected consecutively for 4 days. Half of the animals from each group were killed by decapitation on the 4th day and remainder on the 8th day after the c o m m e n c e m e n t of treatment. The liver and kidneys were removed, washed free of extraneous material and homogenised in 0.25 M sucrose {10%). The blood was collected from the heart for serum separation. Standard procedures were used for the determination of the activities of GOT [15] in urine, serum, kidney and liver GPT [15] in serum and liver LDH [16] and total proteins [17] in urine and ALP [18] in kidney. The protein content of liver and kidney was estimated by the m e t h o d of Lowry et al. [19]. Cadmium, Cu and Zn were estimated in liver and kidney following wet acid digestion using atomic absorption spectrophotometer, PerkinElmer model 303 [ 2 0 ] . RESULTS The administration of Cd significantly enhanced the urinary excretion of
347
LDH (NADH Oxid,zed )
% Z
GOT (Hydrozone formed)
PROTEIN
3,
Cd
c)
Cd*%e " ~, ~
E
%e
Cd
--
--
~ ~
~d -a i
I1
S,
C T,
E c~
DAY
llllllllllH
.:
1234
Fig. 1. The effect of Cd, Cd + Se or Se administration o n the urinary excretions o f LDH, GOT and proteins in rats ( m e a n ± S.E. o f 5 values), ap < 0.001, b p < 0.01, c p < 0.05 w h e n compared to control ( m e a n +- S.E. o f 8 - - 1 0 values, s h o w n b y horizontal line), * * * P < 0.001, * * P < 0.01, *P < 0.05 w h e n compared to Cd-treated group as evaluated by the S t u d e n t ' s t-test.
LDH, GOT and total proteins indicating kidney damage. The simultaneous administration of Se with Cd reduces this effect significantly. The treatment with selenium alone caused slight proteinurea {Fig. 1). Cadmium caused significant decreases in the activities of renal GOT and ALP on the 4th day but, on the 8th day after the commencement of treatment, the levels of these enzymes were the same as those in the kidneys of control rats and thus indicative of recovery from the initial tissue damage {Table I). The levels of Cd, Cu and Zn however, increased significantly on the 4th and the 8th day of exposure. The simultaneous administration of Se reduced the Cd-induced inhibition of these enzymes and the increased levels of Cu on the 4th and the 8th day and of Zn only at the 4th day. The renal Cd, on the other hand, increased from the 4th day to the 8th of the experiment and this increase
GOT
o
GPT C_d Q
u
E
7-1p
Fsl i °
1
=
DAY &
C d+S_e S_ee
.... ft...
II-HI
Fig. 2. The effect of Cd, C d + Se or Se administration on the serum activitiesof G O T and G P T in rats (mean ± S.E. of 5---6 values), ap < 0.001, bp < 0.01, cp < 0.05 w h e n compared to control (mean + S.E. of 9 values shown by horizontal line) * * * P < 0.001, * * P < 0.01, *P < 0.05 w h e n compared to Cd-treated group as evaluated by the Student's t-test.
13.7 0.34 28.1 12.9 26.9
+ ± ± ± ±
0.67 a ( 4 ) 0.03 a ( 6 ) 3.06a(4) 0.675(5) 0.59 a ( 6 )
21.1 0.54 41.1 22.1 25.4
± ± + + ÷
1.01 (5) 0.02 (5) 6.51a(5) 1.26 a ( 5 ) 0.825 (6)
21.6± 0.42 ± 28.6 ± 8.2 ± 19.6 ±
0 . 7 0 * * * (6) 0.02b,* (6) 2.22a(6) 0 . 5 7 * * (6) 1 . 4 1 " * (6)
Day 4
Day 4
Day 8
Cd + Se
Cd
0.52 (6) 0.02 c (6) 0.14 (4) 1.54 (5) 2.23 c (5)
GOTt GPT ~ Cd~: Cu$ Zn-J:
19.1 20.2 3.1 5.7 36.7
+ 1.81 + 1.09 + 0.56 * 0.37 ÷ 2.31
Control
(9) (8) (7) (7) (8)
5.9 14.1 90.7 6.3 73.8
+ 0.45 a (5) ÷ 0.975 (4) +- 6.31 a (6) + 0.48 (6) ÷ 6.425 (6)
13.9 13.5 63.5 7.0 91.0
+ 0.78 a (5) + 1.22 c (5) ± 11.52 a (4) ~ 0.74 (5) ÷ 7.03 a (6)
8.3 19.3 65.5 6.4 46.0
± ± ± ± *
0.59 a,* (5) 0.52* (5) 2.54 a,* (5) 0.57 (6) 3.92* (5)
Day 4
Day 4
Day 8
Cd + Se
Cd
+ ± + ± +
20.0 25.5 1.6 6.4 33.2
0.90 a (5) 0.46 a ( 5 ) 2.54 a (5) 0.175 (5) 5.45 a (5)
14.7 13.8 99.6 8.0 97.2
± ± + + ±
Day 4
Day 8
Se
0.93 (5) 2.06 c (5) 0.20 (5) 1.08 (5) 3.05 (5)
17.4 19.2 1.7 4.9 38.5
-+ 1.26 + 1.21 ± 0.19 + 0.28 ± 2.20
Day 8 (6) (6) (6) (6) (5)
t n m o l h y d r a z o n e f o r m e d l m i n / m g p r o t e i n ; $ug/g fresh tissue. Each figure is m e a n ± S.E. o f n u m b e r o f rats given in parenthesis~ ap < 0.001, 5 p < 0.01, cp < 0.05 c o m p a r e d t o c o n t r o l , * P < 0.05 c o m p a r e d t o Cd g r o u p as evaluated b y S t u d e n t ' s t-test.
+- 1 . 5 6 ( 6 ) ± 0.03 (6) + 0.15(6) ± 0.68 (6) ± 0.80a(6)
19.1 0.54 1.4 8.9 27.9 + ± ± ± ±
21.2 0.47 2.0 8.9 27.8
0.52 (5) 0.23 (5) 0.50 a ( 4 ) 0 . 5 8 * * * (5) 2.40 c (5)
20.8 0.64 53.7 9.6 28.8
± ± ± + +
Day 8
Day 4
Day 8
Se
E F F E C T OF S E L E N I U M ON C A D M I U M - I N D U C E D C H A N G E S IN H E P A T I C E N Z Y M E S A N D M E T A L C O N T E N T S
T A B L E II
GOT t / 2 2 . 8 ± 0 . 3 2 ( 9 ) ALP $ ~ 0.56 ± 0.02 (9) Cd § 1.2 + 0 . 2 7 ( 9 ) Cu § 7.9 ÷ 0.43 (8) Zn § 21.7 ± 0.72 (6)
Control
t n m o l h y d r a z o n e f o r m e d / r a i n / r a g p r o t e i n , S u m o l p - n i t r o p h e n o l l i b . ] m i n / m g p r o t e i n ; § ug]g fresh tissue. E a c h figure is m e a n ± S.E. o f n u m b e r o f rats given in p a r e n t h e s i s ; a p < 0.001, 5 p < 0.01, c p < 0.05 c o m p a r e d to c o n t r o l ; * * * P < 0.001, * * P < 0.01, *P < 0.05 c o m p a r e d t o Cd g r o u p as evaluated by t h e S t u d e n t ' s t-test.
E F F E C T OF S E L E N I U M ON C A D M I U M - I N D U C E D C H A N G E S IN R E N A L E N Z Y M E S A N D M E T A L C O N T E N T S
TABLE I
O0
349 was unaffected by the treatment with Se. The treatment with Se alone however, increased the renal level of Zn significantly (Table I). The activities of serum GOT and GPT increased significantly on exposure to Cd suggesting liver damage. However, hepatotoxicity of Se cannot be suggested with any certainty on the basis of an increase in the activity of only serum GOT on the 8th day. Cadmium-induced increases were less marked in animals treated with Se simultaneously (Fig. 2). The activities of GOT and GPT decreased and the levels of Cd and Zn increased in liver significantly after the administration of Cd (Table II). The concomitant administration of Se prevented these alterations only at the 4th day. However, at the 8th day the activities of both the enzymes remained inhibited and the levels of Cd, Cu and Zn remained significantly higher in animals supplemented with Se indicative of no such protection against the toxic effects of Cd (Table II). DISCUSSION Enzymuria and proteinuria observed in Cd administered rats are the early signs of Cd nephrotoxicity and precede functional disturbances [21,22] (Fig. 1}. Concomitantly, the activities of the renal GOT and ALP decreased initially after Cd administration. The kidney damage being closely related to the induction of metallothionein synthesis by Cd, the uptake and/or retention of Cu and Zn also increased as expected with the progressive accummulation of Cd in the renal tissue [23] (Table I). The increase in the activities of GOT and GPT in serum and decrease in liver on Cd administration are the indices of Cd hepatotoxicity [24] (Fig. 2, Table II). Enzymatic and metabolic alterations in liver and kidney have been observed in rats exposed to Cd [3,6]. The inital uptake of Cd by liver has been far more than that by kidney. However, it decreased subsequently in liver and increased in kidney, the latter being the target organ. The increase in renal and hepatic Zn in Cd administered animals might be related to the intervening effect of Cd in absorption and disposition rate of Zn [4,25] or the induction of metallothionein by Cd [23,26]. The hepatic level of Cu did not increase significantly on exposure to Cd in this study (Table II). Some investigators reported a decrease [4,14], while others observed an increase in the hepatic Cu on Cd administration [27,28]. However, an excessive increase of Zn in liver and of Cu in kidney on exposure to Cd has been well established in the rat [27--29]. In contrast to Cd, the administration of Se had no effect on most of these parameters, except slight proteinuria and an increase in renal content of Zn indicative of some renotoxicity of Se. However, administration of Se concurrently with Cd significantly reduced the Cd induced enzymuria, proteinuria and elevation of serum enzymes. Selenium also ameliorated the inhibition in the activities of renal and hepatic enzymes observed on the 4th day of Cd administration. The increase in the uptake of renal Cu at the 4th a n d the 8th day and of renal and hepatic Zn on the 4th day of Cd
350 administration was also prevented by Se. Interestingly, the accummulation of Cd in kidney and the increase of Zn in liver and kidney observed on the 8th day of Cd administration could not be checked by Se given simultaneously. Furthermore, hepatic uptake of Cd was successfully prevented by Se on the 4th day. However, the level of hepatic Cd and Cu increased from the 4th to the 8th day on simultaneous administration of Se. These observations clearly suggest an initial protection against Cd toxicity by Se, possibly when the tissue levels of Se are high. Selenium may compete with Cd for binding with the functional biological ligands or the target tissue sites [10,30]. Alternatively, Cd and Se might interact with the reversible formation of cadmium selenide (CdSe), thus reducing the 'free' concentration of toxic Cd 2+ ions in the body [30,31]. Subsequently, CdSe is dissociated and the toxic effects of Cd are observed. The present results thus indicate that nephrotoxicity and hepatotoxicity of Cd could be reduced immediately following Se administration, however, later when the body or tissue level of Se decline, the toxic effects of Cd appear, particularly the hepatotoxicity. ACKNOWLEDGEMENTS
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