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Exogenous metallothionein and renal toxicity of cadmium and mercury in rats
Toxicology, 76 (1992) 15-26 Elsevier Scientific Publishers Ireland Ltd.
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Exogenous metallothionein and renal toxicity of cadmium and mercury in rats H i n g M a n C h a n a, M a s a h i k o S a t o h a, R u d o l f s K. Z a l u p s b a n d M. G e o r g e C h e r i a n a aDepartment of Pathology, The University of Western Ontario, London, Ontario (Canada) and bDivision of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207 (USA) (Received April 3rd, 1992; accepted July 20th, 1992)
Summary The relative tissue distribution and toxicity of cadmium (Cd) and mercury (Hg) in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT were studied. Male Spragne-Dawley rats were injected (i.v.) with Cd or Hg inorganic salt of chloride or in a complex of MT at a dose of 0.3 mg/kg body weight. The concentration of MT and metals in plasma and urine was monitored for 7 days, at the end of which the rats were killed. Injection of both HgCI 2 and Hg-MT induced the synthesis of MT only in the kidney but not in the liver, whereas CdCI 2 and Cd-MT injections induced MT synthesis in both liver and kidney, respectively. Plasma MT levels increased 3 days after CdC12 but not after HgCI 2 injection, suggesting that hepatic MT may be an important source of plasma MT under our experimental conditions. Renal toxicity was observed morphologically and by an increase in blood urea nitrogen, plasma creatinine, proteinuria in rats injected with Cd-MT and both forms of Hg. Urinary MT excretion was significantly elevated in Cd-MT injected rats compared with those injected with CdCI 2. However, HgCI 2 and Hg-MT injected rats showed no significant difference in urinary MT excretion. The magnitude in the renal accumulation of Hg is similar after the administration of Hg-MT or HgCI 2, but our findings suggest that the site of epithelial injury may be different. Injury effects of HgMT localized mainly in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule whereas inorganic Hg caused necrosis in pars recta segments of the proximal tubule.
Key words: Metallothionein; Cadmium; Mercury; Plasma; Urine
Introduction Metallothionein (MT), a low molecular weight metal-binding protein, sequesters cadmium (Cd) intracellularly and thereby provides a protective effect against the acute toxicity of Cd [1-3]. In contrast, extracellular cadmium-metallothionein (Cd-MT) has been found to be nephrotoxic [4]. Parenteral administration of Cd as Cd-MT results in the selective accumulation and toxicity in the proximal convoluted Correspondence to." M.G. Cherian, Department of Pathology, The University of Western Ontario, London, Ontario N6A 5C1, Canada. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
16 tubules of the kidney [5]. Cellular injury and necrosis occur in this segment of the nephron at much lower doses of Cd in the form of Cd-MT than in the ionic form [6,7]. The toxicity of exogenous MT is proportional to the Cd:zinc (Zn) ratio and increases as the percentage of Cd is increased. Zn-metallothionein is not toxic [8]. The level of MT in the plasma of blood has been found to correlate with the level of Cd exposure [9-11]. Plasma MT is also believed to be responsible for the redistribution of Cd from the liver to the kidney [12]. The main site of accumulation of inorganic mercury (Hg) in mammals is in the cortex and outer medulla of the kidney [13]. The metal accumulates along the entire length of the proximal tubule, however, the toxic effects of the metal are expressed primarily in the pars recta or straight portion of the proximal tubule [14-16]. Induction of renal synthesis of MT has been shown to have a protective effect against the nephrotoxicity of inorganic Hg but little is known on the metabolism and toxicity of exogenous Hg-MT [17,18]. Urinary excretion of MT increases after chronic exposure to Cd and is shown to be a good indicator of the severity of the nephropathy induced by Cd [19,201. The concentration of MT in the urine has also been shown to increase after injections of Hg [21]. The aim of the present study is to investigate and compare the relative tissue distribution and toxicity of Cd and Hg in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT. Materials and methods
Chemicals 2°3HGC12 (spec. act. 1.8 mCi/mg Hg) was obtained from Buffalo Materials Corporation (Buffalo, New York). All other chemicals were of reagent grade from BDH Inc. (Toronto, Ontario).
Preparation of Cd-MT and Hg-MT Cd-MT was isolated from livers of male Sprague-Dawley rats that had been injected i.p. with CdCI2 (1 mg Cd/kg) daily for 2 weeks. The purification of MT-1 and MT-2 was similar to that described previously [22]. Briefly, liver samples were homogenized in a 50-mM sodium phosphate buffer (pH 7.4) and following centrifugation at 12 000 x g for 10 min at 4°C, the supernatants were heated at 80°C for 2 min. The denatured proteins were removed by centrifugation at 27 000 x g for 10 min (4°C) and the supernatant was applied to a Sephadex G-75 column (5 x 90 cm). Fractions of molecular weight 10 000 containing Cd were pooled and lyophilized. The two MT isoforms were separated by polyacrylamide gel electrophoresis (PAGE) in a 7.5% gel under non-denaturing conditions, eluted from the gel and lyophilized. Only the isoform Cd-MT-2 was used in the present study. Cd/Zn (w/w) ratio in the MT preparation was 14. Hg-MT was prepared by adding HgCI2 to Apo-MT. Apo-MT was prepared as described previously [23]. Rats were injected with ZnSO4 (20 mg/kg s.c.) for 5 days. Zn-MT-2 was prepared as described earlier [22]. Zn was removed from Zn-MT-2 by gel filtration chromatography using a Sephadex G-25 column (5 x 45 cm)
17 equilibrated with 0.01 N HCI. Hg-MT was prepared by dialysing about 50 mg of Apo-MT overnight at 4°C against phosphate buffered saline (0.05 M sodium phosphate, 0.15 M NaC1, pH 7.4, PBS) after addition of 10 mg of Hg as HgC12 spiked with 1.0/~Ci 2°3Hg/mg Hg. Animal treatments Male Sprague-Dawley rats (300-350 g) obtained from Charles River (Montreal, Quebec) were housed in individual plastic Nalgene metabolic cages under controlled temperature conditions and 12-h light and dark cycles. Rats were given water and a standard rat chow ad libitum. Five groups of 6 rats were given the following injections (i.v.): (a) 0.3 ml saline (0.9%) (control); (b) 0.3 mg Cd/kg body weight as CdCI2; (c) 0.3 mg Cd/kg as CdMT; (d) 0.3 mg Hg/kg as HgCI 2 (labelled with 1.0 #Ci 2°3Hg/mg Hg); and (e) 0.3 mg Hg/kg as Hg-MT (labelled with 1.0 /~Ci 2°3Hg/mg Hg). A 0.9% saline solution (0.3 ml) was used as vehicle for all Cd and Hg injections. Two rats from each group were killed 24 h after the injections and their kidneys were excised for morphological analysis. The other four rats were kept under the experimental conditions and urine samples were collected every 24 h for 7 days. Approximately 0.5 ml heparinized plasma was collected from each rat by venipuncture of the tail vein 2, 24, 48, 72, 96 and 168 h after injection. Plasma samples were prepared by centrifugation at 2000 × g for 10 min at 4°C. Rats were killed by decapitation 7 days after injection. Livers and kidneys were processed for metal and MT analysis. Samples of kidney were fixed in 10% neutral buffered formaldehyde for 48 h and then processed for standard light microscopy. Sections (5/~m) of paraffin-embedded tissue were mounted on glass slides, stained with hematoxylin and eosin and then examined with a light microscope. Renal injury was assessed primarily by the presence or absence of indicators of cellular necrosis (i.e. regenerating epithelial cells and remnants of necrotic epithelial cells in the lumen of tubules). Chemical analysis The Cd content of tissues, plasma and urine was determined by atomic absorption spectroscopy (Varian Spectra-30, Varian Canada, Georgetown, Ontario) using either flame (air-acetylene) or graphite furnace depending on Cd concentrations. Hg content of the samples was measured by counting 2°3Hg in an LKB 1270 Rackgamma II gamma-counter (Pharmacia Inc., Baie d'Urfe, Quebec) with a counting efficiency of 70%. MT concentrations in plasma and urine were measured in triplicate by a competitive enzyme-linked immunosorbent assay (ELISA) [24]. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer which cross-reacts with both MT-1 and MT-2, a biotinylated secondary antibody and peroxidase conjugated avidin. The detection limit is about 100 pg MT. Coefficients of variation for reproducibility within and between assays are generally less than 5% and 10%, respectively. Blood urea nitrogen (BUN) in plasma samples were measured using a Sigma Diagnostics Kit (Sigma Chem. Co., St. Louis, MO). Protein concentration in urine was measured by the method of Lowry et al. [25]. Creatinine in plasma and urine were measured colorimetrically by Jaffe reaction using the method of Heinegard and Tiderstrom [26].
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Statistical analysis The data were analyzed by one way analysis of variance (ANOVA) and means of different groups were compared by Student-Newman-Keuls test according to Sokal and Rohlf [27]. Five percent was considered as the level of significance. Results
The concentrations of Cd, Hg and MT in the rat liver and kidney 7 days after the injection are shown in Table I. Significantly higher Cd concentrations were found in the liver of rats injected with CdCI 2 than those injected with an equivalent dose of Cd-MT. However, Cd-MT injected rats had significantly higher Cd accumulation in the kidney than the CdCI2 injected rats. In contrast, no significant difference was observed in the concentrations of Hg in both liver and kidney between the rats injected with same dose of Hg as HgCI2 or Hg-MT, although the Hg concentration in the kidney was higher than that in the liver. In plasma of control rats, the concentration of MT was about 30 ng ml -l (Fig. 1). Plasma MT concentrations of rats injected with CdCI2 increased significantly at about 2 days after injection and remained high for the following 5 days. Rats injected with inorganic Hg, which accumulated mainly in the kidney, showed no significant increase in plasma MT concentrations, when compared with the control. As expected, rats injected with Cd-MT exhibited significantly elevated plasma MT concentrations at the earliest time period measured after injection. It decreased significantly but remained higher than that of the control for the next 2 days and then decreased to the control level after 3 days. In contrast, plasma MT concentra-
TABLE I C O N C E N T R A T I O N S OF METALS (Cd A N D Hg) A N D MT IN RAT LIVER A N D K I D N E Y F O L L O W I N G INJECTION OF Cd A N D Hg COMPOUNDS Treatment a
Control CdCI 2 Cd-MT HgCI 2 Hg-MT
Cd conc.b (ttg/g tissue)
Hg conc. b (/~g/g tissue)
MT conc. b (/~g/g tissue)
Liver
Kidney
Liver
Kidney
Liver
ND 7.9 ± 2.1 c 0.6 ± 0.2 c,d ND ND
ND 1.9 ± 0.3 11 ± 3.2 a ND ND
ND ND ND 0.7 ± 0.2 c 0.5 ± 0.2 c
ND ND ND 16 ± 5 18 ± 4
11 109 61 89 55
+ ± ± ± 4-
Kidney 3c 15 c 32 c 36 c 11 c
23 39 202 208 290
± + ± ± ±
5 11 38 31 43 e
aRats were injected i.v. with saline, 0.3 mg Cd or Hg per kg in the form of CdCI2, Cd-MT, HgCI 2 or HgMT and killed 7 days after injection. b Values represent mean ± S.D. for 4 animals. c Significantly different from those in the kidney (P < 0.05). d Significantly different from those injected with CdCI 2 (P < 0.05). e Significantly different from those injected with HgC12 (P<0.05). N D - - values below detection limit (0.1 #g/g) or not determined.
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t i o n o f the H g - M T i n j e c t e d rats was significantly h i g h e r t h a n t h a t o f the c o n t r o l o n l y at 2 h after the injection. B l o o d u r e a n i t r o g e n ( B U N ) in rats injected w i t h C d - M T a n d b o t h f o r m s o f H g s h o w e d a s i g n i f i c a n t e l e v a t i o n 24 h after the i n j e c t i o n a n d r e t u r n e d to n o r m a l after 48 h (Fig. 2). A t 24 h, there was n o s i g n i f i c a n t difference in B U N b e t w e e n the 50
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20 HgCl2 and Hg-MT rats, but both of them were significantly higher than that of the Cd-MT injected rats. Similar patterns were observed for the levels of plasma creatinine (Fig. 3). Rats injected with Cd-MT and both forms of Hg showed significantly higher plasma creatinine than that of control and rats injected with CdCl 2 2 days after the injections. Rats injected with both forms of Hg had similar plasma creatinine levels. However, the levels were both significantly higher than that of Cd-MT at 24 h, at the dose level used. Volume of urine excreted by all rats was quite uniform and averaged 20-30 ml/day. Mean MT concentration in urine from control rats and rats injected with CdC12 were not significantly different and ranged from 30-150 ng/day (Fig. 4). Urinary excretion of MT in Cd-MT injected rats was elevated during the first 24 h, it then decreased with time but remained significantly higher than that of the control and CdC12 injected rats. Urinary MT was significantly higher in both HgCl 2 and Hg-MT injected rats than the control for the first 3 days after injection. No significant difference was observed in urinary MT levels between the two Hg injected groups of rats. Urinary Cd in the Cd-MT injected rats was significantly higher than that of the control for 4 days after injection (Fig. 5). Injected CdCI2, on the other hand, did not cause any increase in the urinary excretion of Cd. Significantly more Hg was excreted in the urine by the HgCl 2 injected rats than the Hg-MT injected rats. Total protein concentration in the urine of rats injected with Cd-MT significantly increased during 24 h after injection and remained higher than those of the control and CdCl 2 injected rats for 72 h (Fig. 6). Similarly, urinary protein concentrations of rats injected with both forms of Hg also showed a significant elevation during 24 h after the injection and returned to normal after 48 h. There was no significant
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difference in urinary protein concentrations between the Hg injected and the Cd-MT injected rats. Results of morphological analysis of kidney by light microscopy are summarized in Table II. A mild level of renal injury occurred in the rats injected with 0.3 mg/kg dose of HgC12 at 24 h after the injection. The injury consisted of necrosis in isolated pars recta segments of the proximal tubule located at the cortico-medullary junction. Renal injury also occurred at 24 h after injection of Hg-MT and consisted of cellular necrosis that appeared to be localized in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule situated at the cortico-medullary junction. In this case, the level of renal injury appeared to be greater than that in the rats injected with HgC12. Injection of 0.3 mg Cd/kg as CdMT to rats induced a moderately severe nephropathy at 24 h. It was characterized by necrosis in proximal convoluted tubules throughout the cortex. No apparent renal injury was observed in rats 24 h after injection of a similar dose of Cd as CdCI 2. The sections of kidney from the rats injected with HgC12 revealed that epithelial regeneration had occurred in the pars recta of proximal tubules in the outer stripe of the outer medulla and medullary rays of the cortex in 7 days after the injection. In contrast, no obvious signs of cellular or tubular necrosis or regeneration were found in the pars recta of proximal tubules in sections of kidney from the rats treated 7 days prior to sacrifice with Hg-MT. There were, however, a few sets of proximal convoluted tubules that displayed a rudimentary epithelium, which is indicative of cellular regeneration. Epithelial regeneration had occurred in most of the proximal convoluted tubules 7 days after the injection of Cd-MT indicating that cellular
24 h 7 days 24 h 7 days 24 h 7 days 24 h 7 days
CdCI 2
No No Yes Yes Yes Yes Yes Yes
Evidence of Cellular necrosis
--Cortex Cortex CorterdOM Cortex + OS Cortex/OM Cortex/OM
Zonal location of cellular necrosis --PCT PCT PST PCT PCT/PST PCT/PST
Tubular site of cellular necrosis
--Moderate Moderate Mild Mild Mild-Mod. Mild-Mod.
Level of severity of cellular necrosis No No No Yes No Yes No Yes
Evidence of epithelial regeneration
---Cortex -Cortex + OM -Cortex
Zonal localization of epithelial regeneration ---PCT -PST -PCT/PST
Tubular site of epithelial regeneration
---Almost complete -Almost complete -Almost complete
Stage o f epithelial regeneration
Cortex/OM, junction of the cortex and outer medulla; OS, outer stripe of the outer medulla; PCT, proximal convoluted tubules; PST, proximal straight tubules; PCT/PST, involving terminal portions of the proximal convoluted tubules and the initial portions of the proximal straight tubules.
Hg-MT
HgCI2
Cd-MT
Time
Treatment
HISTOLOGICAL ASSESSMENT OF K I D N E Y S
TABLE II
24 necrosis occurred in this part of the proximal tubule soon after the injection. Remnants of epithelial cells that had undergone necrosis were observed in the lumen of some of the tubules. In addition, pyknotic nuclei were present in some of the epithelial cells indicating that they were in the process of dying. No obvious signs of cellular injury were observed in the kidney of rats injected with CdCI2. Discussion
The relative tissue distribution and toxicity of cadmium chloride (CdCI2), cadmium-metallothionein (Cd-MT), mercury chloride (HgC12) and mercurymetallothionein (Hg-MT) are reported in this study. Concentrations of Cd were higher in the kidney and lower in the liver in rats injected with Cd-MT compared with those of rats injected with CdCI2 and were similar to previous findings [6,7]. In contrast, Hg selectively accumulated in the kidney regardless of the injected form. The reason for the difference between Cd and Hg tissue distribution after injection of CdC12 and HgCI2 is not known, but may be related to the binding affinity of these metals to carrier proteins in the blood [11]. Moreover, Gerson and Shaikh [28] found that liver cells in vitro accumulate more Cd than Hg when incubated with corresponding salts. The selective accumulation of both Cd-MT and Hg-MT in the kidney may be because the low molecular weight MT is easily filtered and reabsorbed in the kidney. Induction of MT synthesis by metals has been extensively studied. Accumulation of Hg in rat kidney after injection of HgCI 2 increased renal MT concentrations, particularly in the cortex and outer medulla [17]. Rats injected with Hg-MT showed higher renal MT concentration than those injected with HgCI2. Part of this increased MT may result from the uptake of injected intact Hg-MT, which contained about 200 #g MT. Nevertheless, the induction of MT synthesis in the kidney may still be a major factor for the increase in renal MT concentrations and this may have a protective effect on Hg nephrotoxicity, although the mechanism is still unknown [18]. The increase in plasma MT concentrations in rats injected with CdCI2 and the lack of increase in plasma MT concentrations of rats after the injection of HgCI2 suggest that the increased plasma MT in the CdCI2 injected rats may originate from the liver. These results agree with the generally accepted hypothesis that Cd-MT synthesized in the liver may be released and transported in the blood to the kidney and at high doses, may cause renal injury [29,30]. Rats injected with both Cd-MT and Hg-MT showed an increase in plasma MT concentrations and the clearance rate of Hg-MT appeared to be higher than that of Cd-MT. However, since 0.3 mg of either Cd or Hg were injected in each rat, assuming that the molar ratio 7 for both Cd and Hg for binding to MT, the amount of MT injected in the case of Hg would be twice that of Cd because the difference in their atomic weight. Therefore, the clearance rate between Cd-MT and Hg-MT may not be directly comparable. It has been shown that injected Cd-MT is transported in the plasma to the kidney, where it is freely filtered by the glomerulus and reabsorbed by proximal convoluted tubules [31,32]. The high urinary MT level observed in the first day may be due to incomplete reabsorption of the injected Cd-MT by the tubular cells [33]. Injection of both Hg-MT and HgC12 caused a similar level of increase in urinary excretion of MT suggesting
25 that the nephrotoxic effects of the two forms of Hg may be similar. This also supported the view that urinary MT can be used as a marker for renal toxicity of Cd and Hg. Injection of Cd-MT and both forms of Hg resulted in nephrotoxicity as shown by the increase in blood urea nitrogen, total urinary protein concentrations and urinary MT excretion. Histopathological results also show that injection of Cd-MT caused cellular injury in the $1 segment, whereas HgC12 caused damage in the $2 and $3 segments of the proximal tubule, confirming previous reports [5,7]. It is interesting that Hg-MT seems to cause cellular damage in the convoluted tubules which resembles the toxicity of Cd-MT and not that of HgCI2. Even though injections of both forms of Hg caused increase in BUN, plasma creatinine, urinary protein and MT, higher level of urinary Hg excretion was observed after injection of HgCI2. These results indicate that the two forms of Hg are taken up by different mechanisms. In previous studies [6,7], the direct comparison of toxicities of metal compounds such as CdC12 and Cd-MT has been difficult because of the differences in tissue distribution of metals. However, in the present study, Hg from both HgC12 and HgMT was deposited mainly in the kidney and this similar renal accumulation of Hg enabled us to compare their toxicities in the kidney. Results of this biochemical and preliminary morphological study suggest that although both forms of Hg are nephrotoxic, their renal uptake mechanisms and site of toxicity in the kidney may be different. Implications of these differences require further investigation.
Reference 1 J.H.R.K~giand B.L. Vallee, Metallothionein: a cadmium-and zinc-containingprotein from equine renal cortex. J. Biol. Chem., 235 (1960) 3460. 2 P.L. Goering and C.D. Klaassen, Tolerance to cadmium-inducedhepatoxicityfollowingcadmium pretreatment. Toxicol. Appl. Pharmacol., 24 (1984) 308. 3 W.S. Din and J.M. Frazier, Protective effect of metallothionein on cadmium toxicity in isolated rat hepatocytes. Biochem. J., 230 (1985) 395. 4 M. Webb, Toxicological significance of metallothionein, in J.H.R. K/igi and Y. Kojima (Eds.), MetaUothionein II, Birkhauser Verlag, Basel, 1987, p. 109. 5 B.A.Fowlerand G.F. Nordberg, The renal toxicityof cadmiummetallothionien: morphometricand x-rays micro-analytical studies. Toxicol. Appl. Pharmacol., 46 (1978) 609. 6 G.F. Nordberg, R.A. Goyer and M. Nordberg, Comparative toxicity of cadmium-metallothionein and cadmium chloride on mouse kidney. Arch. Pathol., 99 (1975) 192. 7 M.G. Cherian, R.A. Goyer and L. Delaquerriere-Richardson, Cadmium metallothionein induced nephropathy. Toxicol. Appl. Pharmacol., 38 (1976) 399. 8 K.T.Suzuki, S. Takenata and K. Kubota, Fate and apparent toxicityof MT with different cadmium and zinc ratios in rat kidney. Arch. Environ. Contam. Toxicol., 8 (1979) 85. 9 J.S. Garveyand C.C. Chang, Detection of circulating metallothionein in rats injected with zinc or cadmium. Science, 214 (1981) 805. 10 C. Tohyamaand Z.A. Shaikh, Metattothionein in plasma and urine of cadmium-exposedrats determined by a single-antibodyradioimmunoassay. Fund. Appl. Toxicol., 1 (1981) 1-7. 11 R.K. Mehra and I. Brenmer, Development of a radioimmunoassayfor rat liver metallothionein-I and its application to the analysis of rat plasma and kidneys. Biochem. J., 213 (1983) 459. 12 J.M. Frazier, The role of metallothionein in the system distribution of cadmium, in E.C. Foulkes (Ed.), Biological Roles of Metallothionein, Elsevier, New York, 1982, p. 141.
26 13 14 15 16
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R.K. Zalups and G.L. Diamond, Intrarenal distribution of mercury in the rats: Effect of administered dose of mercuric chloride. Bull. Environ. Contam. Toxicol., 38 (1987) 67. R.K. Zalups and D.W. Barfuss, Accumulation of inorganic mercury along the renal proximal tubule of the rabbit. Toxicol. Appl. Pharmacol., 106 (1990) 245. T.L. Gritzka and B.F. Trump, Renal tubular leisons caused by mercuric chloride: Electron microscopic observation: degeneration of the pars recta. Am. J. Pathol., 52 (1968) 1225. E.M. McDowell, R.B. Nagle, R.C. Zalme, J.S. McNeil, W. Flamenbaum, B.F. Trump, Studies on the pathophysiology of acute renal failure. I. Correlation of ultrastructure and function in the proximal tubule of the rat following administration of mercuric chloride. Virchows. Arch. [Cell Pathol.], 22 (1976) 173. R.K. Zalups and M.G. Cherian, Renal metallothionein after a reduction of renal mass. I. Effect of unilateral nephrectomy and compensatory renal growth on basal and metal-induced renal metallothionein metabolism. Toxicology, 71 (1992) 83. R.K. Zalups and M.G. Cherian, Renal metallothionein after a reduction of renal mass. I. Effect of zinc pretreatment on the renal toxicity and intrarenal accumulation of inorganic mercury. Toxicology, 71 (1992) 103. C. Tohyama, Y. Mitane, E. Kobayashi, N. Sugihira, A. Nakano and H. Saito, The relationship of urinary metallothionein with other indicators of renal dysfunction in people living in a cadmiumpolluted area in Japan. J. Appl. Toxicol., 8 (1988) 15. Z.A. Shaikh, K.J. Ellis, K.S. Subramanian and A. Greenberg, Biological monitoring for occupational cadmium exposure: the urinary metallothionein. Toxicology, 63 (1990) 53. Y. Mitane and C. Tohyama, Urinary excretion of metallothionein in cadmium- and mercury-treated rats. Proc. Asian-Pac. Symp. Environ. Occup. Toxicol., 8 (1988) 163. D.M. Templeton and M.G. Cherian, Chemical modification of metallothionein. Biochem. J., 221 (1984) 569. M.J. Stillman, W. Cai and A.J. Zelazowski, Cadmium binding to metallothioneins. J. Biol. Chem., 262 (1987) 4538. H.M. Chan, M. Satoh, G.A. Pringle and M.G. Cherian, Quantification ofmetallothionein in tissues and biological fluids by an enzyme-linked immunosorbent assay (ELISA). Toxicologist, 12 (1992) 141. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265. D. Heinegard and G. Tiderstrom, Determination of serum creatinine by a direct colorimetric method. Clin. Chim. Acta, 43 (1973) 305. R.R. Sokal and F.J. Rolf, Biometry, W.H. Freeman & Co., San Francisco, 1969. R.J. Gerson and Z.A. Shaikh, Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures. Biochem. J., 208 (1982) 465. R.E. Dudley, L.M. Gammal and C.D. Klaassen, Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol., 77 (1985) 414. I. Brenmer, Nutritional and physiological significance of metallothionein, in J.H.R. K/igi and Y. Kojima (Eds.), Metallothionein II, Birkhauser Verlag, Basel, 1987, p. 81. E.C. Foulkes, Renal tubular transport of cadmium-metallothionein. Toxicol. Appl. Pharmacol., 45 (1978) 505. M.G. Cherian, Studies on toxicity of metaUothionein in rat kidney epithelial cell culture, in E.C. Foulkes (Ed.), Biological Roles of Metallothionein, Elsevier, Amsterdam, 1982, p. 193. T. Jin, P. Leffler and G.F. Nordberg, Cadmium-metallothionein in nephrotoxicity in the rat: transient calcuria and proteinuria. Toxicology, 45 (1987) 307.
15
Exogenous metallothionein and renal toxicity of cadmium and mercury in rats H i n g M a n C h a n a, M a s a h i k o S a t o h a, R u d o l f s K. Z a l u p s b a n d M. G e o r g e C h e r i a n a aDepartment of Pathology, The University of Western Ontario, London, Ontario (Canada) and bDivision of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207 (USA) (Received April 3rd, 1992; accepted July 20th, 1992)
Summary The relative tissue distribution and toxicity of cadmium (Cd) and mercury (Hg) in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT were studied. Male Spragne-Dawley rats were injected (i.v.) with Cd or Hg inorganic salt of chloride or in a complex of MT at a dose of 0.3 mg/kg body weight. The concentration of MT and metals in plasma and urine was monitored for 7 days, at the end of which the rats were killed. Injection of both HgCI 2 and Hg-MT induced the synthesis of MT only in the kidney but not in the liver, whereas CdCI 2 and Cd-MT injections induced MT synthesis in both liver and kidney, respectively. Plasma MT levels increased 3 days after CdC12 but not after HgCI 2 injection, suggesting that hepatic MT may be an important source of plasma MT under our experimental conditions. Renal toxicity was observed morphologically and by an increase in blood urea nitrogen, plasma creatinine, proteinuria in rats injected with Cd-MT and both forms of Hg. Urinary MT excretion was significantly elevated in Cd-MT injected rats compared with those injected with CdCI 2. However, HgCI 2 and Hg-MT injected rats showed no significant difference in urinary MT excretion. The magnitude in the renal accumulation of Hg is similar after the administration of Hg-MT or HgCI 2, but our findings suggest that the site of epithelial injury may be different. Injury effects of HgMT localized mainly in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule whereas inorganic Hg caused necrosis in pars recta segments of the proximal tubule.
Key words: Metallothionein; Cadmium; Mercury; Plasma; Urine
Introduction Metallothionein (MT), a low molecular weight metal-binding protein, sequesters cadmium (Cd) intracellularly and thereby provides a protective effect against the acute toxicity of Cd [1-3]. In contrast, extracellular cadmium-metallothionein (Cd-MT) has been found to be nephrotoxic [4]. Parenteral administration of Cd as Cd-MT results in the selective accumulation and toxicity in the proximal convoluted Correspondence to." M.G. Cherian, Department of Pathology, The University of Western Ontario, London, Ontario N6A 5C1, Canada. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
16 tubules of the kidney [5]. Cellular injury and necrosis occur in this segment of the nephron at much lower doses of Cd in the form of Cd-MT than in the ionic form [6,7]. The toxicity of exogenous MT is proportional to the Cd:zinc (Zn) ratio and increases as the percentage of Cd is increased. Zn-metallothionein is not toxic [8]. The level of MT in the plasma of blood has been found to correlate with the level of Cd exposure [9-11]. Plasma MT is also believed to be responsible for the redistribution of Cd from the liver to the kidney [12]. The main site of accumulation of inorganic mercury (Hg) in mammals is in the cortex and outer medulla of the kidney [13]. The metal accumulates along the entire length of the proximal tubule, however, the toxic effects of the metal are expressed primarily in the pars recta or straight portion of the proximal tubule [14-16]. Induction of renal synthesis of MT has been shown to have a protective effect against the nephrotoxicity of inorganic Hg but little is known on the metabolism and toxicity of exogenous Hg-MT [17,18]. Urinary excretion of MT increases after chronic exposure to Cd and is shown to be a good indicator of the severity of the nephropathy induced by Cd [19,201. The concentration of MT in the urine has also been shown to increase after injections of Hg [21]. The aim of the present study is to investigate and compare the relative tissue distribution and toxicity of Cd and Hg in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT. Materials and methods
Chemicals 2°3HGC12 (spec. act. 1.8 mCi/mg Hg) was obtained from Buffalo Materials Corporation (Buffalo, New York). All other chemicals were of reagent grade from BDH Inc. (Toronto, Ontario).
Preparation of Cd-MT and Hg-MT Cd-MT was isolated from livers of male Sprague-Dawley rats that had been injected i.p. with CdCI2 (1 mg Cd/kg) daily for 2 weeks. The purification of MT-1 and MT-2 was similar to that described previously [22]. Briefly, liver samples were homogenized in a 50-mM sodium phosphate buffer (pH 7.4) and following centrifugation at 12 000 x g for 10 min at 4°C, the supernatants were heated at 80°C for 2 min. The denatured proteins were removed by centrifugation at 27 000 x g for 10 min (4°C) and the supernatant was applied to a Sephadex G-75 column (5 x 90 cm). Fractions of molecular weight 10 000 containing Cd were pooled and lyophilized. The two MT isoforms were separated by polyacrylamide gel electrophoresis (PAGE) in a 7.5% gel under non-denaturing conditions, eluted from the gel and lyophilized. Only the isoform Cd-MT-2 was used in the present study. Cd/Zn (w/w) ratio in the MT preparation was 14. Hg-MT was prepared by adding HgCI2 to Apo-MT. Apo-MT was prepared as described previously [23]. Rats were injected with ZnSO4 (20 mg/kg s.c.) for 5 days. Zn-MT-2 was prepared as described earlier [22]. Zn was removed from Zn-MT-2 by gel filtration chromatography using a Sephadex G-25 column (5 x 45 cm)
17 equilibrated with 0.01 N HCI. Hg-MT was prepared by dialysing about 50 mg of Apo-MT overnight at 4°C against phosphate buffered saline (0.05 M sodium phosphate, 0.15 M NaC1, pH 7.4, PBS) after addition of 10 mg of Hg as HgC12 spiked with 1.0/~Ci 2°3Hg/mg Hg. Animal treatments Male Sprague-Dawley rats (300-350 g) obtained from Charles River (Montreal, Quebec) were housed in individual plastic Nalgene metabolic cages under controlled temperature conditions and 12-h light and dark cycles. Rats were given water and a standard rat chow ad libitum. Five groups of 6 rats were given the following injections (i.v.): (a) 0.3 ml saline (0.9%) (control); (b) 0.3 mg Cd/kg body weight as CdCI2; (c) 0.3 mg Cd/kg as CdMT; (d) 0.3 mg Hg/kg as HgCI 2 (labelled with 1.0 #Ci 2°3Hg/mg Hg); and (e) 0.3 mg Hg/kg as Hg-MT (labelled with 1.0 /~Ci 2°3Hg/mg Hg). A 0.9% saline solution (0.3 ml) was used as vehicle for all Cd and Hg injections. Two rats from each group were killed 24 h after the injections and their kidneys were excised for morphological analysis. The other four rats were kept under the experimental conditions and urine samples were collected every 24 h for 7 days. Approximately 0.5 ml heparinized plasma was collected from each rat by venipuncture of the tail vein 2, 24, 48, 72, 96 and 168 h after injection. Plasma samples were prepared by centrifugation at 2000 × g for 10 min at 4°C. Rats were killed by decapitation 7 days after injection. Livers and kidneys were processed for metal and MT analysis. Samples of kidney were fixed in 10% neutral buffered formaldehyde for 48 h and then processed for standard light microscopy. Sections (5/~m) of paraffin-embedded tissue were mounted on glass slides, stained with hematoxylin and eosin and then examined with a light microscope. Renal injury was assessed primarily by the presence or absence of indicators of cellular necrosis (i.e. regenerating epithelial cells and remnants of necrotic epithelial cells in the lumen of tubules). Chemical analysis The Cd content of tissues, plasma and urine was determined by atomic absorption spectroscopy (Varian Spectra-30, Varian Canada, Georgetown, Ontario) using either flame (air-acetylene) or graphite furnace depending on Cd concentrations. Hg content of the samples was measured by counting 2°3Hg in an LKB 1270 Rackgamma II gamma-counter (Pharmacia Inc., Baie d'Urfe, Quebec) with a counting efficiency of 70%. MT concentrations in plasma and urine were measured in triplicate by a competitive enzyme-linked immunosorbent assay (ELISA) [24]. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer which cross-reacts with both MT-1 and MT-2, a biotinylated secondary antibody and peroxidase conjugated avidin. The detection limit is about 100 pg MT. Coefficients of variation for reproducibility within and between assays are generally less than 5% and 10%, respectively. Blood urea nitrogen (BUN) in plasma samples were measured using a Sigma Diagnostics Kit (Sigma Chem. Co., St. Louis, MO). Protein concentration in urine was measured by the method of Lowry et al. [25]. Creatinine in plasma and urine were measured colorimetrically by Jaffe reaction using the method of Heinegard and Tiderstrom [26].
18
Statistical analysis The data were analyzed by one way analysis of variance (ANOVA) and means of different groups were compared by Student-Newman-Keuls test according to Sokal and Rohlf [27]. Five percent was considered as the level of significance. Results
The concentrations of Cd, Hg and MT in the rat liver and kidney 7 days after the injection are shown in Table I. Significantly higher Cd concentrations were found in the liver of rats injected with CdCI 2 than those injected with an equivalent dose of Cd-MT. However, Cd-MT injected rats had significantly higher Cd accumulation in the kidney than the CdCI2 injected rats. In contrast, no significant difference was observed in the concentrations of Hg in both liver and kidney between the rats injected with same dose of Hg as HgCI2 or Hg-MT, although the Hg concentration in the kidney was higher than that in the liver. In plasma of control rats, the concentration of MT was about 30 ng ml -l (Fig. 1). Plasma MT concentrations of rats injected with CdCI2 increased significantly at about 2 days after injection and remained high for the following 5 days. Rats injected with inorganic Hg, which accumulated mainly in the kidney, showed no significant increase in plasma MT concentrations, when compared with the control. As expected, rats injected with Cd-MT exhibited significantly elevated plasma MT concentrations at the earliest time period measured after injection. It decreased significantly but remained higher than that of the control for the next 2 days and then decreased to the control level after 3 days. In contrast, plasma MT concentra-
TABLE I C O N C E N T R A T I O N S OF METALS (Cd A N D Hg) A N D MT IN RAT LIVER A N D K I D N E Y F O L L O W I N G INJECTION OF Cd A N D Hg COMPOUNDS Treatment a
Control CdCI 2 Cd-MT HgCI 2 Hg-MT
Cd conc.b (ttg/g tissue)
Hg conc. b (/~g/g tissue)
MT conc. b (/~g/g tissue)
Liver
Kidney
Liver
Kidney
Liver
ND 7.9 ± 2.1 c 0.6 ± 0.2 c,d ND ND
ND 1.9 ± 0.3 11 ± 3.2 a ND ND
ND ND ND 0.7 ± 0.2 c 0.5 ± 0.2 c
ND ND ND 16 ± 5 18 ± 4
11 109 61 89 55
+ ± ± ± 4-
Kidney 3c 15 c 32 c 36 c 11 c
23 39 202 208 290
± + ± ± ±
5 11 38 31 43 e
aRats were injected i.v. with saline, 0.3 mg Cd or Hg per kg in the form of CdCI2, Cd-MT, HgCI 2 or HgMT and killed 7 days after injection. b Values represent mean ± S.D. for 4 animals. c Significantly different from those in the kidney (P < 0.05). d Significantly different from those injected with CdCI 2 (P < 0.05). e Significantly different from those injected with HgC12 (P<0.05). N D - - values below detection limit (0.1 #g/g) or not determined.
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t i o n o f the H g - M T i n j e c t e d rats was significantly h i g h e r t h a n t h a t o f the c o n t r o l o n l y at 2 h after the injection. B l o o d u r e a n i t r o g e n ( B U N ) in rats injected w i t h C d - M T a n d b o t h f o r m s o f H g s h o w e d a s i g n i f i c a n t e l e v a t i o n 24 h after the i n j e c t i o n a n d r e t u r n e d to n o r m a l after 48 h (Fig. 2). A t 24 h, there was n o s i g n i f i c a n t difference in B U N b e t w e e n the 50
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20 HgCl2 and Hg-MT rats, but both of them were significantly higher than that of the Cd-MT injected rats. Similar patterns were observed for the levels of plasma creatinine (Fig. 3). Rats injected with Cd-MT and both forms of Hg showed significantly higher plasma creatinine than that of control and rats injected with CdCl 2 2 days after the injections. Rats injected with both forms of Hg had similar plasma creatinine levels. However, the levels were both significantly higher than that of Cd-MT at 24 h, at the dose level used. Volume of urine excreted by all rats was quite uniform and averaged 20-30 ml/day. Mean MT concentration in urine from control rats and rats injected with CdC12 were not significantly different and ranged from 30-150 ng/day (Fig. 4). Urinary excretion of MT in Cd-MT injected rats was elevated during the first 24 h, it then decreased with time but remained significantly higher than that of the control and CdC12 injected rats. Urinary MT was significantly higher in both HgCl 2 and Hg-MT injected rats than the control for the first 3 days after injection. No significant difference was observed in urinary MT levels between the two Hg injected groups of rats. Urinary Cd in the Cd-MT injected rats was significantly higher than that of the control for 4 days after injection (Fig. 5). Injected CdCI2, on the other hand, did not cause any increase in the urinary excretion of Cd. Significantly more Hg was excreted in the urine by the HgCl 2 injected rats than the Hg-MT injected rats. Total protein concentration in the urine of rats injected with Cd-MT significantly increased during 24 h after injection and remained higher than those of the control and CdCl 2 injected rats for 72 h (Fig. 6). Similarly, urinary protein concentrations of rats injected with both forms of Hg also showed a significant elevation during 24 h after the injection and returned to normal after 48 h. There was no significant
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difference in urinary protein concentrations between the Hg injected and the Cd-MT injected rats. Results of morphological analysis of kidney by light microscopy are summarized in Table II. A mild level of renal injury occurred in the rats injected with 0.3 mg/kg dose of HgC12 at 24 h after the injection. The injury consisted of necrosis in isolated pars recta segments of the proximal tubule located at the cortico-medullary junction. Renal injury also occurred at 24 h after injection of Hg-MT and consisted of cellular necrosis that appeared to be localized in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule situated at the cortico-medullary junction. In this case, the level of renal injury appeared to be greater than that in the rats injected with HgC12. Injection of 0.3 mg Cd/kg as CdMT to rats induced a moderately severe nephropathy at 24 h. It was characterized by necrosis in proximal convoluted tubules throughout the cortex. No apparent renal injury was observed in rats 24 h after injection of a similar dose of Cd as CdCI 2. The sections of kidney from the rats injected with HgC12 revealed that epithelial regeneration had occurred in the pars recta of proximal tubules in the outer stripe of the outer medulla and medullary rays of the cortex in 7 days after the injection. In contrast, no obvious signs of cellular or tubular necrosis or regeneration were found in the pars recta of proximal tubules in sections of kidney from the rats treated 7 days prior to sacrifice with Hg-MT. There were, however, a few sets of proximal convoluted tubules that displayed a rudimentary epithelium, which is indicative of cellular regeneration. Epithelial regeneration had occurred in most of the proximal convoluted tubules 7 days after the injection of Cd-MT indicating that cellular
24 h 7 days 24 h 7 days 24 h 7 days 24 h 7 days
CdCI 2
No No Yes Yes Yes Yes Yes Yes
Evidence of Cellular necrosis
--Cortex Cortex CorterdOM Cortex + OS Cortex/OM Cortex/OM
Zonal location of cellular necrosis --PCT PCT PST PCT PCT/PST PCT/PST
Tubular site of cellular necrosis
--Moderate Moderate Mild Mild Mild-Mod. Mild-Mod.
Level of severity of cellular necrosis No No No Yes No Yes No Yes
Evidence of epithelial regeneration
---Cortex -Cortex + OM -Cortex
Zonal localization of epithelial regeneration ---PCT -PST -PCT/PST
Tubular site of epithelial regeneration
---Almost complete -Almost complete -Almost complete
Stage o f epithelial regeneration
Cortex/OM, junction of the cortex and outer medulla; OS, outer stripe of the outer medulla; PCT, proximal convoluted tubules; PST, proximal straight tubules; PCT/PST, involving terminal portions of the proximal convoluted tubules and the initial portions of the proximal straight tubules.
Hg-MT
HgCI2
Cd-MT
Time
Treatment
HISTOLOGICAL ASSESSMENT OF K I D N E Y S
TABLE II
24 necrosis occurred in this part of the proximal tubule soon after the injection. Remnants of epithelial cells that had undergone necrosis were observed in the lumen of some of the tubules. In addition, pyknotic nuclei were present in some of the epithelial cells indicating that they were in the process of dying. No obvious signs of cellular injury were observed in the kidney of rats injected with CdCI2. Discussion
The relative tissue distribution and toxicity of cadmium chloride (CdCI2), cadmium-metallothionein (Cd-MT), mercury chloride (HgC12) and mercurymetallothionein (Hg-MT) are reported in this study. Concentrations of Cd were higher in the kidney and lower in the liver in rats injected with Cd-MT compared with those of rats injected with CdCI2 and were similar to previous findings [6,7]. In contrast, Hg selectively accumulated in the kidney regardless of the injected form. The reason for the difference between Cd and Hg tissue distribution after injection of CdC12 and HgCI2 is not known, but may be related to the binding affinity of these metals to carrier proteins in the blood [11]. Moreover, Gerson and Shaikh [28] found that liver cells in vitro accumulate more Cd than Hg when incubated with corresponding salts. The selective accumulation of both Cd-MT and Hg-MT in the kidney may be because the low molecular weight MT is easily filtered and reabsorbed in the kidney. Induction of MT synthesis by metals has been extensively studied. Accumulation of Hg in rat kidney after injection of HgCI 2 increased renal MT concentrations, particularly in the cortex and outer medulla [17]. Rats injected with Hg-MT showed higher renal MT concentration than those injected with HgCI2. Part of this increased MT may result from the uptake of injected intact Hg-MT, which contained about 200 #g MT. Nevertheless, the induction of MT synthesis in the kidney may still be a major factor for the increase in renal MT concentrations and this may have a protective effect on Hg nephrotoxicity, although the mechanism is still unknown [18]. The increase in plasma MT concentrations in rats injected with CdCI2 and the lack of increase in plasma MT concentrations of rats after the injection of HgCI2 suggest that the increased plasma MT in the CdCI2 injected rats may originate from the liver. These results agree with the generally accepted hypothesis that Cd-MT synthesized in the liver may be released and transported in the blood to the kidney and at high doses, may cause renal injury [29,30]. Rats injected with both Cd-MT and Hg-MT showed an increase in plasma MT concentrations and the clearance rate of Hg-MT appeared to be higher than that of Cd-MT. However, since 0.3 mg of either Cd or Hg were injected in each rat, assuming that the molar ratio 7 for both Cd and Hg for binding to MT, the amount of MT injected in the case of Hg would be twice that of Cd because the difference in their atomic weight. Therefore, the clearance rate between Cd-MT and Hg-MT may not be directly comparable. It has been shown that injected Cd-MT is transported in the plasma to the kidney, where it is freely filtered by the glomerulus and reabsorbed by proximal convoluted tubules [31,32]. The high urinary MT level observed in the first day may be due to incomplete reabsorption of the injected Cd-MT by the tubular cells [33]. Injection of both Hg-MT and HgC12 caused a similar level of increase in urinary excretion of MT suggesting
25 that the nephrotoxic effects of the two forms of Hg may be similar. This also supported the view that urinary MT can be used as a marker for renal toxicity of Cd and Hg. Injection of Cd-MT and both forms of Hg resulted in nephrotoxicity as shown by the increase in blood urea nitrogen, total urinary protein concentrations and urinary MT excretion. Histopathological results also show that injection of Cd-MT caused cellular injury in the $1 segment, whereas HgC12 caused damage in the $2 and $3 segments of the proximal tubule, confirming previous reports [5,7]. It is interesting that Hg-MT seems to cause cellular damage in the convoluted tubules which resembles the toxicity of Cd-MT and not that of HgCI2. Even though injections of both forms of Hg caused increase in BUN, plasma creatinine, urinary protein and MT, higher level of urinary Hg excretion was observed after injection of HgCI2. These results indicate that the two forms of Hg are taken up by different mechanisms. In previous studies [6,7], the direct comparison of toxicities of metal compounds such as CdC12 and Cd-MT has been difficult because of the differences in tissue distribution of metals. However, in the present study, Hg from both HgC12 and HgMT was deposited mainly in the kidney and this similar renal accumulation of Hg enabled us to compare their toxicities in the kidney. Results of this biochemical and preliminary morphological study suggest that although both forms of Hg are nephrotoxic, their renal uptake mechanisms and site of toxicity in the kidney may be different. Implications of these differences require further investigation.
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