Enhanced Renal Toxicity by Inorganic Mercury in Metallothionein-Null Mice

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1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 3 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283: , 1997 Enhanced Renal Toxicity by Inorganic Mercury in Metallothionein-Null Mice MASAHIKO SATOH, NORIKO NISHIMURA, YOSHITAKA KANAYAMA, AKIRA NAGANUMA, TSUGUYOSHI SUZUKI and CHIHARU TOHYAMA Environmental Health Sciences Division (M.S., C.T.), National Institute for Environmental Studies (T.S.), Tsukuba, Ibaraki 305, Japan; Department of Veterinary Pathology (N.N.), University of Sydney, Sydney 2006, Australia; and Department of Molecular and Biochemical Toxicology (Y.K., A.N.), Faculty of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai , Japan Accepted for publication August 7, 1997 ABSTRACT To elucidate a protective role of metallothionein (MT) in the manifestation of inorganic mercury toxicity, we studied the susceptibility of MT-null mice to the renal toxicity of mercuric chloride. Because the MT-null (J) mice are a genetic background of 129/Sv strain, the 129/Sv mice were used as wildtype controls. Nine-week-old male MT-null (J) and 129/Sv mice were given subcutaneous injections of mercuric chloride at doses of 10 to 40 mol/kg. The basal MT level in the kidney of MT-null (J) mice was undetectable ( 0.2 g/g of tissue) and 2.5 g/g of tissue in 129/Sv mice. The sensitivity to the renal Inorganic mercury present in the environment is a wellestablished toxicant to human health (Clarkson et al., 1988; WHO, 1991). It is well known that inorganic mercury causes severe kidney damage after acute and chronic exposure (Ganote et al., 1974; McDowell et al., 1976; Zalups and Diamond, 1987). Because inorganic mercury has high affinity for sulfhydryl groups, the toxicity of inorganic mercury has been suppressed by administration of various thiol compounds such as d-penicillamine (Aposhian and Aposhian, 1959), dithiothreitol (Kleinman et al., 1977; Klonne and Johnson, 1983; Suzuki and Ozaki, 1984) and glutathione monoisopropyl ester (Naganuma et al., 1990). MT is a cysteine-rich low-molecular-weight protein with a high affinity for metals such as mercury, zinc, copper and cadmium, and it is induced by these metals and many other factors, such as glucocorticoids and cytokines (reviewed by Kägi and Schäffer, 1988). MT is found ubiquitously in the tissues of many animal species and plays roles in the homeostasis of essential metals such as zinc and copper. Elevation of MT in the cells was shown to protect against the toxicities of heavy metals, mutagens, anticancer agents and radical-inducing substances (Basu and Lazo, 1990; Kägi and Schäffer, 1988; Sato and Bremner, 1993). The kidney damage Received for publication March 17, toxicity of mercuric chloride was markedly enhanced in the MT-null (J) mice compared with the 129/Sv mice. The renal mercury level was similar for the MT-null (J) and 129/Sv mice at 4 hr after the injection of mercuric chloride (20 mol/kg) but became significantly lower in MT-null (J) mice than in 129/Sv mice at 24 and 72 hr. Based on the present results, we conclude that MT is an important protective factor against the renal toxicity caused by inorganic mercury and that it may play a major role in the retention of mercury in the kidney. caused by inorganic mercury is prevented by preinduction of renal MT because intracellular mercury in the kidney is firmly trapped by the MT (Webb and Magos, 1976; Zalups and Cherian, 1992). However, a putative role of endogenous MT to detoxify inorganic mercury remains unclear because there has not been experimental methods of depressing the intracellular MT level. Recently, transgenic mice that are deficient in the MT-I and MT-II genes (MT-null mice) have been established (Masters et al., 1994; Michalska and Choo, 1993). In the present study, we examined the sensitivity to the renal toxicity of inorganic mercury in the MT-null mice and investigated the involvement of MT in the distribution of inorganic mercury in the kidney. Materials and Methods Animals and chemicals. We used two different types of MT-null mice, MT-null (Aus) and MT-null (J) mice, who had null mutation of MT-I and -II genes. MT-null (Aus) mice, which were of a mixed genetic background of 129 Ola and C57BL/6 strains, were kindly provided by Dr. Choo (Michalska and Choo, 1993). To produce these MT-null mice, F1 hybrid mice were mated with C57BL/6 mice and their offsprings were back-crossed to C57BL/6 for three generations. Another type of MT-null mice, MT-null (J) mice (Masters et al., 1994), which were of a genetic background of 129/Sv strain, were ABBREVIATIONS: MT, metallothionein; MT-null mice, metallothionein-i and -II knock-out mice; NP-SH, nonprotein SH; BUN, blood urea nitrogen; NIES, National Institute for Environmental Studies; -GTP, -glutamyltranspeptidase. 1529

2 1530 Satoh et al. Vol. 283 purchased from Jackson Laboratory (Bar Harbor, ME). For wild-type control mice, 8-week-old male C57BL/6J and 129/Sv mice were purchased from Japan Clea (Tokyo, Japan). Mice were handled with humane care throughout this study according to NIES guidelines. These MT-null mice were randomly bred in the vivarium, in which temperature and humidity were maintained at 23 1 C and 55 10%, respectively with a 12-hr light/dark cycle. Mice were given free access to food and tap water. Microbiological and viral examinations performed for different colonies over a 1-year period did not provide pathogenic infections or significant phenotypic abnormalities. Biotinylated goat anti-rabbit IgG, goat control serum and an avidin-biotin horseradish peroxidase complex (ABC) kit were purchased from Vector Laboratories (Burlingame, CA). HistoChoice was obtained from Amresco (Solon, OH). Metal compounds and other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Treatments. Nine-week-old male MT-null (Aus), MT-null (J), C57BL/6J and 129/Sv mice were randomized into control and experimental groups, respectively. The groups of mice (4 mice/each group) were given a single subcutaneous injection of mercuric chloride at a dose of 10 to 40 mol/kg. To determine renal MT and mercury concentrations and evaluate renal toxicity, blood and kidney samples were collected under diethylether anesthesia from each mouse 3 days after the injection. MT-null (J) and 129/Sv mice were subcutaneously administered mercuric chloride at a dose of 20 mol/kg to characterize the distribution of mercury in the kidney. The kidney was removed with the mice under diethylether anesthesia at 4, 24 and 72 hr after the injection for mercury determination. Immunohistochemical staining. For immunohistochemical evaluation of MT, kidney tissues were fixed in HistoChoice solution and embedded in paraffin. Deparaffinized tissue sections of 5- m thickness were stained according to the previously described ABC method using rabbit anti-rat MT antiserum (Nishimura et al., 1989). Gel filtration. The kidney removed from MT-null (J) and 129/Sv mice was homogenized in 9 volumes of 10 mm Tris HCl buffer (ph 7.4), and the homogenate was ultracentrifuged at 4 C for 60 min at 105,000 g. The resultant supernatant was subsequently applied to a Sephadex G-75 column ( mm) and eluted with 10 mm Tris HCl (ph 7.4). The eluate was collected in fractions of 3 ml each, and mercury amounts in the fractions were determined. Analysis. MT concentrations in the kidney were measured using radioimmunoassay (Tohyama and Shaikh, 1981) as modified by Nishimura et al. (1990). To estimate glutathione level, renal NP-SH was determined according to the modified method of Ellman using 5,5 -dithiobis-2-nitrobenzoic acid (Costa and Murphy, 1986). Blood urea nitrogen and creatinine in plasma were determined with automatic dry-chemistry analyzer system (Spotchem SP-4410; Kyoto Daiichi-kagaku, Kyoto, Japan). Mercury concentrations in the kidney were measured with a cold vapor atomic absorption spectrophotometer (RA-2A Mercury Analyzer; Nippon Instruments, Tokyo, Japan) after digestion with concentrated acid mixture [HNO 3 /HClO 4 1:3 (v/v)]. Statistical analysis. Data are expressed as mean S.D. for four mice. Statistical analysis was performed using one-way analysis of variance followed by Fisher s least significant difference tests for post hoc comparison. Data were analyzed with StatView software for Macintosh computers. Differences between groups were considered significant at P.05. TABLE 1 MT and NP-SH concentrations in the kidney of untreated mice Mice MT NP-SH g/g of tissue mol/g of tissue C57BL/6J /Sv MT-null (Aus) MT-null (J) Values are mean S.D. for four mice. the basal NP-SH levels in the kidney of all of these mice were not significantly different from one another. We studied the renal toxicity of inorganic mercury in the MT-null (Aus), MT-null (J), C57BL/6J and 129/Sv mice (fig. 1). BUN (fig. 1, top) and creatinine (fig. 1, bottom) values in MT-null (Aus) mice were elevated at and above a mercury dose of 30 mol/kg and significantly higher than those in C57BL/6J mice. A similar observation was obtained in 129/Sv mice, whose BUN and creatinine values were increased in a dose-dependent manner at and above 30 mol/ kg. C57BL/6J mice were most resistant among these three Results Table 1 summarizes MT and NP-SH concentrations in the kidney of untreated mice. Wild-type mice (both C57BL/6 and 129/Sv) harbored basal levels of MT, whereas both MT-null (Aus) and MT-null (J) mice did not have a detectable level (0.2 g/g of tissue) of MT in the kidney. On the other hand, Fig. 1. Renal toxicity of mercuric chloride in MT-null (Aus), MT-null (J), C57BL/6J and 129/Sv mice. Values are mean S.D. for four mice. * Significantly different from the corresponding untreated group (P.05). # Significantly different from the specified group (P.05). n.d., not determined.

3 1997 Hg Nephrotoxicity in MT-Null Mice 1531 strains; BUN but not creatinine value was significantly increased by mercury injection at a dose of 40 mol/kg. On the other hand, renal toxicity of mercury was more conspicuous in MT-null (J) mice at a dose of 20 mol/kg than in 129/Sv mice, in which the BUN (fig. 1, top) and creatinine (fig. 1, bottom) values were elevated at and above 30 mol/kg. All MT-null (J) mice died within 3 days from a mercury dose of 40 mol/kg, whereas all the 129/Sv mice survived. Figure 2 shows mercury and MT concentrations in the kidney of MT-null (Aus), MT-null (J), C57BL/6J and 129/Sv mice after the mercury injection. Renal mercury concentrations (fig. 2, top) in the four types of mice increased in a dose-dependent manner. 129/Sv mice accumulated significantly larger amounts of mercury than C57BL/6J or MT-null (Aus) mice, both of which accumulated one third to one half of the mercury levels of 129/Sv mice. Moreover, the mercury level in the kidney of 129/Sv mice was significantly higher than that of MT-null (J) mice (fig. 2, top). On the other hand, Fig. 2. Renal Hg and MT concentrations in MT-null (Aus), MT-null (J), C57BL/6J and 129/Sv mice 3 days after mercuric chloride injection at various doses. Values are mean S.D. for four mice. * Significantly different from the corresponding untreated group (P.001). # Significantly different from the specified group (P.05). n.d., not determined; N.D., not detected. renal MT levels (fig. 2, bottom) in both C57BL/6J and 129/Sv mice markedly increased according to the accumulated mercury level, and the MT level in 129/Sv mice was significantly higher than that in C57BL/6J mice. In contrast, mercury administration could not induce MT in the kidney of both MT-null (Aus) and MT-null (J) mice. The susceptibility of the kidney of MT-null (J) mice to inorganic mercury was further confirmed by histopathological observations (fig. 3), which are consistent with the result on BUN and creatinine values: in MT-null (J) mice, marked morphological changes such as the degeneration and necrosis of the proximal tubular cells and dilation of the tubular lumen were observed by the mercury injection at a dose of 20 mol/kg (fig. 3B). In contrast, almost no damage was observed in the renal tubules of 129/Sv mice by the same mercury dose (fig. 3D). Localization of MT in the kidneys of MT-null (J) and 129/Sv mice was assessed by ABC staining (fig. 3). The renal tubules of untreated 129/Sv mice showed minimal amounts of MT immunostaining (fig. 3C), whereas mercury injection markedly increased MT in proximal tubular epithelial cells in these mice (fig. 3D). No MT immunostaining was detected in the renal tubules of MT-null (J) mice indifferent from mercury administration (fig. 3, A and B). Next, we examined the time course of renal mercury concentrations in MT-null (J) and 129/Sv mice after a minimum toxic dose (20 mol/kg) of mercury (fig. 4). In both MT-null (J) and 129/Sv mice, renal mercury levels considerably increased by 4 hr after the injection with a subsequent decrease. At 4 hr, similar amounts of mercury were retained in the kidney of MT-null (J) and 129/Sv mice. At 24 and 72 hr after the injection, however, the mercury accumulation in the kidney was significantly lower in MT-null (J) mice than in 129/Sv mice. Typical Sephadex G-75 chromatograms of the kidney cytosols from MT-null (J) and 129/Sv mice are shown in figure 5. Almost all of the mercury in the cytosol was associated with high-molecular-weight fraction in the both MT-null (J) and 129/Sv mice at 4 hr after the injection (fig. 5, top). However, at 72 hr after the mercury injection, a difference in the distribution pattern of mercury was observed between MT-null (J) and 129/Sv mice (fig. 5, bottom): in the 129/Sv mice, the cytosolic mercury ( 65%) was redistributed to an MT fraction, whereas mercury was found only in the highmolecular-weight fraction in the MT-null (J) mice. Discussion In the present study, the use of MT gene knock-out mice clearly indicates that endogenous and postinduced MT in the kidney can protect mice against the renal toxicity of inorganic mercury. The sensitivity to mercury toxicity was markedly increased in the MT-null (J) mice compared with 129/Sv, the wild-type control mice (figs. 1 and 3). Several studies have shown that preinduced MT in the kidney by treatment with cadmium or zinc compounds prevents the renal toxicity caused by inorganic mercury (Webb and Magos, 1976; Zalups and Cherian, 1992). The detoxification of inorganic mercury by preinduced MT can be explained through the sequestering of mercury ions by MT molecules because mercuric ions have a stronger affinity for MT than cadmium or zinc. The present results indicate that MT does not affect the

1532 Satoh et al. Vol. 283 Fig. 3. Localization of MT by immunohistochemical staining and histopathological changes in the renal cortex of MT-null (J) and 129/Sv mice treated with mercuric chloride.

B, Mercuric chloride (20 mol/kg)-treated MT-null (J) mice. C, Untreated 129/Sv mice. D, Mercuric chloride (20 mol/kg)- treated 129/Sv mice. Fig. 4.

4 1532 Satoh et al. Vol. 283 Fig. 3. Localization of MT by immunohistochemical staining and histopathological changes in the renal cortex of MT-null (J) and 129/Sv mice treated with mercuric chloride. MT was detected with rabbit anti-rat MT antiserum and the kidney was lightly stained with hematoxylin. Bar, 50 m. A, Untreated MT-null (J) mice. B, Mercuric chloride (20 mol/kg)-treated MT-null (J) mice. C, Untreated 129/Sv mice. D, Mercuric chloride (20 mol/kg)- treated 129/Sv mice. Fig. 4. Renal mercury concentrations in MT-null (J) and 129/Sv mice at various times after a mercuric chloride (20 mol/kg) injection. Values are mean S.D. for four mice. * Significantly different from the corresponding untreated group (P.005). initial distribution of mercury to the kidney but instead participates in the retention of this metal in the kidney (fig. 4). This fact may be explained by the induction of MT synthesis in the kidney and the subsequent binding of mercury to MT (fig. 5). Because MT plays a key role in the retention of other heavy metals, kinetics of distribution of the metals in MTnull mice remain to be studied. In the most commonly used procedure for the production of gene knock-out mice, embryonic stem cells derived from 129 Ola strain and blastcysts from C57BL/6 strain are used to produce chimeric mice, which are crossed with C57BL/6, and their offsprings are mated further with C57BL/6. As a wildtype control, the C57BL/6 strain has been used. However, in the present study, we found a remarkable strain difference between C57BL/6J and 129/Sv mice in the renal toxicity of inorganic mercury (fig. 1) as well as mercury accumulation and induced MT amounts in the kidney (fig. 2). We thought that MT-null (J) mice would be an appropriate animal model in which to study the biological role of MT because these mice are of the genetic background of the 129/Sv strain only. We thus used MT-null (J) and 129/Sv mice for further study. MT-null (J) mice have a much higher sensitivity to inorganic mercury than MT-null (Aus) mice (fig. 1); we believe that this difference is probably due to the absence of a C57BL/6 genetic trait in the MT-null (J) mice. As the underlying mechanisms for the above-mentioned strain difference between C57BL/6J and 129/Sv mice, a -GTP-dependent transport system in the kidney (Tanaka et al., 1990) is thought to play a role. Tanaka et al. (1990) reported that Hg-glutathione complex formed in the plasma after intravenous injections of inorganic mercury is filtered through the glomerulus and that mercury ions are incorporated into renal tubular cells as an Hg-CysGly and/or Hg-Cys complex after degradation of glutathione by -GTP. They also found differences in renal -GTP activity by strain as well as by sex (Tanaka et al., 1991). In addition, Zalups and Barfuss (1995) demonstrated that pretreatment with p-aminohippurate, which is a well-established competitive inhibitor of proximal tubular transport of a number of organic anions, inhibits the renal uptake of inorganic mercury, although no evidence is currently provided with regard to strain difference in this transport system. Further studies are needed to prove these hypotheses for the MT-null mice and their controls. In conclusion, the use of MT-null mice and appropriate control mice revealed that the endogenous MT and preinduced MT can protect mice against the renal toxicity of inorganic mercury. These mice should provide deeper insights into toxic mechanisms of heavy metals, including inorganic mercury. This would be particularly true when living organisms are overwhelmed by amounts of heavy metals,

5 1997 Hg Nephrotoxicity in MT-Null Mice 1533 Fig. 5. Gel filtration (Sephadex G-75) profiles of mercury in the kidney cytosol of MT-null (J) and 129/Sv mice at 4 and 72 hr after a mercuric chloride (20 mol/kg) injection. The mercury eluted in Fraction and Fraction is considered to be bound to high-molecular-weight proteins and MT, respectively. which cannot be sequestered by MT. Moreover, it was clear by the use of MT-null (J) mice that MT plays an important role in the retention of mercury in the kidney but not in the uptake of this metal. Acknowledgments The authors thank Mr. T. Oda at Animal Care Company (Tokyo, Japan) for his excellent assistance in the maintenance of transgenic mice at NIES. References APOSHIAN, H. V. AND APOSHIAN, M. M.: N-Acetyl-DL-penicillamine, a new oral protective agent against the lethal effects of mercuric chloride. J. Pharmacol. Exp. Ther. 126: , BASU, A. AND LAZO, J. S.: A hypothesis regarding the protective role of metallothioneins against the toxicity of DNA interactive anticancer drugs. Toxicol. Lett. 50: , CLARKSON, T. W., HURSH, J. B., SAGER, P.R.AND SYVERSEN, T. L. M.: Mercury. In Biological Monitoring of Toxic Metals, eds. by T. W. Clarkson, L. Friberg, G. F. Nordberg and P. R. Sager, pp , Plenum Press, New York, COSTA, L. G. AND MURPHY, S. D.: Effect of diethylmaleate and other glutathione depletors on protein synthesis. Biochem. Pharmacol. 35: , GANOTE, C. E., REIMER, K. A. AND JENNINGS, R. B.: Acute mercuric chloride nephrotoxicity: An electron microscopic and metabolic study. Lab. Invest. 31: , KÄGI, J.H.R.AND SCHÄFFER, A.: Biochemistry of metallothionein. Biochemistry 27: , KLEINMAN, J. G., MCNEIL, J. S., SCHWARTZ, J. H., HAMBURGER, R. J. AND FLAMEN- BAUM, W.: Effect of dithiothreitol on mercuric chloride- and uranyl nitrateinduced acute renal failure in the rat. Kidney Int., 12: , KLONNE, D. R. AND JOHNSON, D. R.: Amelioration of mercuric chloride-induced acute renal failure by dithiothreitol. Toxicol. Appl. Pharmacol. 29: , MASTERS, B. A., KELLY, E. J., QUAIFE, C. J., BRINSTER, R. L. AND PALMITER, R. D.: Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. USA 91: , MCDOWELL, E. M., NAGLE, R. B., ZALME, R. C., MCNEIL, J. S., FLAMENBAUM, W. AND TRUMP, B. F.: 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: , MICHALSKA, A. E. AND CHOO, K. H. A.: Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. USA 90: , NAGANUMA, A., ANDERSON, M. E. AND MEISTER, A.: Cellular glutathione as a determinant of sensitivity to mercuric chloride toxicity: Prevention of toxicity by giving glutathione monoester. Biochem. Pharmacol. 40: , NISHIMURA, H., NISHIMURA, N. AND TOHYAMA, C.: Immunohistochemical localization of metallothionein in developing rat tissues. J. Histochem. Cytochem. 37: , NISHIMURA, H., NISHIMURA, N. AND TOHYAMA, C.: Localization of metallothionein in the genital organs of the male rat. J. Histochem. Cytochem. 38: , SATO, M.AND BREMNER, I.: Oxygen free radicals and metallothionein. Free Rad. Biol. Med. 14: , SUZUKI, S. AND OZAKI, N.: The protective effects of thiol-containing compounds on mercuric chloride-induced acute inhibition of enzymes from mouse kidney. Toxicology 29: , TANAKA, T., NAGANUMA, A.AND IMURA, N.: Role of -glutamyltranspeptidase in renal uptake and toxicity of inorganic mercury in mice. Toxicology 60: , TANAKA, T., NAGANUMA, A., KOBAYASHI, K. AND IMURA, N.: An explanation for strain and sex differences in renal uptake of methylmercury in mice. Toxicology 69: , TOHYAMA, C. AND SHAIKH, Z. A.: Metallothionein in plasma and urine of cadmium-exposed rats determined by a single antibody radioimmunoassay. Fundam. Appl. Toxicol. 1: 1 7, WEBB, M. AND MAGOS, L.: Cadmium-thionein and the protection by cadmium against the nephrotoxicity of mercury. Chem.-Biol. Interact. 14: , WORLD HEALTH ORGANIZATION: Inorganic mercury. In Environmental Health Criteria 118, pp , Geneva, Switzerland, ZALUPS, R. K. AND BARFUSS, D. W.: Pretreatment with p-aminohippurate inhibits the renal uptake and accumulation of injected inorganic mercury in the rat. Toxicology 103: 23 35, ZALUPS, R. K. AND CHERIAN, M. G.: Renal metallothionein metabolism after a reduction of renal mass. II. Effect of zinc pretreatment on the renal toxicity and intrarenal accumulation of inorganic mercury. Toxicology 71: , ZALUPS, R. K. AND DIAMOND, G. L.: Mercuric chloride-induced nephrotoxicity in the rat following unilateral nephrectomy and compensatory renal growth. Virchows Arch. B 53: , Send reprint requests to: Masahiko Satoh, Ph.D., Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan. masahiko@nies.go.jp.

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