9 1987 by The Humana Press Inc. All rights of any nature, whatsoever, reserved.
0163-4984/87/1200-0419502.00
Induction of Metallothionein in Rat Tissues Following Subchronic Exposure to Mercury Shown by Radioimmunoassay CANICE V. NOLAN AND ZAHIR A. SHAIKH*
Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI 02881-0809 ABSTRACT Although the analysis of metallothionein (MT) by radioimmunoassay (R1A) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 Ixg/g), kidney (68 tx/g), brain (0.8 jxg/g), spleen (1.0 ~xg/g), heart (5.4 ~xg/g), plasma (11 ng/ml), and urine (200-300 gg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 ixmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in tt'te kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity. Index Entries: Metallothionein, induction in/by mercury, rat tissues; mercury, subchronic exposure; radioimmunoassay; metallothionein, determination by radioimmunoassay; mercury rne'cabolism and toxicity; copper; zinc; rat. *Author to whom all correspondence and reprint requests should be addressed. Biological TraceElement Research
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INTRODUCTION Following chronic exposure of animals to inorganic mercury, the highest concentration of the metal is found in the kidney (1). Nephrotoxicity is often seen, and the principal target site appears to be the cells of the proximal tubular epithelium (2). In the soluble cytoplasmic fraction of this tissue, the metal has been shown to be associated with both highmolecular-weight protein (HMWP) and metallothionein (MT) (3-6). The MT is induced in this tissue and, to a lesser extent, in the liver of animals exposed to mercury (3, 7-12). Changes in the plasma levels of MT following exposure to mercury have also been noted (13). The synthesis of this protein in liver and kidney tissue may also be induced by other metals (8). Based on these and other findings, it has been speculated that the induction of MT synthesis following exposure to heavy metals may be a detoxification mechanism that reduces their potential to interact with sensitive cellular components (14). Altered tissue levels of copper and zinc following exposure to mercury have also been documented by some authors (13,15,16). It would therefore be of interest to determine whether induction of MT synthesis and changes in the tissue levels of the essential metals copper and zinc take place and, if so, to what extent, following subchronic exposure of animals to a very low, nontoxic level of mercury.
METHODS
Experimental Protocol Forty-five female Sprague-Dawley rats (mean body weight, 175 g) were randomly divided into nine groups of five animals each. They were given food and water ad libitum. Four of these groups were administered ip with a solution of HgCI2 (0.2 ~mol/kg/d) 5 d in every 7 for periods of up to 4 wk. Paired control groups were administered physiological saline. The ninth group served as a zero-time control. At the end of each week, 24 h after the last injection, the animals in one exposed and one control group were weighed and sacrificed by exsanguination under ether anesthesia. The blood, brain, heart, kidney, liver, and spleen were removed, weighed, and stored at -80~ pending analysis. For the 6 h immediately preceding sacrifice, all animals were separately maintained in metabolic cages, without food or water, and their urine collected in a beaker at 4~ Blood and urine samples were also collected from the 4 wk control and exposed groups at weekly intervals. The samples were stored frozen at -80~ and thawed immediately prior to analyses.
Analytical Techniques Tissue MT was analyzed by radioimmunoassay (RIA), as described by Nolan and Shaikh (17). The RIA of urine and plasma were performed Biological Trace Element Research
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according to the method of Tohyama and Shaikh (18). Urinary protein and creatinine were determined by the methods of Bradford (19) and Heingard and Tiderstrom (20), respectively. Subcellular fractionation of the kidney was performed by homogenization of the tissue in distilled water (16.7%, w/v), with a Tekmar tissue homogenizer, followed by centrifugation of the homogenate at 10,5000g for 90 min in a Beckman Model L ultracentrifuge equipped with a swinging bucket rotor. Gel filtration chromatography of aliquots of the soluble supernatant fractions prepared above was performed on Sephadex G-75 columns. The conditions of the chromatographic separations are described in legend to Fig. 2. The eluted fractions were assayed for absorbance (254 nm) in a Gilford spectrophotometer. Fractions were analyzed without prior acid digestion for copper (graphite furnace), zinc (flame), and mercury (cold vapor) in a Varian t475 atomic absorption spectrophotometer linked to a Varian GTA-95 graphite furnace according to the manufacturers' instructions. Urine and whole blood plasma samples were similarly assayed for mercury without digestion. For the determination of metals in tissues, acid digestion was performed prior to analysis. Typically, to 200 mg tissue, homogenized in i mL distilled water, was added i mL of an acid mix (50 parts HNO3:48 parts HCIO4:2 parts H2SO4), and digestion was allowed to proceed for 2 d at 80~ (to minimize losses of Hg). A two sample t-test was used to compare paired control and exposed groups. One way analysis of variance (ANOVA) was used when a paired control was not available.
RESULTS No significant differences in growth rate of animals were seen over the experimental period. Similarly, with the exception of the kidneys of animals exposed to mercury for 4 wk (1.79 _+0.10 g as opposed to 1.55 +_ 0.09 g in controls), no differences in organ weights were noted. Urinary protein levels also remained constant (8-16 ~g/g creatinine) in both control and exposed groups during the course of this experiment and did not show a significant change. No significant change in MT levels in the tissues of control groups was noted during the study, except for an increase in renal MT in the 4-wk control group. This increase in renal MT was probably related to the stress incurred by this group during weekly blood sampling and urine collection. Among the mercury-exposed groups, no significant changes in the MT levels were noted in heart (mean value 3.2 ~g/g), or in urine (range, 200-300 ~g/g creatinine). Since the blood samples were partially hemolyzed, not much emphasis can be placed on the plasma MT values (mean 11 ng/mL), which did not show a significant difference between the groups. In the spleen and brain (Fig. 1), increases in the MT levels peaking at 2 wk were noted (1.8-6.3 ~g MT/g spleen and 0.8-4.3 bLgMT/g Biological Trace Element Research
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422
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Fig. 1. MT concentration in brain, spleen, liver, and kidney of rats injected with saline (o o) or inorganic mercury (o-~--o) for periods of up to 4 wk. N = 5; mean _+ SD *Significantly different from paired control group at p 0.05. brain). Dramatic increases with time in the MT concentration of liver (10.5-84.2 tJ,g/g) and kidney (92-538 ~g/g) were noted (Fig. 1). The concentrations of zinc and copper in control tissues did not change during the period of study, but, in exposed groups, several changes were notable. Mercury was not detected (detection limit, 0.07 ~g Hg/g tissue) in any samples other than the kidneys, where levels in-
Biological Trace Element Research
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Nolan and Shaikh
creased linearly up to 39.8 txg Hg/g after 4 wk (Table 1). Concomitant to this increase in mercury, the levels of copper in the kidney also rose more than threefold. No change in the copper content in the liver, spleen, or brain of exposed animals was noted (heart not analyzed). The concentration of zinc increased slightly, but significantly in the kidney (Table 1). A significant rise in zinc concentration was also seen in the liver (23.7-37.4 Ixg/g) and spleen (7.8-10.5 txg/g), but not in the brain, where levels remained stable (mean 6.4 Ixg/g). Again, the heart was not analyzed. It can be seen from Table 1 that about half or more of the kidney copper and zinc was found in the soluble cytoplasmic fraction. Furthermore, the accumulation of excess copper, zinc, and mercury in the kidney following exposure to inorganic mercury was seen to occur predominantly in this fraction, though increases in the levels of copper and mercury in the 105,000g pellet fraction were also significant. In Fig. 2 the distributions of mercury, copper, and zinc, among the proteins of the soluble cytoplasmic fraction of the kidney, following gel filtration chromatography on Sephadex G75, are shown for the zero-time control group and the 4-wk mercury-exposed group. In control animals, copper was associated almost exclusively with HMWP (molecular weight > 10000), but it can be seen that, in those animals exposed to mercury, 90% of the excess copper was bound to MT, with 10% in the lowermolecular-weight (LMW) fractions. Zinc was associated mainly with HMWP in control animals, with some also in the LMW region. In exposed animals, the excess zinc was bound to HMWP, MT, and LMW components. Mercury was detected only in the exposed group, in which it was associated predominantly with MT (70%), but also with HMWP (30%). No mercury was detected in the LMW fractions. Although not shown, there was no difference in the UV absorbance (254 nm) of various fractions between the groups.
DISCUSSION Indicators of chronic mercury toxicity in rats include decreased growth rates, increased kidney weight, diuresis, and proteinuria (21). In this study the only toxic effect noted was the slightly increased kidney weight of those animals exposed to mercury for 4 wk. Elevated urinary MT levels have been proposed to be a good indicator of cadmiuminduced nephrotoxicity (22). Probably, it would also be a good indicator of mercury-induced toxicity, as noted by Lee et al. (13), but no increases in urinary MT were seen here. Therefore, at the exposure levels used, it seemed that the rats in this study did not succumb to the toxic effects of mercury. The basal levels of MT in the tissue of these animals are similar to those determined previously by RIA (17). With the exception of kidney tissue, they lie below the detection limits of the metal-saturation assays (17,23). Furthermore, since the induced MT in the kidney tissue in this Biological Trace Element Research
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F r a c t i o n Number Fig. 2. Gel filtration chromatography of the solubte supernatant of rat kidney before (.... ) and after ( - - ) 4-wk exposure to inorganic mercury. The concentrations of mercury, copper, and zinc in each fraction are plotted. Chromatographic conditions: volume of supernatant applied, 6 mL; column, 1.6 x 85 cm Sephadex G-75; eluting buffer, 0.005M Tris-HCl, pH 8.6; flow rate, 25 mL/h; fraction size, 2.2 mL. Fraction numbers 49-72 are designated as MT. study contains mainly mercury and copper, which have higher affinities for the protein than c a d m i u m (24), the determination of the induced MT by cadmium-saturation m e t h o d w o u l d probably result in underestimation of the total a m o u n t (25). The RIA offers the advantages of superior specificity and sensitivity and indifference to metal content and is, therefore, the preferred technique. The induction of MT synthesis in rat liver and kidney following acute or chronic exposure to high levels of mercury has been noted by Biological Trace Element Research
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blolan and Shaikh
several authors (7-12), but there is scant information in the literature on the levels of this protein in brain, spleen, and heart after exposure to mercury. Similarly, little information is available on the metabolism of this protein following exposure to toxicologically insignificant levels of the metal. The lack of any effect of mercury on MT levels in the heart is a reflection of the insignificant role that this organ plays in mercury metabolism. The transient increases in brain and spleen MT levels followed by a return to normal levels may indicate an initial detoxication response, which disappears over time as the kidneys acclimatize to the metal. There is, however, a need for further data in this respect. The induction of MT synthesis in liver and kidney is probably a detoxication mechanism to prevent detrimental interaction of mercury with sensitive cellular components, but any discussion on the signficance of observed changes must also explain the altered distribution of the essential elements zinc and copper in these tissues. Although copper levels in the liver did not change, a significant increase in zinc was observed concomitant to that of MT. Mercury was not detectable in this organ. Increased liver zinc concentration following exposure to mercury has also been noted by other authors (10,13,15). The excess zinc was associated with MT, but no mercury was seen in this fraction. Similar results were seen in our study, even though smaller doses of mercury were used. The mechanism of MT induction in this organ following mercury exposure and its significance in mercury detoxification are not clear. Elevated renal copper concentrations following administration of mercury to rats have been variously described (13,16,26). This mercuryinduced increased copper content is often associated with increased levels of MT in this tissue (3,11,12,26,27). It is not clear, however, whether the MT synthesis is induced by mercury or copper. The MT contains two separate metal-binding regions, the o~- and f3-domains (28). Mercury may induce the synthesis of the apoprotein and bind to the oL-region. Since copper binds preferentially to the g-domain (29), the newly synthesized protein would sequester copper along with mercury. Yet another possibility is that copper and/or zinc induce(s) the synthesis of metallothionein. Mercury displaces copper and/or zinc from the o~-domain, the freed metal(s) then induce(s) more protein, and a cycle is set up. It is clear then that exposure to mercury can have profound effects on the metabolism of zinc, copper, and MT in animals, even at toxicologically insignificant doses. The association of the metal with high-molecular-weight components, even at the low levels noted here, indicates that some clinically undetectable toxic processes may be taking place, probably through nonspecific interactions with the sulfhydryl groups on proteins.
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SUMMARY Rats were subchronically exposed to a low dose of inorganic mercury. The injected mercury accumulated primarily in the kidney, w h e r e the highest MT levels were noted. Copper and, to a lesser extent, zinc levels also increased in this tissue. Chromatographic analyses s h o w e d that the excess copper and zinc, like mercury, were b o u n d mainly to MT. The tissue MT levels increased significantly in liver, brain, and spleen, but did not change in heart, urine, or plasma. No toxicological effects were noted, thus, the induction of MT in the tissues may be construed as a detoxication mechanism for this metal.
ACKNOWLEDGMENTS This research was supported by Public Health Service Grant No. ES 03187. Technical assistance for part of this project was provided by Mrs. Sara Nelson.
REFERENCES 1. M. Berlin, in Handbook on the Toxicolocgy of Metals, L. Friberg, G. F. Nordberg, and V. B. Vouk, eds., Elsevier, Amsterdam, 1979, pp. 503-530. 2. C. E. Ganote, K. A. Reimer, and R. B. Jennings, Lab. Invest. 31, 633 (1974). 3. J. K. Piotrowski, B. Trojanowska, J. M. Wisniewska-Knypl, and W. Bola nowska, Toxicol. Appl. Pharmacol. 27, 11 (1974). 4. Z. A. Shaikh, O. J. Lucis, and R. L. Coleman, Proc. 7th Conf. Trace Substances in Environmental Health, D. D. Hemphill, ed., University of Missouri, Columbia, MO, 1973, pp. 313-321. 5. M. Webb and L. Magos, Chem. Biol. Interact. 21, 215 (1978). 6. G. Lorenzon, E. Marafante, and E. Sabbioni, Euro. Appl. Res. Reps. (in press). 7. Z. A. S h a i k h a n d J. C. Smith, Abstracts, lnternat. Conf. Heavy Metals in the
Envinmment, Toronto, 1975, pp. B108-109. 8. D. R. Winge, R. Premakumar, and K. V. Rajagopalan, Arch. Bh~chem. Biophys. 170, 242 (1975). 9. M. G. Cherian and T. W. Clarkson, Chem. Biol. Interact. 12, 109 (1976). 10. M. Martin and F. O. Brady, Proc. South Dakota Acad. Sci. 56, 72 (1977). 11. J. K. Piotrowski, B. Trojanowska, and A. Sapota, Arch. Tocicol. 32, 351 (1974). 12. P. D. Whanger and J. T. Deagen, Environ. Res. 30, 372 (1983). 13. Y. H. Lee, Z. A., Shaikh and C. Tohyama, Toxicology 27, 337 (1983). 14. J. H. R. Kagi, and M. Nordberg, eds. Metallothionein, Birkhauser Verlag, Basel, 1979, pp. 41-124. 15. Y. Suzuki, Ind. Health 10, 56 (1972). 16. J. D. Bogden, F. W. Kemp, R. A. Troiano, B. S. Jortner, C. Timpone, and D. Guiliani, Environ. Res. 21, 350 (1980).
Biological Trace Element Research
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blolan and Shaikh C. V. Nolan and Z. A. Shaikh, Anal. Biochem. 154, 213 (1986). C. Tohyama and Z. A. Shaikh, Fundam. Appl. Toxicol. 1, 1 (1981). M. M. Bradford, Anal. Biochem. 72, 248 (1976). D. Heingard and G. Tiderstrom, Clin. Chim. Acta. 43, 305 (1973). G. P. Daston, R. J. Kavlock, E. M. Rogers, and B. Carver, Toxicol. Appl. Pharmacol. 71, 24 (1983). Z. A. Shaikh and C. Tohyama, Environ. Health. Perspect. 54, 171 (1984). S. Onasaka and M. G. Cherian, Toxicol. Appl. Pharmacol. 63, 270 (1982). M. P. Waalkes and C. D. Klaasen, Toxicol. Appl. Pharmacol. 74, 314 (1984). D. L. Eaton, Toxicol. Appl. Pharmacol. 78, 158 (1985). J. A. Szymanska and A. J. Zelazowski, Environ. Res. 19, 121 (1974). E. Sabbioni and E. Marafante, Environ. Physiol. Biochem. 5, 465 (1975). J. D. Otvos and I. M. Armitage, Proc. Natl. Acad. Sci. USA, 77, 7094 (1980). K. B. Nielson and D. R. Winge, J. Biol. Chem. 259, 4941 (1984).
Biological Trace Element Research
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Induction of Metallothionein in Rat Tissues Following Subchronic Exposure to Mercury Shown by Radioimmunoassay CANICE V. NOLAN AND ZAHIR A. SHAIKH*
Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI 02881-0809 ABSTRACT Although the analysis of metallothionein (MT) by radioimmunoassay (R1A) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 Ixg/g), kidney (68 tx/g), brain (0.8 jxg/g), spleen (1.0 ~xg/g), heart (5.4 ~xg/g), plasma (11 ng/ml), and urine (200-300 gg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 ixmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in tt'te kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity. Index Entries: Metallothionein, induction in/by mercury, rat tissues; mercury, subchronic exposure; radioimmunoassay; metallothionein, determination by radioimmunoassay; mercury rne'cabolism and toxicity; copper; zinc; rat. *Author to whom all correspondence and reprint requests should be addressed. Biological TraceElement Research
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INTRODUCTION Following chronic exposure of animals to inorganic mercury, the highest concentration of the metal is found in the kidney (1). Nephrotoxicity is often seen, and the principal target site appears to be the cells of the proximal tubular epithelium (2). In the soluble cytoplasmic fraction of this tissue, the metal has been shown to be associated with both highmolecular-weight protein (HMWP) and metallothionein (MT) (3-6). The MT is induced in this tissue and, to a lesser extent, in the liver of animals exposed to mercury (3, 7-12). Changes in the plasma levels of MT following exposure to mercury have also been noted (13). The synthesis of this protein in liver and kidney tissue may also be induced by other metals (8). Based on these and other findings, it has been speculated that the induction of MT synthesis following exposure to heavy metals may be a detoxification mechanism that reduces their potential to interact with sensitive cellular components (14). Altered tissue levels of copper and zinc following exposure to mercury have also been documented by some authors (13,15,16). It would therefore be of interest to determine whether induction of MT synthesis and changes in the tissue levels of the essential metals copper and zinc take place and, if so, to what extent, following subchronic exposure of animals to a very low, nontoxic level of mercury.
METHODS
Experimental Protocol Forty-five female Sprague-Dawley rats (mean body weight, 175 g) were randomly divided into nine groups of five animals each. They were given food and water ad libitum. Four of these groups were administered ip with a solution of HgCI2 (0.2 ~mol/kg/d) 5 d in every 7 for periods of up to 4 wk. Paired control groups were administered physiological saline. The ninth group served as a zero-time control. At the end of each week, 24 h after the last injection, the animals in one exposed and one control group were weighed and sacrificed by exsanguination under ether anesthesia. The blood, brain, heart, kidney, liver, and spleen were removed, weighed, and stored at -80~ pending analysis. For the 6 h immediately preceding sacrifice, all animals were separately maintained in metabolic cages, without food or water, and their urine collected in a beaker at 4~ Blood and urine samples were also collected from the 4 wk control and exposed groups at weekly intervals. The samples were stored frozen at -80~ and thawed immediately prior to analyses.
Analytical Techniques Tissue MT was analyzed by radioimmunoassay (RIA), as described by Nolan and Shaikh (17). The RIA of urine and plasma were performed Biological Trace Element Research
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according to the method of Tohyama and Shaikh (18). Urinary protein and creatinine were determined by the methods of Bradford (19) and Heingard and Tiderstrom (20), respectively. Subcellular fractionation of the kidney was performed by homogenization of the tissue in distilled water (16.7%, w/v), with a Tekmar tissue homogenizer, followed by centrifugation of the homogenate at 10,5000g for 90 min in a Beckman Model L ultracentrifuge equipped with a swinging bucket rotor. Gel filtration chromatography of aliquots of the soluble supernatant fractions prepared above was performed on Sephadex G-75 columns. The conditions of the chromatographic separations are described in legend to Fig. 2. The eluted fractions were assayed for absorbance (254 nm) in a Gilford spectrophotometer. Fractions were analyzed without prior acid digestion for copper (graphite furnace), zinc (flame), and mercury (cold vapor) in a Varian t475 atomic absorption spectrophotometer linked to a Varian GTA-95 graphite furnace according to the manufacturers' instructions. Urine and whole blood plasma samples were similarly assayed for mercury without digestion. For the determination of metals in tissues, acid digestion was performed prior to analysis. Typically, to 200 mg tissue, homogenized in i mL distilled water, was added i mL of an acid mix (50 parts HNO3:48 parts HCIO4:2 parts H2SO4), and digestion was allowed to proceed for 2 d at 80~ (to minimize losses of Hg). A two sample t-test was used to compare paired control and exposed groups. One way analysis of variance (ANOVA) was used when a paired control was not available.
RESULTS No significant differences in growth rate of animals were seen over the experimental period. Similarly, with the exception of the kidneys of animals exposed to mercury for 4 wk (1.79 _+0.10 g as opposed to 1.55 +_ 0.09 g in controls), no differences in organ weights were noted. Urinary protein levels also remained constant (8-16 ~g/g creatinine) in both control and exposed groups during the course of this experiment and did not show a significant change. No significant change in MT levels in the tissues of control groups was noted during the study, except for an increase in renal MT in the 4-wk control group. This increase in renal MT was probably related to the stress incurred by this group during weekly blood sampling and urine collection. Among the mercury-exposed groups, no significant changes in the MT levels were noted in heart (mean value 3.2 ~g/g), or in urine (range, 200-300 ~g/g creatinine). Since the blood samples were partially hemolyzed, not much emphasis can be placed on the plasma MT values (mean 11 ng/mL), which did not show a significant difference between the groups. In the spleen and brain (Fig. 1), increases in the MT levels peaking at 2 wk were noted (1.8-6.3 ~g MT/g spleen and 0.8-4.3 bLgMT/g Biological Trace Element Research
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Fig. 1. MT concentration in brain, spleen, liver, and kidney of rats injected with saline (o o) or inorganic mercury (o-~--o) for periods of up to 4 wk. N = 5; mean _+ SD *Significantly different from paired control group at p 0.05. brain). Dramatic increases with time in the MT concentration of liver (10.5-84.2 tJ,g/g) and kidney (92-538 ~g/g) were noted (Fig. 1). The concentrations of zinc and copper in control tissues did not change during the period of study, but, in exposed groups, several changes were notable. Mercury was not detected (detection limit, 0.07 ~g Hg/g tissue) in any samples other than the kidneys, where levels in-
Biological Trace Element Research
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Induction of /Vletallothionein by/Vlercury
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Nolan and Shaikh
creased linearly up to 39.8 txg Hg/g after 4 wk (Table 1). Concomitant to this increase in mercury, the levels of copper in the kidney also rose more than threefold. No change in the copper content in the liver, spleen, or brain of exposed animals was noted (heart not analyzed). The concentration of zinc increased slightly, but significantly in the kidney (Table 1). A significant rise in zinc concentration was also seen in the liver (23.7-37.4 Ixg/g) and spleen (7.8-10.5 txg/g), but not in the brain, where levels remained stable (mean 6.4 Ixg/g). Again, the heart was not analyzed. It can be seen from Table 1 that about half or more of the kidney copper and zinc was found in the soluble cytoplasmic fraction. Furthermore, the accumulation of excess copper, zinc, and mercury in the kidney following exposure to inorganic mercury was seen to occur predominantly in this fraction, though increases in the levels of copper and mercury in the 105,000g pellet fraction were also significant. In Fig. 2 the distributions of mercury, copper, and zinc, among the proteins of the soluble cytoplasmic fraction of the kidney, following gel filtration chromatography on Sephadex G75, are shown for the zero-time control group and the 4-wk mercury-exposed group. In control animals, copper was associated almost exclusively with HMWP (molecular weight > 10000), but it can be seen that, in those animals exposed to mercury, 90% of the excess copper was bound to MT, with 10% in the lowermolecular-weight (LMW) fractions. Zinc was associated mainly with HMWP in control animals, with some also in the LMW region. In exposed animals, the excess zinc was bound to HMWP, MT, and LMW components. Mercury was detected only in the exposed group, in which it was associated predominantly with MT (70%), but also with HMWP (30%). No mercury was detected in the LMW fractions. Although not shown, there was no difference in the UV absorbance (254 nm) of various fractions between the groups.
DISCUSSION Indicators of chronic mercury toxicity in rats include decreased growth rates, increased kidney weight, diuresis, and proteinuria (21). In this study the only toxic effect noted was the slightly increased kidney weight of those animals exposed to mercury for 4 wk. Elevated urinary MT levels have been proposed to be a good indicator of cadmiuminduced nephrotoxicity (22). Probably, it would also be a good indicator of mercury-induced toxicity, as noted by Lee et al. (13), but no increases in urinary MT were seen here. Therefore, at the exposure levels used, it seemed that the rats in this study did not succumb to the toxic effects of mercury. The basal levels of MT in the tissue of these animals are similar to those determined previously by RIA (17). With the exception of kidney tissue, they lie below the detection limits of the metal-saturation assays (17,23). Furthermore, since the induced MT in the kidney tissue in this Biological Trace Element Research
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Mercury
0
Copper
~3
x
0.11-
0.2""z
0
'
/
9
"
Zinc
O.t 0.2 ,
I
....
30
50
~ -I- -"
70
"
~t~'---
90
F r a c t i o n Number Fig. 2. Gel filtration chromatography of the solubte supernatant of rat kidney before (.... ) and after ( - - ) 4-wk exposure to inorganic mercury. The concentrations of mercury, copper, and zinc in each fraction are plotted. Chromatographic conditions: volume of supernatant applied, 6 mL; column, 1.6 x 85 cm Sephadex G-75; eluting buffer, 0.005M Tris-HCl, pH 8.6; flow rate, 25 mL/h; fraction size, 2.2 mL. Fraction numbers 49-72 are designated as MT. study contains mainly mercury and copper, which have higher affinities for the protein than c a d m i u m (24), the determination of the induced MT by cadmium-saturation m e t h o d w o u l d probably result in underestimation of the total a m o u n t (25). The RIA offers the advantages of superior specificity and sensitivity and indifference to metal content and is, therefore, the preferred technique. The induction of MT synthesis in rat liver and kidney following acute or chronic exposure to high levels of mercury has been noted by Biological Trace Element Research
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blolan and Shaikh
several authors (7-12), but there is scant information in the literature on the levels of this protein in brain, spleen, and heart after exposure to mercury. Similarly, little information is available on the metabolism of this protein following exposure to toxicologically insignificant levels of the metal. The lack of any effect of mercury on MT levels in the heart is a reflection of the insignificant role that this organ plays in mercury metabolism. The transient increases in brain and spleen MT levels followed by a return to normal levels may indicate an initial detoxication response, which disappears over time as the kidneys acclimatize to the metal. There is, however, a need for further data in this respect. The induction of MT synthesis in liver and kidney is probably a detoxication mechanism to prevent detrimental interaction of mercury with sensitive cellular components, but any discussion on the signficance of observed changes must also explain the altered distribution of the essential elements zinc and copper in these tissues. Although copper levels in the liver did not change, a significant increase in zinc was observed concomitant to that of MT. Mercury was not detectable in this organ. Increased liver zinc concentration following exposure to mercury has also been noted by other authors (10,13,15). The excess zinc was associated with MT, but no mercury was seen in this fraction. Similar results were seen in our study, even though smaller doses of mercury were used. The mechanism of MT induction in this organ following mercury exposure and its significance in mercury detoxification are not clear. Elevated renal copper concentrations following administration of mercury to rats have been variously described (13,16,26). This mercuryinduced increased copper content is often associated with increased levels of MT in this tissue (3,11,12,26,27). It is not clear, however, whether the MT synthesis is induced by mercury or copper. The MT contains two separate metal-binding regions, the o~- and f3-domains (28). Mercury may induce the synthesis of the apoprotein and bind to the oL-region. Since copper binds preferentially to the g-domain (29), the newly synthesized protein would sequester copper along with mercury. Yet another possibility is that copper and/or zinc induce(s) the synthesis of metallothionein. Mercury displaces copper and/or zinc from the o~-domain, the freed metal(s) then induce(s) more protein, and a cycle is set up. It is clear then that exposure to mercury can have profound effects on the metabolism of zinc, copper, and MT in animals, even at toxicologically insignificant doses. The association of the metal with high-molecular-weight components, even at the low levels noted here, indicates that some clinically undetectable toxic processes may be taking place, probably through nonspecific interactions with the sulfhydryl groups on proteins.
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SUMMARY Rats were subchronically exposed to a low dose of inorganic mercury. The injected mercury accumulated primarily in the kidney, w h e r e the highest MT levels were noted. Copper and, to a lesser extent, zinc levels also increased in this tissue. Chromatographic analyses s h o w e d that the excess copper and zinc, like mercury, were b o u n d mainly to MT. The tissue MT levels increased significantly in liver, brain, and spleen, but did not change in heart, urine, or plasma. No toxicological effects were noted, thus, the induction of MT in the tissues may be construed as a detoxication mechanism for this metal.
ACKNOWLEDGMENTS This research was supported by Public Health Service Grant No. ES 03187. Technical assistance for part of this project was provided by Mrs. Sara Nelson.
REFERENCES 1. M. Berlin, in Handbook on the Toxicolocgy of Metals, L. Friberg, G. F. Nordberg, and V. B. Vouk, eds., Elsevier, Amsterdam, 1979, pp. 503-530. 2. C. E. Ganote, K. A. Reimer, and R. B. Jennings, Lab. Invest. 31, 633 (1974). 3. J. K. Piotrowski, B. Trojanowska, J. M. Wisniewska-Knypl, and W. Bola nowska, Toxicol. Appl. Pharmacol. 27, 11 (1974). 4. Z. A. Shaikh, O. J. Lucis, and R. L. Coleman, Proc. 7th Conf. Trace Substances in Environmental Health, D. D. Hemphill, ed., University of Missouri, Columbia, MO, 1973, pp. 313-321. 5. M. Webb and L. Magos, Chem. Biol. Interact. 21, 215 (1978). 6. G. Lorenzon, E. Marafante, and E. Sabbioni, Euro. Appl. Res. Reps. (in press). 7. Z. A. S h a i k h a n d J. C. Smith, Abstracts, lnternat. Conf. Heavy Metals in the
Envinmment, Toronto, 1975, pp. B108-109. 8. D. R. Winge, R. Premakumar, and K. V. Rajagopalan, Arch. Bh~chem. Biophys. 170, 242 (1975). 9. M. G. Cherian and T. W. Clarkson, Chem. Biol. Interact. 12, 109 (1976). 10. M. Martin and F. O. Brady, Proc. South Dakota Acad. Sci. 56, 72 (1977). 11. J. K. Piotrowski, B. Trojanowska, and A. Sapota, Arch. Tocicol. 32, 351 (1974). 12. P. D. Whanger and J. T. Deagen, Environ. Res. 30, 372 (1983). 13. Y. H. Lee, Z. A., Shaikh and C. Tohyama, Toxicology 27, 337 (1983). 14. J. H. R. Kagi, and M. Nordberg, eds. Metallothionein, Birkhauser Verlag, Basel, 1979, pp. 41-124. 15. Y. Suzuki, Ind. Health 10, 56 (1972). 16. J. D. Bogden, F. W. Kemp, R. A. Troiano, B. S. Jortner, C. Timpone, and D. Guiliani, Environ. Res. 21, 350 (1980).
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428 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
blolan and Shaikh C. V. Nolan and Z. A. Shaikh, Anal. Biochem. 154, 213 (1986). C. Tohyama and Z. A. Shaikh, Fundam. Appl. Toxicol. 1, 1 (1981). M. M. Bradford, Anal. Biochem. 72, 248 (1976). D. Heingard and G. Tiderstrom, Clin. Chim. Acta. 43, 305 (1973). G. P. Daston, R. J. Kavlock, E. M. Rogers, and B. Carver, Toxicol. Appl. Pharmacol. 71, 24 (1983). Z. A. Shaikh and C. Tohyama, Environ. Health. Perspect. 54, 171 (1984). S. Onasaka and M. G. Cherian, Toxicol. Appl. Pharmacol. 63, 270 (1982). M. P. Waalkes and C. D. Klaasen, Toxicol. Appl. Pharmacol. 74, 314 (1984). D. L. Eaton, Toxicol. Appl. Pharmacol. 78, 158 (1985). J. A. Szymanska and A. J. Zelazowski, Environ. Res. 19, 121 (1974). E. Sabbioni and E. Marafante, Environ. Physiol. Biochem. 5, 465 (1975). J. D. Otvos and I. M. Armitage, Proc. Natl. Acad. Sci. USA, 77, 7094 (1980). K. B. Nielson and D. R. Winge, J. Biol. Chem. 259, 4941 (1984).
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