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[67] Biological indicators of cadmium exposure
592
[67]
I N D U C T I O N OF M E T A L L O T H I O N E I N S
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Biological Indicators of Cadmium
Exposure
By JUDITH A. GLAVEN, ROBIN E. GANDLEY, a n d BRUCE A. FOWLER Introduction Cellular exposure to cadmium is known to elicit a number of responses in addition to metallothionein induction. A mechanistic understanding of these processes may permit development of biological indicators of cadmium exposure. This approach may be applied to both mammalian and nonmammalian tissues. This chapter will examine a number of these methods and attempt to evaluate their utility for monitoring how cells respond to this metal at the organelle and molecular levels of biological organization. Ideally, such indicators should provide early, sensitive, and readily measurable responses that are specific for cadmium. In order for a potential indicator to be accepted as an indicator of effect, it must ultimately show a mechanistic correlation with the biological activity of this metal in living tissues. As discussed below, there are a number of potential candidate indicators of exposure and effect for cadmium that have each shown some promise for monitoring the actions of cadmium in cells. Indicators of Cadmium Exposure Total Tissue Burden In addition to cadmium induction of metallothionein (MT), monitoring of total tissue cadmium burdens via either atomic absorption spectroscopy ~,2or in vivo neutron activation analyses3,4 may provide useful data on cadmium exposure. The atomic absorption spectroscopy methods involve sampling and digestion of tissues prior to analysis and hence are of limited value for monitoring living organisms. These analyses are frequently conducted on autopsy samples of target tissues such as the kidney5 or for R. Lauwerys, H. Roels, M. Regniers, J. P. Buchet, A. Bernard, and A. Goret, Environ. Res. 20, 375 (1979). : R. Lauwerys, J. P. Buchet, and H. Roehls, Inst. Arch. Occup. Environ. Health 26, 275 (1976). 3 K. J. Ellis, W. D. Morgan, I. Zanai, S. Yasumura, D. Vartsky, and S. H. Cohn, J. Toxicol. Environ. Health 7, 691 (1981). 4 H. A. Roels, R. R. Lauwerys, J. P. Buchet, A. Bernrd, D. R. Chettle, T. C. Harvey, and I. K. AI-Haddad, Environ. Res. 26, 217 (1981). C. G. Elinder, T. Kjellstrom, L. Friberg, B. Lind, and L. Linnman, Arch. Environ. Health 31,292 (1976).
METHODS IN ENZYMOLOGY, VOL. 205
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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nonmammalian aquatic organisms such as mussels, 6 where field sampling methods may be employed to assess cadmium exposure of populations. In vivo neutron activation analyses of renal cadmium concentrations in exposed workers is another potentially useful methodology 3,4 but further studies are needed to validate this approach with regard to direct wet chemical analyses, because the technique appears to be sensitive to geometric considerations. Mineral Concretions X-Ray microanalysis and atomic absorption spectroscopy are potentially useful monitoring tools for detecting cadmium in mineral concretions of marine mollusks. 7- i1 In these species, the concretions represent a larger intracellular storage compartment than the metallothionein pool 7,8 and have the potential advantage of being extensively excreted from the kidney when cadmium exposure exceeds the tissue capacity to store this metal. 8,1~ This response may thus also represent an indicator of cadmium toxicity. Disadvantages of monitoring concretions are that exposure to other metals such as copper will also cause this phenomenon, 1~ so that there is a lack of specificity for cadmium in this response if other metals are also present. Stress Proteins Prokaryotic and eukaryotic cells respond to heat shock and various other forms of stress, including metal exposure, by synthesizing a discrete set of proteins.12-16 These molecules were first observed in cells exposed to conditions of thermal stress and as a result have been termed heat-shock proteins (hsps). 12'~3 Subsequently, it has been found that various forms of 6 E. D. Goldberg, M. Koide, V. Hodge, A. R. Fleagal, and J. H. Martin, Estuarine Coastal ShelfSci. 16, 69 (1983). 7 N. G. Carmichael, K. S. Squibb, and B. A. Fowler, J. Fish. Res. Bd. Can. 36, 1149 (1979). 8 N. G. Carmichael and B. A. Fowler, Mar. Biol. 65, 35 (1981). 9 S. G. George and B. J. S. Pirie, Bioehem. Biophys. Acta 5811, 234 (1979). ~oS. G. George, B. J. S. Pirie, and T. L. Combs, J. Exp. Mar. Biol. EcoL 42, 143 (1980). tmB. A. Fowler and E. Gould, Mar. Biol. 96, 207 (1988), t2 A. Tissieres, H. K. Mitchell, and V. M. Tracey, J. MoL Biol. 84, 389 (1974). ~3 M. Ashburner and J. J. Bonner, Cell. (Cambridge, Mass.) 17, 241 (1979). ~4S. Lindquist, Annu. Rev. Biochem. 55, 1151 (1986). z5 W. J. Welch, J. I. Garrels, and J. R. Fermaisco, in "Heat Shock from Bacteria to Man" (M. J. Schlesinger, M. Ashburner, and A. Tissieres, eds.), p. 257. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ~6W. J. Levinson, H. Opperman, and J. Jackson, Bioehem. Biophys. Acta 606, 170 (1980).
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INDUCTION OF METALLOTHIONEINS
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stress, including drugs, 17 amino acid analogs ~7,~s and metals, 16 induce the expression of these molecules, which are now more commonly termed the stress proteins. This response is highly conserved and presumed to be universal, serving a general protective function in the stressed cell. Principal stress proteins have molecular masses ranging from 28,000174,000 Da. Of these, those between 70,000 and 90,000 are seen in response to most situations of stress; the other stress proteins appear to be induced more selectively. The occurrence of specific protein responses to specific conditions of stress may have great utility as biological indicators of exposure, and in elucidating the mechanisms of toxicity at the subcellular level. Specific patterns of protein synthesis can be detected through metabolic labeling of proteins and standard techniques of two-dimensional gel electrophoresis. ~9-2~ The technique consists of an isoelectric separation in the first dimension and separation based on molecular weight in the second dimension. With the development of computerized digitizing image analysis systems, autoradiographs of the gels may be evaluated in a more quantitative manner. Because of the environmental significance of cadmium there is an interest in identifying early indicators of exposure and delineating their relationships to mechanisms of action. Levinson et al. described the induction of four proteins in chicken and human cell lines in response to several transition series metals, including cadmium. ,6 Along with the induction of hsp70, several low-molecular-mass proteins between 25,000 and 35,000 were induced. In a study examining the effect of sublethal acute doses of cadmium on Drosophila cells, it was shown that some of the induced proteins were the same as those seen in heat-shocked cells. However, cadmium also stimulated the synthesis of several other unique low-molecular-weight proteins. 2° These proteins are thus candidates as biomarkers of exposure to this element. There are several other examples of stress proteins detected in response to cadmium. Induced gene expression in two fish cell lines exposed to cadmium has been investigated by Price-Haughy and Gedamu22; they described the induction of metaUothionein (MT), a 14,000 metal-inducible protein, and several stress proteins with molecular masses between 28,000 and 29,000 in response to cadmium. A number of studies have been focused on the biological function of those molecules in an effort to better understand their roles in cell survival ~7L. E. Hightower, J. Cell. Physiol. 102, 407 (1980). 18 p. M. Kelley and M. J. Schlesinger, Cell (Cambridge, Mass.) 15, 1277 (1978). 19 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). 20 A. Courageon, C. Maisonhaute, and M. Best-Belpomme, Exp. CellRes. 153, 515 (1984). 21 y. Aoki, M. M. Lipsky, and B. A. Fowler, Toxicol. Appl. PharmacoL 106, 462 (1990). 22 j. Price-Haughey and L. Gedamu, Experientia, Suppl. 52, 465 (1987).
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during chemical or physical stress. Riabowol et al. demonstrated the importance of stress proteins to the cell while examining the function of the heat shock protein with molecular mass 70,000 (hsp70) in rat fibroblasts. 23 Cells injected with monoclonal antibodies raised against hsp70 were unable to withstand heat-shock treatment. This work shows that the protein has an important protective function in the stressed cell. A stress protein seen in response to cadmium with a relative molecular mass of 34,000 has been identified as heme oxygenase. 24 Specific antibodies for human heme oxygenase were used to identify the protein by an immunoblot technique. 25 These data suggest that the rate-limiting enzyme in the oxidative metabolism of heme 26 may be of importance during early response to sublethal injury. The direction of future research in this area will be to identify molecular triggers for stress response following exposure to cadmium, and to relate specific stress proteins to adaptation/protection in the stressed cell. Research that may provide further information in this direction includes DNA footprinting techniques to examine the trigger for the induction of a specific stress protein by identifying DNA-binding proteins, 27 and various immunological techniques to determine the intracellular location of specific stress proteins during and after the stress. 15,28As specific stress proteins induced in response to cadmium exposure are identified and examined from the perspective of the role they play in the protection of the cell, a more basic understanding of the mechanisms of cadmium biological activity will emerge.
Metallothionein Gene-Binding Factors The significance of metallothionein (MT) as an indicator of cadmium exposure is discussed later in this chapter. Briefly, we would like to describe cellular factors involved in the regulation of MT, because they may serve as indicators of cadmium exposure prior to MT induction. Investigation into the structure and function of genes encoding for MT have identified trans-acting nuclear factors (NF) involved in the regulation of MT expression. 29,3° Cadmium induces the formation of complexes of NF with metal regulatory elements (MRE) of the M T gene. Initially it was hoped that exposure to cadmium would cause detectable, diagnostic increases in the 23 K. T. Riabowol, L. A. Mizzen, and W. J. Welch, Science 242, 433 (1988). 24 S. Taketani, H. Kohno, T. Yoshinga, and R. Tounaga, FEBS Lett. 245, 173 (1989). 25 H. Towbin, T. Stahelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 26 M. D. Maines, FASEBJ. 2, 2557 (1988). 27 D. J. Galas and A. Schmitz, Nucleic Acids Res. 5, 3157 (1978). 28 W. J. Welch and L. A. Mizzen, J. Cell Biol. 106, 117 (1988). 29 C. Seguin, B. K. Felber, A. D. Carter, and D. Hamer, Nature(London) 312, 781 (1984). 30 C. Seguin and D. Hamer, Science 235, 1383 (1987).
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cellular concentration of NF; unfortunately, however, comparisons of cellular concentrations of MRE-binding NF in cells grown in the presence and absence of cadmium showed that cadmium does not act by increasing the synthesis of these proteins) ° Instead, cadmium may act on NFs directly to increase their affinity for the MRE of the gene, or cadmium may act by altering protein-protein interactions between the NF and some as yet unidentified "coactivator" protein? ° If this is the case it would be necessary to detect the cadmium-induced change in the NF, or the induction of the "coactivator." The use of DNA footprinting 3°,27 to reveal MREs of the M T gene protected from exonuclease activity due to bound NF, and synthetic oligonucleotides from the MRE sequence to detect DNA-binding proteins immobilized on blots, 32,33 has resulted in the description of two M T genebinding NFs with molecular masses of 74,000 and 108,000. 31,32 One of these, the 74K molecule, has been purified by affinity chromatography on trout MRE31; further characterization of this protein and its interaction with regulatory regions of the mouse M T gene are awaited. Proteinuria
Cadmium-induced tubular proteinuria can be used as an early indicator of cadmium exposure and effect. Alterations in renal function produced by cadmium can be due to alterations in tubular reabsorption or glomerular filtration. A distinctive low-molecular-weight proteinuria has been associated with elevated cadmium exposure and is characterized by an increase in low-molecular-weight urinary proteins and a decrease in high-molecular-weight proteins, suggesting damage to the renal proximal tubule. 34 Alternatively, another possible effect of cadmium exposure is a decrease in glomerular permeability to larger proteins. 35,36 Studies by Squibb et al. 37 provided a mechanistic basis for development of cadmiuminduced proteinuria and its use as an early indlcator of cadmium exposure following parenteral administration of cadmium-metallothionein (CdMT) to rats. Since Cd-MT is the major form of cadmium in the circula3, j. Imbert, M. Zafarullah, V. C. Culotta, L. Gedamu, and D. Hamer, Mol. Cell. Biol. 9, 5315 (1989). 32C. Seguinand J. Prevost, Nucleic Acids Res. 22, 1052 (1988). 33W. K. Miskimins,M. P. Roberts, A. McClelland,and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 82, 6741 (1985). 34p. L. Goering,K. S. Squibb, and B. A. Fowler, TraceSubst. Environ. Health 19, 22 (1985). 35A. Bernard, H. Rods, G. Hubermont, J. P. Buchet, P. L. Masson, and R. Lauwerys,Int. Arch. Occup. Environ. Health 38, 19 (1976). 36A. Bernard, J. P. Buchet,H. Roels,P. Masson,and R. Lauwerys,Eur. J. Clin. Invest. 9, 11 (1979). 37K. S. Squibb, J. B. Pritchard, and B. A. Fowler,J. PharmacoL Exp. Ther. 229, 311 (1984).
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tion, this model mimicks the actual in vivo condition. Within 8 hr of Cd-MT administration, an increase in low-molecular-weight proteins was detected in the urine, via sodium dodecyl sulfate (SDS) gel electrophoresis, 37 that was temporally correlated to altered lysosome structure and function in renal proximal tubule cells. This study demonstrated the utility of proteinuria as a biological indicator and the relationship of the proteinuria to pathophysiological changes in target cell populations. Specific Urinary Proteins. The proteinuria induced by cadmium may not increase the overall urine protein content significantly until the cadmium exposure is quite extensive. Clinical diagnosis of proteinuria is based on detection of an increase in total protein in the urine. The methods used for early detection of cadmium-induced proteinuria must discriminate between high- and low-molecular-weight proteins in the urine. Immunological assays for fl2-microglobulin, retinol-binding protein, and urinary metallothionein are used to detect low-molecular-weight proteinuria. fl2-Microglobulin, fl2-Microglobulin is used to monitor tubule reabsorption capabilities. There are several problems with using this protein to detect proteinuria. First, there is an increase in fl2-microglobulin in the urine of adults, beginning at the age of 50 years. 3s Second, fl2-microglobulin is unstable at urine pH < 5.5. The most common assay for fl2-microglobulin is a radioimmunoassay based on latex particle agglutination. Samples are chromatographed on Sephadex G-75 and fractions are pooled and concentrated, using ultrafiltration. The sample is then analyzed via immunoassay using an antiserum and immunoglobulin G-coated latex particles. 39 The samples can be read via an automated latex immunoassay system.4° Another common protein assay used for cadmium exposure is an electrophoretic technique using silver staining with sodium dodecyl sulfate-polyacrylamide gel. 41 A detection level of 0.8 mg protein/liter urine can be achieved with this technique. Retinol-Binding Protein. Retinol-binding protein (RBP) differs from fl2-microglobulin in that it is stable in urine of pH 4.5 and higher, and its degradation is slower.42 Like fl2-microglobulin, RBP is present at elevated levels in older human subjects due to an overall loss of renal function. Enzyme-linked immunosorbent assay (ELISA) can also be used to detect urinary retinol-binding protein. 43 It has a range of detection of 25- 250/~g 3s K. Nomiyama, M. Yotoriyama, and H. Nomiyama, Arch. Environ. Contain. Toxicol. 12, 147 (1983). 39 C. Viau, A. Bernard, and R. Lauwerys, J. AppL ToxicoL 6, 185 (1986). 40 A. Bernard and R. Lauwerys, Clin. Chem. 29, 1007 (1983). 41 K. Nomiyama, H. Nomiyama, M. Yototiyama, and K. Matsui, Ind. Health 20, 11 (1982). 42 A. M. Bernard, D. Moreau, and R. Lauwerys, Clin. Chim. Acta 126, 1 (1982). 43 M. D. Topping, H. W. Forster, C. Dolman, C. M. Luczynska, and A. M. Bernard, Clin. Chem. 32, 1863 (1986).
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protein/liter urine. This method compares favorably to the commercially available test kits for fl2-microglobulin. 44 The assays described above can be utilized in both the clinical or laboratory setting. Metallothionein. A third protein that may be used as an indicator of cadmium exposure is metallothionein (MT). 42 A significant correlation between urinary cadmium and urinary MT levels has been seen as a more accurate indicator than either fl2-microglobulin or retinol-binding protein because MT binds the cadmium directly and plays a central role in the biology of this metal in vivo. 45 Preliminary studies have considered several routes of exposure to cadmium (oral and injection), while trying to determine the utility of urinary MT as a biological indicator of cadmium exposure. Studies considering both routes have found favorable results for the use of urinary MT as a biomonitoring too1. 46,47 The assays recommended for MT are radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). Monoclonal and polyclonal antibodies are used for both assays. The RIA method has been found to be slightly more accurate, while the ELISA is faster. The RIA method is also recommended for detection of low levels of MT. 48 This method is a double-antibody assay using radiolabeled MT. Competition between labeled and unlabeled MT is measured via scintillation counting to determine a curve that is compared to a standard to determine the amount of MT present in a sample. 49 The ELISA is a similar procedure that is not as accurate but can be useful for faster results. 5° Another possible biological indicator of cadmium exposure is N-acetylfl-D-glucosaminidase (NAG). Work to determine the usefulness of NAG is still in preliminary stages. NAG, like MT, is more closely related to the cadmium exposure than to the damage done by the exposure. In this sense, these indicators may be more specific to cadmium than fl2-microglobulin and retinol-binding protein levels which may be caused by any agent causing renal tubular damage. 5~,52 Problems with the use of proteinuria as a biological indicator of cadmium exposure are the possibility of delayed development and uncertain 44 A. M. Bernard, D. Moreau, and R. Lauwerys, Clin. Chem. 28, 1167 (1982). 45 R. R. Lauwerys, A. Bernard, H. A. Roels, J. P. Buchet, and C. Viau, Environ. Health Perspect. 54, 147 (1984). 46 Z. A. Shaikh, K. M. Harnett, S. A. Perlin, and P. C. Huang, Experientia 45, 146 (1989). 47 M. Sato, Y. Nagai, and I. Bermner, Toxicology6, 23 (1989). 48 M. P. Waalkes, J. S. Garvey, and C. D. Klaassen, ToxicoL Appl. PharmacoL 79, 524 (1985). 49 j. S. Garvey, R. J. Vander Mallie, and C. C. Chang, this series, Vol. 84, p. 121. 50 D. G. Thomas, H. J. Linton, and J. S. Garvey, J. Immunol. Methods 89, 239 (1986). 5~ T. Kawand, C. Tohyama, and S. Suzuki, Int. Arch. Occup. Environ. Health 62, 95 (1990). 52 C. Tohyama, Y. Mitane, E. Kobayashi, N. Sugihira, A. Nakano, and H. Saito, J. Appl. Toxicol. 8, 15 (1988).
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relationship to other manifestations of renal disease. Proteinuria is an early sign of cadmium exposure; however, at subchronic levels it is far more difficult to initially detect due to other signs of renal dysfunction that may be occurring concomitantly. The physiological mechanism of cadmium toxicity in the sense of renal damage seems to be progressive. Many epidemiological studies at a variety of exposure levels have noted an increase in the degree of proteinuria 53 during followup evaluations. This problem appears to be more a complication of progressive cadmium toxicity than a problem with the use ofproteinuria as an indicator. In the future this problem may be bypassed if assays for MT and NAG prove to be as accurate and early at indicating cadmium exposure as they appear at present. By more fully understanding the mechanism of toxicity of cadmium, biological indicators of exposure will be much easier to develop. 53 H. A. Rods, R. R. Lauwerys, J. P. Buchet, A. M. Bernard, A. Vos, and M. Oversteyns, Br. J. Ind. Med. 46, 755 (1989).
[67]
I N D U C T I O N OF M E T A L L O T H I O N E I N S
[67]
Biological Indicators of Cadmium
Exposure
By JUDITH A. GLAVEN, ROBIN E. GANDLEY, a n d BRUCE A. FOWLER Introduction Cellular exposure to cadmium is known to elicit a number of responses in addition to metallothionein induction. A mechanistic understanding of these processes may permit development of biological indicators of cadmium exposure. This approach may be applied to both mammalian and nonmammalian tissues. This chapter will examine a number of these methods and attempt to evaluate their utility for monitoring how cells respond to this metal at the organelle and molecular levels of biological organization. Ideally, such indicators should provide early, sensitive, and readily measurable responses that are specific for cadmium. In order for a potential indicator to be accepted as an indicator of effect, it must ultimately show a mechanistic correlation with the biological activity of this metal in living tissues. As discussed below, there are a number of potential candidate indicators of exposure and effect for cadmium that have each shown some promise for monitoring the actions of cadmium in cells. Indicators of Cadmium Exposure Total Tissue Burden In addition to cadmium induction of metallothionein (MT), monitoring of total tissue cadmium burdens via either atomic absorption spectroscopy ~,2or in vivo neutron activation analyses3,4 may provide useful data on cadmium exposure. The atomic absorption spectroscopy methods involve sampling and digestion of tissues prior to analysis and hence are of limited value for monitoring living organisms. These analyses are frequently conducted on autopsy samples of target tissues such as the kidney5 or for R. Lauwerys, H. Roels, M. Regniers, J. P. Buchet, A. Bernard, and A. Goret, Environ. Res. 20, 375 (1979). : R. Lauwerys, J. P. Buchet, and H. Roehls, Inst. Arch. Occup. Environ. Health 26, 275 (1976). 3 K. J. Ellis, W. D. Morgan, I. Zanai, S. Yasumura, D. Vartsky, and S. H. Cohn, J. Toxicol. Environ. Health 7, 691 (1981). 4 H. A. Roels, R. R. Lauwerys, J. P. Buchet, A. Bernrd, D. R. Chettle, T. C. Harvey, and I. K. AI-Haddad, Environ. Res. 26, 217 (1981). C. G. Elinder, T. Kjellstrom, L. Friberg, B. Lind, and L. Linnman, Arch. Environ. Health 31,292 (1976).
METHODS IN ENZYMOLOGY, VOL. 205
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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nonmammalian aquatic organisms such as mussels, 6 where field sampling methods may be employed to assess cadmium exposure of populations. In vivo neutron activation analyses of renal cadmium concentrations in exposed workers is another potentially useful methodology 3,4 but further studies are needed to validate this approach with regard to direct wet chemical analyses, because the technique appears to be sensitive to geometric considerations. Mineral Concretions X-Ray microanalysis and atomic absorption spectroscopy are potentially useful monitoring tools for detecting cadmium in mineral concretions of marine mollusks. 7- i1 In these species, the concretions represent a larger intracellular storage compartment than the metallothionein pool 7,8 and have the potential advantage of being extensively excreted from the kidney when cadmium exposure exceeds the tissue capacity to store this metal. 8,1~ This response may thus also represent an indicator of cadmium toxicity. Disadvantages of monitoring concretions are that exposure to other metals such as copper will also cause this phenomenon, 1~ so that there is a lack of specificity for cadmium in this response if other metals are also present. Stress Proteins Prokaryotic and eukaryotic cells respond to heat shock and various other forms of stress, including metal exposure, by synthesizing a discrete set of proteins.12-16 These molecules were first observed in cells exposed to conditions of thermal stress and as a result have been termed heat-shock proteins (hsps). 12'~3 Subsequently, it has been found that various forms of 6 E. D. Goldberg, M. Koide, V. Hodge, A. R. Fleagal, and J. H. Martin, Estuarine Coastal ShelfSci. 16, 69 (1983). 7 N. G. Carmichael, K. S. Squibb, and B. A. Fowler, J. Fish. Res. Bd. Can. 36, 1149 (1979). 8 N. G. Carmichael and B. A. Fowler, Mar. Biol. 65, 35 (1981). 9 S. G. George and B. J. S. Pirie, Bioehem. Biophys. Acta 5811, 234 (1979). ~oS. G. George, B. J. S. Pirie, and T. L. Combs, J. Exp. Mar. Biol. EcoL 42, 143 (1980). tmB. A. Fowler and E. Gould, Mar. Biol. 96, 207 (1988), t2 A. Tissieres, H. K. Mitchell, and V. M. Tracey, J. MoL Biol. 84, 389 (1974). ~3 M. Ashburner and J. J. Bonner, Cell. (Cambridge, Mass.) 17, 241 (1979). ~4S. Lindquist, Annu. Rev. Biochem. 55, 1151 (1986). z5 W. J. Welch, J. I. Garrels, and J. R. Fermaisco, in "Heat Shock from Bacteria to Man" (M. J. Schlesinger, M. Ashburner, and A. Tissieres, eds.), p. 257. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ~6W. J. Levinson, H. Opperman, and J. Jackson, Bioehem. Biophys. Acta 606, 170 (1980).
594
INDUCTION OF METALLOTHIONEINS
[67]
stress, including drugs, 17 amino acid analogs ~7,~s and metals, 16 induce the expression of these molecules, which are now more commonly termed the stress proteins. This response is highly conserved and presumed to be universal, serving a general protective function in the stressed cell. Principal stress proteins have molecular masses ranging from 28,000174,000 Da. Of these, those between 70,000 and 90,000 are seen in response to most situations of stress; the other stress proteins appear to be induced more selectively. The occurrence of specific protein responses to specific conditions of stress may have great utility as biological indicators of exposure, and in elucidating the mechanisms of toxicity at the subcellular level. Specific patterns of protein synthesis can be detected through metabolic labeling of proteins and standard techniques of two-dimensional gel electrophoresis. ~9-2~ The technique consists of an isoelectric separation in the first dimension and separation based on molecular weight in the second dimension. With the development of computerized digitizing image analysis systems, autoradiographs of the gels may be evaluated in a more quantitative manner. Because of the environmental significance of cadmium there is an interest in identifying early indicators of exposure and delineating their relationships to mechanisms of action. Levinson et al. described the induction of four proteins in chicken and human cell lines in response to several transition series metals, including cadmium. ,6 Along with the induction of hsp70, several low-molecular-mass proteins between 25,000 and 35,000 were induced. In a study examining the effect of sublethal acute doses of cadmium on Drosophila cells, it was shown that some of the induced proteins were the same as those seen in heat-shocked cells. However, cadmium also stimulated the synthesis of several other unique low-molecular-weight proteins. 2° These proteins are thus candidates as biomarkers of exposure to this element. There are several other examples of stress proteins detected in response to cadmium. Induced gene expression in two fish cell lines exposed to cadmium has been investigated by Price-Haughy and Gedamu22; they described the induction of metaUothionein (MT), a 14,000 metal-inducible protein, and several stress proteins with molecular masses between 28,000 and 29,000 in response to cadmium. A number of studies have been focused on the biological function of those molecules in an effort to better understand their roles in cell survival ~7L. E. Hightower, J. Cell. Physiol. 102, 407 (1980). 18 p. M. Kelley and M. J. Schlesinger, Cell (Cambridge, Mass.) 15, 1277 (1978). 19 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). 20 A. Courageon, C. Maisonhaute, and M. Best-Belpomme, Exp. CellRes. 153, 515 (1984). 21 y. Aoki, M. M. Lipsky, and B. A. Fowler, Toxicol. Appl. PharmacoL 106, 462 (1990). 22 j. Price-Haughey and L. Gedamu, Experientia, Suppl. 52, 465 (1987).
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during chemical or physical stress. Riabowol et al. demonstrated the importance of stress proteins to the cell while examining the function of the heat shock protein with molecular mass 70,000 (hsp70) in rat fibroblasts. 23 Cells injected with monoclonal antibodies raised against hsp70 were unable to withstand heat-shock treatment. This work shows that the protein has an important protective function in the stressed cell. A stress protein seen in response to cadmium with a relative molecular mass of 34,000 has been identified as heme oxygenase. 24 Specific antibodies for human heme oxygenase were used to identify the protein by an immunoblot technique. 25 These data suggest that the rate-limiting enzyme in the oxidative metabolism of heme 26 may be of importance during early response to sublethal injury. The direction of future research in this area will be to identify molecular triggers for stress response following exposure to cadmium, and to relate specific stress proteins to adaptation/protection in the stressed cell. Research that may provide further information in this direction includes DNA footprinting techniques to examine the trigger for the induction of a specific stress protein by identifying DNA-binding proteins, 27 and various immunological techniques to determine the intracellular location of specific stress proteins during and after the stress. 15,28As specific stress proteins induced in response to cadmium exposure are identified and examined from the perspective of the role they play in the protection of the cell, a more basic understanding of the mechanisms of cadmium biological activity will emerge.
Metallothionein Gene-Binding Factors The significance of metallothionein (MT) as an indicator of cadmium exposure is discussed later in this chapter. Briefly, we would like to describe cellular factors involved in the regulation of MT, because they may serve as indicators of cadmium exposure prior to MT induction. Investigation into the structure and function of genes encoding for MT have identified trans-acting nuclear factors (NF) involved in the regulation of MT expression. 29,3° Cadmium induces the formation of complexes of NF with metal regulatory elements (MRE) of the M T gene. Initially it was hoped that exposure to cadmium would cause detectable, diagnostic increases in the 23 K. T. Riabowol, L. A. Mizzen, and W. J. Welch, Science 242, 433 (1988). 24 S. Taketani, H. Kohno, T. Yoshinga, and R. Tounaga, FEBS Lett. 245, 173 (1989). 25 H. Towbin, T. Stahelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 26 M. D. Maines, FASEBJ. 2, 2557 (1988). 27 D. J. Galas and A. Schmitz, Nucleic Acids Res. 5, 3157 (1978). 28 W. J. Welch and L. A. Mizzen, J. Cell Biol. 106, 117 (1988). 29 C. Seguin, B. K. Felber, A. D. Carter, and D. Hamer, Nature(London) 312, 781 (1984). 30 C. Seguin and D. Hamer, Science 235, 1383 (1987).
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cellular concentration of NF; unfortunately, however, comparisons of cellular concentrations of MRE-binding NF in cells grown in the presence and absence of cadmium showed that cadmium does not act by increasing the synthesis of these proteins) ° Instead, cadmium may act on NFs directly to increase their affinity for the MRE of the gene, or cadmium may act by altering protein-protein interactions between the NF and some as yet unidentified "coactivator" protein? ° If this is the case it would be necessary to detect the cadmium-induced change in the NF, or the induction of the "coactivator." The use of DNA footprinting 3°,27 to reveal MREs of the M T gene protected from exonuclease activity due to bound NF, and synthetic oligonucleotides from the MRE sequence to detect DNA-binding proteins immobilized on blots, 32,33 has resulted in the description of two M T genebinding NFs with molecular masses of 74,000 and 108,000. 31,32 One of these, the 74K molecule, has been purified by affinity chromatography on trout MRE31; further characterization of this protein and its interaction with regulatory regions of the mouse M T gene are awaited. Proteinuria
Cadmium-induced tubular proteinuria can be used as an early indicator of cadmium exposure and effect. Alterations in renal function produced by cadmium can be due to alterations in tubular reabsorption or glomerular filtration. A distinctive low-molecular-weight proteinuria has been associated with elevated cadmium exposure and is characterized by an increase in low-molecular-weight urinary proteins and a decrease in high-molecular-weight proteins, suggesting damage to the renal proximal tubule. 34 Alternatively, another possible effect of cadmium exposure is a decrease in glomerular permeability to larger proteins. 35,36 Studies by Squibb et al. 37 provided a mechanistic basis for development of cadmiuminduced proteinuria and its use as an early indlcator of cadmium exposure following parenteral administration of cadmium-metallothionein (CdMT) to rats. Since Cd-MT is the major form of cadmium in the circula3, j. Imbert, M. Zafarullah, V. C. Culotta, L. Gedamu, and D. Hamer, Mol. Cell. Biol. 9, 5315 (1989). 32C. Seguinand J. Prevost, Nucleic Acids Res. 22, 1052 (1988). 33W. K. Miskimins,M. P. Roberts, A. McClelland,and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 82, 6741 (1985). 34p. L. Goering,K. S. Squibb, and B. A. Fowler, TraceSubst. Environ. Health 19, 22 (1985). 35A. Bernard, H. Rods, G. Hubermont, J. P. Buchet, P. L. Masson, and R. Lauwerys,Int. Arch. Occup. Environ. Health 38, 19 (1976). 36A. Bernard, J. P. Buchet,H. Roels,P. Masson,and R. Lauwerys,Eur. J. Clin. Invest. 9, 11 (1979). 37K. S. Squibb, J. B. Pritchard, and B. A. Fowler,J. PharmacoL Exp. Ther. 229, 311 (1984).
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tion, this model mimicks the actual in vivo condition. Within 8 hr of Cd-MT administration, an increase in low-molecular-weight proteins was detected in the urine, via sodium dodecyl sulfate (SDS) gel electrophoresis, 37 that was temporally correlated to altered lysosome structure and function in renal proximal tubule cells. This study demonstrated the utility of proteinuria as a biological indicator and the relationship of the proteinuria to pathophysiological changes in target cell populations. Specific Urinary Proteins. The proteinuria induced by cadmium may not increase the overall urine protein content significantly until the cadmium exposure is quite extensive. Clinical diagnosis of proteinuria is based on detection of an increase in total protein in the urine. The methods used for early detection of cadmium-induced proteinuria must discriminate between high- and low-molecular-weight proteins in the urine. Immunological assays for fl2-microglobulin, retinol-binding protein, and urinary metallothionein are used to detect low-molecular-weight proteinuria. fl2-Microglobulin, fl2-Microglobulin is used to monitor tubule reabsorption capabilities. There are several problems with using this protein to detect proteinuria. First, there is an increase in fl2-microglobulin in the urine of adults, beginning at the age of 50 years. 3s Second, fl2-microglobulin is unstable at urine pH < 5.5. The most common assay for fl2-microglobulin is a radioimmunoassay based on latex particle agglutination. Samples are chromatographed on Sephadex G-75 and fractions are pooled and concentrated, using ultrafiltration. The sample is then analyzed via immunoassay using an antiserum and immunoglobulin G-coated latex particles. 39 The samples can be read via an automated latex immunoassay system.4° Another common protein assay used for cadmium exposure is an electrophoretic technique using silver staining with sodium dodecyl sulfate-polyacrylamide gel. 41 A detection level of 0.8 mg protein/liter urine can be achieved with this technique. Retinol-Binding Protein. Retinol-binding protein (RBP) differs from fl2-microglobulin in that it is stable in urine of pH 4.5 and higher, and its degradation is slower.42 Like fl2-microglobulin, RBP is present at elevated levels in older human subjects due to an overall loss of renal function. Enzyme-linked immunosorbent assay (ELISA) can also be used to detect urinary retinol-binding protein. 43 It has a range of detection of 25- 250/~g 3s K. Nomiyama, M. Yotoriyama, and H. Nomiyama, Arch. Environ. Contain. Toxicol. 12, 147 (1983). 39 C. Viau, A. Bernard, and R. Lauwerys, J. AppL ToxicoL 6, 185 (1986). 40 A. Bernard and R. Lauwerys, Clin. Chem. 29, 1007 (1983). 41 K. Nomiyama, H. Nomiyama, M. Yototiyama, and K. Matsui, Ind. Health 20, 11 (1982). 42 A. M. Bernard, D. Moreau, and R. Lauwerys, Clin. Chim. Acta 126, 1 (1982). 43 M. D. Topping, H. W. Forster, C. Dolman, C. M. Luczynska, and A. M. Bernard, Clin. Chem. 32, 1863 (1986).
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protein/liter urine. This method compares favorably to the commercially available test kits for fl2-microglobulin. 44 The assays described above can be utilized in both the clinical or laboratory setting. Metallothionein. A third protein that may be used as an indicator of cadmium exposure is metallothionein (MT). 42 A significant correlation between urinary cadmium and urinary MT levels has been seen as a more accurate indicator than either fl2-microglobulin or retinol-binding protein because MT binds the cadmium directly and plays a central role in the biology of this metal in vivo. 45 Preliminary studies have considered several routes of exposure to cadmium (oral and injection), while trying to determine the utility of urinary MT as a biological indicator of cadmium exposure. Studies considering both routes have found favorable results for the use of urinary MT as a biomonitoring too1. 46,47 The assays recommended for MT are radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). Monoclonal and polyclonal antibodies are used for both assays. The RIA method has been found to be slightly more accurate, while the ELISA is faster. The RIA method is also recommended for detection of low levels of MT. 48 This method is a double-antibody assay using radiolabeled MT. Competition between labeled and unlabeled MT is measured via scintillation counting to determine a curve that is compared to a standard to determine the amount of MT present in a sample. 49 The ELISA is a similar procedure that is not as accurate but can be useful for faster results. 5° Another possible biological indicator of cadmium exposure is N-acetylfl-D-glucosaminidase (NAG). Work to determine the usefulness of NAG is still in preliminary stages. NAG, like MT, is more closely related to the cadmium exposure than to the damage done by the exposure. In this sense, these indicators may be more specific to cadmium than fl2-microglobulin and retinol-binding protein levels which may be caused by any agent causing renal tubular damage. 5~,52 Problems with the use of proteinuria as a biological indicator of cadmium exposure are the possibility of delayed development and uncertain 44 A. M. Bernard, D. Moreau, and R. Lauwerys, Clin. Chem. 28, 1167 (1982). 45 R. R. Lauwerys, A. Bernard, H. A. Roels, J. P. Buchet, and C. Viau, Environ. Health Perspect. 54, 147 (1984). 46 Z. A. Shaikh, K. M. Harnett, S. A. Perlin, and P. C. Huang, Experientia 45, 146 (1989). 47 M. Sato, Y. Nagai, and I. Bermner, Toxicology6, 23 (1989). 48 M. P. Waalkes, J. S. Garvey, and C. D. Klaassen, ToxicoL Appl. PharmacoL 79, 524 (1985). 49 j. S. Garvey, R. J. Vander Mallie, and C. C. Chang, this series, Vol. 84, p. 121. 50 D. G. Thomas, H. J. Linton, and J. S. Garvey, J. Immunol. Methods 89, 239 (1986). 5~ T. Kawand, C. Tohyama, and S. Suzuki, Int. Arch. Occup. Environ. Health 62, 95 (1990). 52 C. Tohyama, Y. Mitane, E. Kobayashi, N. Sugihira, A. Nakano, and H. Saito, J. Appl. Toxicol. 8, 15 (1988).
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relationship to other manifestations of renal disease. Proteinuria is an early sign of cadmium exposure; however, at subchronic levels it is far more difficult to initially detect due to other signs of renal dysfunction that may be occurring concomitantly. The physiological mechanism of cadmium toxicity in the sense of renal damage seems to be progressive. Many epidemiological studies at a variety of exposure levels have noted an increase in the degree of proteinuria 53 during followup evaluations. This problem appears to be more a complication of progressive cadmium toxicity than a problem with the use ofproteinuria as an indicator. In the future this problem may be bypassed if assays for MT and NAG prove to be as accurate and early at indicating cadmium exposure as they appear at present. By more fully understanding the mechanism of toxicity of cadmium, biological indicators of exposure will be much easier to develop. 53 H. A. Rods, R. R. Lauwerys, J. P. Buchet, A. M. Bernard, A. Vos, and M. Oversteyns, Br. J. Ind. Med. 46, 755 (1989).