Journal of Toxicology and Environmental Health

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Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression G. N. Cosma , D. Currie , K. S. Squibb , C. A. Snyder & S. J. Carte To cite this article: G. N. Cosma , D. Currie , K. S. Squibb , C. A. Snyder & S. J. Carte (1991) Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression, Journal of Toxicology and Environmental Health, 34:1, 39-49, DOI: 10.1080/15287399109531547 To link to this article: http://dx.doi.org/10.1080/15287399109531547

Published online: 19 Oct 2009.

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DETECTION OF CADMIUM EXPOSURE IN RATS BY INDUCTION OF LYMPHOCYTE METALLOTHIONEIN GENE EXPRESSION G. N. Cosma, D. Currie, K. S. Squibb, C. A. Snyder, S. J. Carte

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Institute of Environmental Medicine, New York University Medical Center, New York, New York

The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute and chronic CdCl2 exposures resulted in the induction of the MT-1 gene in lymphocytes as measured by standard RNA Northern blot analysis. Twenty-four hours following an ip injection of 3.4 mg/kg CdCl2, a ninefold increase in MT gene expression was observed in lymphocytes, as well as five- and sevenfold increases in liver and kidney, respectively. Oral exposure of rats to 1-100 ppm CdCl2 via drinking water resulted in an approximate twofold enhanced MT signal in lymphocytes after 6 wk, and a threefold increase after 13 wk of exposure to 100 ppm Cd. No increases in lymphocyte MT gene expression were observed after 3 wk of Cd exposure. Liver MT gene expression was substantially induced following chronic Cd exposure, while kidney was not, although this organ had a higher basal expression of the MT-1 gene. Analysis of tissue Cd burdens demonstrated a dose-response Cd accumulation in liver and kidney, but only kidney burdens increased substantially with prolonged Cd exposure. These results demonstrate the utility of lymphocyte gene expression assays to detect in vivo toxicant exposure, and thus their applicability as molecular biomarker assays for human exposure assessment.

INTRODUCTION The development of biological markers capable of detecting exposure to toxicants is an important focus of current environmental research (Perera, 1987). Our laboratory has been involved recently in the development of biomarkers of toxicant exposure in animal models, with the ultimate goal of applying these assays to the relatively noninvasive screening of human populations exposed to environmental toxicants. Our approach has been to apply the principles of molecular biology in Support for this work came from NIH grants ES 04895, ES 00260, and CA 13343. Requests for reprints should be sent to S. J. Carte, Institute of Environmental Medicine, New York University Medical Center, 550 First Ave., New York, NY 10016.

39 Journal of Toxicology and Environmental Health, 34:39-49, 1991 Copyright © 1991 by Hemisphere Publishing Corporation

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C. N. COSMA ET AL.

order to develop sensitive measures of exposure assessment, and to determine the genetic susceptibility within an exposed population. The induction of genes whose products function in detoxification processes is a well-characterized cellular response to chemical challenge (Amsbaugh et al., 1986; Jaiswal et al., 1985). We have taken advantage of this response by developing molecular assays that will detect inducible gene expression in peripheral blood lymphocytes. These cells have obvious advantages for use in the development of noninvasive assays to screen human populations for toxicant exposures. Furthermore, the applicability of these cells as a surrogate cell type in which to measure a biological end point that is indicative of toxicant exposure in vivo has been amply documented in the recent scientific literature (Lucier and Thompson, 1987; Harris, 1989). Lymphocytes have been shown to possess a full complement of genes whose protein products are involved in the metabolism and detoxification of xenobiotic compounds. For example, several members of the cytochrome P-450 gene family are expressed by both animal and human lymphocytes, including the P-450IA family (Amsbaugh et al., 1986; Nebert and Jaiswal, 1987), as well as the glutathione Stransferase gene family, such as the GST-pi gene (Kano et al., 1987), and the GST-mu gene (Coles and Ketterer, 1990). A cellular response to heavy metal exposure that has been studied extensively in vivo and in vitro is the induction of a class of metalbinding proteins, the metallothioneins (MT) (Klaassen and LehmanMcKeeman, 1989). There are two major classes of MT proteins, MT-I and MT-II, and they have been found to be encoded for by separate genes (Searle et al., 1984). These proteins function in the detoxification of metals such as cadmium and mercury by avidly binding them in the tissues in which they accumulate. The major sites of cadmium deposition following in vivo exposure in mammals are the liver and kidney (Klaassen and Lehman-McKeeman, 1989). Many reports have documented the synthesis of metallothionein proteins in these, as well as other, organs following Cd exposure (Lehman-McKeeman and Klaassen, 1987). Furthermore, studies conducted in vitro have demonstrated the induction of MT gene expression in a variety of cell types in response to Cd treatment Gahroudi et al., 1990). In addition to Cd accumulation in liver and kidney, there is a measurable amount of Cd found in whole blood following chronic exposure that is associated principally with nucleated blood cells (Nordberg et al., 1971). In vitro studies have shown that these cells, particularly lymphocytes, are capable of induction of MT gene expression in response to cadmium treatment (Enger et al., 1983; Harley et al., 1989). The purpose of this study was to determine whether Cd exposure in vivo can be detected by measuring the induction of MT gene expression in lymphocytes of exposed animals. The relative sensitivity of this response was compared to liver and kidney responses and Cd burdens in these samples.

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MATERIALS AND METHODS Cadmium Exposures

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Male Fischer 344 rats, approximately 8 wk old (Charles River, Wilmington, Del.), were exposed to CdCI2 acutely by ip injection, or chronically in their drinking water, ad libitum. Chronic Cd exposures were routinely monitored by water consumption and body weights. These indices were not rigorously analyzed; rather, they were used as sentinels for possible interindividual differences in exposures, along with differences between exposure groups. Cell and Tissue Isolation Splenic and peripheral blood lymphocytes were isolated by Percoll separation media (Histopaque, Sigma, St. Louis, Mo.). Briefly, whole blood or splenic cell suspensions were layered on top of Percoll media and lymphocytes were isolated by centrifugation at 1000 x g for 20 min. Cells were washed once with RPMI nutrient media (Gibco, Long Island, N.Y.) and then frozen at -70°C until RNA extraction. Livers and kidneys were likewise frozen before RNA extraction. RNA Analysis Total cellular RNA was isolated from lymphocytes and tissues by a rapid guanidinium-phenol extraction method (Chirgwin et al., 1979) (RNAzol, Cinna/Biotecx; Tex.). RNA quantity and purity was determined by spectrophotometric absorbances. For Northern blot analysis, RNA was denatured by glyoxalation (Thomas, 1980), electrophoresed through 1% agarose gels, and blotted onto nylon membrane filters (Zeta-Bind, AMF-Cuno, Meriden, Conn.) by capillary action. For slot-blot analysis, the glyoxalated RNA was applied directly to nylon filters in a vacuum manifold (Schleicher and Schull, Keene, Conn.). Membrane-bound RNA was hybridized to nick-translated 32P-Iabeled cDNA probes (Thomas, 1980). The MT-1 probe was provided by Dr. H. Herschman, UCLA, and /3-actin was purchased from ATCC, Bethesda, Md. Following hybridization, filters were washed to a final stringency of 0.5 x standard saline citrate (SSC) plus 0.1% sodium dodecyl sulfate (SDS) at 65°C for 30 min. RNA was visualized by film autoradiography (Kodak XAR-5) at — 70°C using intensifying screens (DuPont Cronex Lightning-Plus). Autoradiogram signal strengths were quantitated by the measurement of optical densities with a scanning laser densitometer (LKB Ultroscan). All MT gene expression results were normalized to actin expression, which served as an internal control to ensure that artifacts such as unequal loading of RNA onto gels or filters were not responsible for any observed differences in signal strengths. Statistical analysis of lymphocyte MT gene expression was accomplished by analysis of variance followed by Dunnett's two-sided test.

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Cadmium Analysis Lymphocytes (~50 x 106 cells) and tissues (0.5-3.0 g) were wet-ashed using concentrated nitric acid, and Cd concentrations were measured by atomic absorption spectrophotometry at a wavelength of 228.8 nm using an air/acetylene flame and deuterium background correction.

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RESULTS To determine the feasibility of using gene expression assays in lymphocytes to detect in vivo exposure to cadmium, we first exposed rats acutely by ip injection of 3.4 mg/kg CdCI2, and 24 h later isolated splenic and peripheral lymphocytes along with liver and kidney samples. Figure 1 demonstrates the induction of MT gene expression as a result of acute Cd exposure in vivo in splenic lymphocytes by Northern blot analysis. An approximate ninefold induction of MT gene expression was observed in both splenic and peripheral lymphocytes. These initial studies demon-

Lymphocyte MT Gene Expression Oral: 6wk Cd

i p : 24h Cd

MT-

ppm

0

1

1

10 10 30 30 100 100

0

3.4

mg/kg

3.4

mg/kg

Actin

ppm

FIGURE 1. Induction of MT-1 gene expression in lymphocytes from Cd-exposed rats as determined by Northern blot RNA analysis. Following extraction and glyoxal denaturation of RNA, 15-pg samples were electrophoresed through 1% agarose gels, transferred and immobilized onto nylon membrane filters, and then hybridized to the MT-1 cDNA probe. Filters were washed to a final stringency of 0.5X SSC + 0.1% SDS before autoradiography was performed at -70°C for 3-7 d using Kodak XAR-5 film plus Cronex Lightning-Plus intensifying films (DuPont). Following autoradiography, filters were stripped and rehybridized to 0-actin.

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E 2

•a

"

o +i ~ c 75 .05). No increases in MT gene expression were observed after 3 wk of Cd exposure (data not shown). In addition to measuring MT gene expression in lymphocytes following Cd exposure, liver and kidney MT gene expression was determined after chronic (oral) and acute (ip) Cd exposures by Northern and slot-blot RNA analyses as shown in Figures 3 and 4, respectively. It was apparent that kidney displayed a higher basal MT-1 gene expression level than did liver, regardless of Cd exposure. Both organs demonstrated inducible MT gene expression following acute Cd exposure as shown in Figure 3 (fiveand sevenfold induction for liver and kidney, respectively). Table 1 summarizes tissue MT responses to chronic Cd exposure by providing normalized MT gene expression values for liver and kidney following 6- and 13-wk exposures. In contrast to acute exposure, only liver demonstrated a substantial induction of MT gene expression, with an approximate 12fold induction after 6 wk of exposure to 100 ppm Cd and a fourfold induction following 13 wk of exposure. The decrease in liver MT induction following prolonged Cd exposure appeared related primarily to an increase in control gene expression, as is evidenced by comparing 0 ppm liver MT gene responses at 6 versus 13 wk of exposure from Table 1. Table 2 provides elemental analysis of Cd burdens by atomic absorption spectroscopy in livers and kidneys following chronic exposure. Both organs displayed a dose-response accumulation of Cd following exposure; however, only kidney Cd levels increased substantially with exposure duration (6 vs. 13 wk). In addition to organ Cd burdens, we attempted to measure Cd levels in lymphocytes by AA spectroscopy but were unable to detect Cd as a result of these exposures (detection limit = 0.005 ^g Cd/106 lymphocytes). DISCUSSION The purpose of these studies was to determine the feasibility of using gene expression assays to detect toxicant exposure in vivo. To this end, we chose to measure the induction of the metallothionein gene in lymphocytes of rats exposed in vivo to cadmium. The MT gene expression results that were obtained from both acute and chronic Cd exposures demonstrated that the assay is capable of detecting toxicant exposure in lymphocytes. Our ultimate goal is to apply these assays to the noninvasive screening of human populations with a suspected exposure to environmental toxicants. Several conclusions can be drawn from the comparison of responses that were obtained in these studies as a result of chronic and acute Cd exposures: (1) Lymphocyte as well as organ MT gene expression was induced to a greater extent as a result of acute versus chronic Cd exposure. (2) Prolonged chronic Cd exposure (13 vs. 6 wk) did not result in a

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Tissue MT Gene Expression Oral: 11wkCd

ip :24hCd

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0.5 kb

ppm

O L

O 100 100 K L K

3.4mg/kg L K

FIGURE 3. Northern blot RNA analysis of MT-1 gene expression in liver (L) and kidney (K) of Cdexposed rats. Northern analysis was carried out as described in Fig. 1.

substantial increase in either lymphocyte or organ MT gene expression, and in fact liver responses decreased after 13 wk of exposure. (3) Induction of the MT gene did not necessarily correlate with Cd concentrations present in the tissues, as is evidenced by comparison of Cd levels to MT gene expression between liver and kidney from chronically exposed animals. In regard to lymphocyte MT gene expression levels, pharmacokinetic studies of Cd deposition have demonstrated a plateau effect of Cd concentrations in blood following chronic exposure (Nordberg et al., 1971; Kjellstrom and Nordberg, 1978). These data may explain why we did not observe larger increases in lymphocyte MT gene expression after prolonged Cd exposure. On the other hand, lymphocytes have a finite life span in vivo (Sprent, 1989), and the cells, or at least a subpopulation of lymphocytes, may have undergone cell turnover during the course of these studies. Finally, some molecular mechanism of adaptation to the metal-induced gene expression response may have occurred in these cells, as was also evident from liver MT gene induction following prolonged Cd exposure. Taken as a whole, our data suggest a complex regulation of the MT gene by tissue-specific, as well as exposure routespecific, mechanisms. A further complication in understanding the mechanism of MT gene regulation in response to cadmium exposure is the lack of knowledge of the function and control of the synthesis of this protein in normal cell

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Tissue MT Gene Expression (ppm Cd) 0

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6 week

13 week

Kidney

Liver

FIGURE 4. Induction of MT-1 gene expression in liver and kidney of Cd-exposed rats as determined by slot-blot RNA analysis. Ten-microgram glyoxalated RNA samples were applied directly to nylon membrane filters under vacuum in a slot-blot manifold (Schleicher and Schull). Hybridization and autoradiography were performed as described for Northern analysis.

processes (Klaassen and Lehman-McKeeman, 1989). It is thought to be involved in zinc and copper homeostasis (Cousins, 1985), but it also appears to be regulated by immunological factors, such as the interleukins (Schroeder and Cousins, 1990), as well as by humoral factors such as glucocorticoids (Karin et al., 1980). The diversity of mediators of MT response make it difficult to assign a precise role for Cd in the induction of the MT gene; however, the present studies, along with in vitro studies (Enger et al., 1983), have clearly shown its involvement in the regulation of lymphocyte MT gene expression. Finally, to better understand the tissue specificity of the MT gene expression responses to Cd exposure, one must consider the kinetics of metallothionein turnover from protein that

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TABLE 1. Tissue MT Gene Expression Normalized MT mRNA (mean ± SD)a Liver

Kidney

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Cd dose

(ppm)

6 wk

0 1 10 30 100

0.08 0.10 0.18 0.33 1.02

13 wk ± ± ± ± ±

0.01 0.03 0.04 0.25 0.38

0.22 0.16 0.24 0.32 0.86

± ± ± ± ±

6 wk 0.03 0.07 0.05 0.11 0.08

0.34 0.26 0.32 0.59 0.54

13 wk ± ± ± ± ±

0.03 0.13 0.03 0.20 0.22

0.34 0.37 0.42 0.34 0.84

± ± ± ± ±

0.02 0.02 0.14 0.18 0.12

a Data were calculated as densitometry absorbances of MT-1 gene expression relative to actin expression (internal control); n = 2-3 animals/group.

may have been present prior to exposure. Our results demonstrate a significantly higher level of basal MT-1 gene expression in kidney versus liver, but kidney gene expression was not substantially induced by chronic Cd exposure, despite its accumulation of the metal. Similar results have been observed by the measurement of metallothionein protein in other laboratories (Sendelbach and Klaassen, 1988). It is possible that the level of preexisting MT protein, as a result of higher basal gene expression in the kidney, was adequate to bind and sequester the additional cadmium burden. (This same hypothesis may also explain the apparent decrease in liver MT gene induction after prolonged Cd exposure, since basal MT gene expression was also higher.) On the other hand, the relative lack of MT gene induction in kidney may also explain why this organ is a target of Cd-induced toxicity following chronic Cd exposure (Friberg et al., 1986). We chose oral exposure to Cd in drinking water as the route of adTABLE 2. Tissue Cadmium Concentrations a ^g Cd/g wet weight (mean ± SD)

Liver

Kidney

Cd dose (ppm)

6wk

13 wk

6wk

13 wk

0 1 10 30 100

n.d." n.d. 2.8 ± 0.2 7.7 ± 0.3 20.6 ± 1.6

n.d. n.d. 3.7 ± 0.7 12.4 ± 1.1 23.3 + 2.4

n.d.c n.d. 1.9 ± 0.5 7.4 ± 0.3 19.6 ± 1.5

n.d. n.d. 5.3 ± 0.7 17.0 ± 1.2 32.7 ± 4.7

a

n — 2-4 Animals/group. Not detectable; < 0.3 ng/g liver. c Not detectable; < 1.2 /*g/g kidney.

b

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ministration for our chronic studies in order to simulate a common human exposure route to cadmium. While there is more potential variability in this dose regimen, our objective in these studies was to develop and validate these molecular biomarker assays for their ultimate use as toxicant screening assays in human populations. It has been estimated that only 1% of orally administered Cd is gastrointestinally absorbed in the rat; humans absorb 3-6 times more (Decker et ah, 1957; Kjellstrom and Nordberg, 1978). Therefore, the sensitivity of our assay to detect Cd exposure in rats by measuring induction of MT gene expression in lymphocytes is underestimated by the evaluation of dose alone, and the potential to detect lower-level oral Cd exposure in humans may be even greater. In this regard it should be noted that the detection of Cd exposure in these studies by the induction of lymphocyte MT gene expression occurred well below the accumulation of 200 ppm Cd in kidney, which is considered the threshold for the overt onset of Cd-induced nephrotoxicity (Friberg et al., 1986). We are currently adapting these gene expression assays for use in screening both control and exposed human populations as a first step in the emerging discipline of molecular epidemiology.

REFERENCES Amsbaugh, S. C., Ding, J., Swan, D. C., Popescu, N. C., and Chen, Y. 1986. Expression and chromosomal localization of the cytochrome P450 gene in human mitogen-stimulated lymphocytes. Cancer Res. 46:2423-2427. Chirgwin, J. M., Pryzbla, A. E., MacDonald, R. J., and Rutter, W. J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. Coles, B., and Ketterer, B. 1990. The role of glutathione and glutathione transferases in chemical carcinogenesis. Crit. Rev. Biochem. Mol. Biol. 25:47-70. Cousins, R. J. 1985. Absorption, transport, and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65:238-309. Decker, C. F., Byerrum, R. U., and Hoppert, C. A. 1957. A study of the distribution and retention of cadmium-115 in the albino rat. Arch. Biochem. Biophys. 66:140-157. Enger, M. D., Hildebrand, C. E., and Stewart, C. C. 1983. Cd responses of cultured human blood cells. Toxicol. Appl. Pharmacol. 69:214-224. Friberg, L., Elinder, C. C., Kjellstrom, T., and Nordberg, G. F. 1986. Critical organs, critical concentrations, and whole-body dose-response relationships. In Cadmium and Health: A Toxicological and Epidemiological Appraisal, eds. C. G. Elinder, L. Friberg, T. Kjellstrom, and G. F. Nordberg, vol. II, pp. 247-255. Boca Raton, Fla.: CRC Press. Harley, C. B., Menon, C. R., Rachubinski, R. A., and Nieboer, E. 1989. Metallothionein mRNA and protein induction by cadmium in peripheral blood lymphocytes. Biochem. J. 262:873-879. Harris, C. C. 1989. Interindividual variation among humans in carcinogen metabolism, DNA adduct formation, and DNA repair. Carcinogenesis 10:1563-1566. Jahroudi, N., Foster, R., Haughey, J. P., Beitel, G., and Gedamu, L. 1990. Cell-type specific and differential regulation of the human metallothionein genes. J. Biol. Chem. 265:6506-6511. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. 1985. Human P1450 gene sequence and correlation of mRNA with genetic differences in benzo[a]pyrene metabolism. Nucleic Acid Res. 13:45034520. Kano, T., Sakai, M., and Muramatsu, M. 1987. Structure and expression of a human class pi glutathione S-transferase messenger RNA. Cancer Res. 47:5626-5630.

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Karin, M., Andersen, R. D., Slater, E., Smith, K., and Herschman, H. H. 1980. Metallothionein mRNA induction in HeLa cells in response to zinc or dexamethasone is a primary induction response. Nature (Lond.) 286:295-297. Kjellstrom, T., and Nordberg, G. F. 1978. A kinetic model of cadmium metabolism in the human being. Environ. Res. 16:248-269. Klaassen, C. D., and Lehman-McKeeman, L. D. 1989. Induction of metallothionein. J. Am. Coll. Toxicol. 8:1315-1321. Lehman-McKeeman, L. D., and Klaassen, C. D. 1987. Induction of metallothionein-I and metallothionein-II in rats by cadmium and zinc. Toxicol. Appl. Pharmacol. 88:195-202. Lucier, G. W., and Thompson, C. L. 1987. Issues in biochemical applications to risk assessment: When can lymphocytes be used as surrogate markers? Environ. Health Perspect. 76:187-191. Nebert, D. W., and Jaiswal, A. K. 1987. Human drug metabolism polymorphisms. Pharmacol. Ther. 33:11-17. Nordberg, G. F., Piscator, M., and Nordberg, M. 1971. On the distribution of cadmium in blood. Acta Pharmacol. Toxicol. 30:189-295. Perera, F. 1987. The potential usefulness of biological markers of risk assessment. Environ. Health Perspect. 76:141-145. Schroeder, J. J., and Cousins, R. J. 1990. Interleukin-6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc. Natl. Acad. USA 87:3137-3141. Searle, P. F., Davison, B. L., Stuart, G. W., Wilkie, T. M., Norstedt, G., and Palmiter, R. D. 1984. Regulation, linkage, and sequence of mouse metallothionein I and II genes. Mol. Cell Biol. 4:1221-1230. Sendelbach, L. E., and Klaassen, C. D. 1988. Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmium-metallothionein. Toxicol. Appl. Pharmacol. 92:95-101. Sprent, J. 1989. T lymphocytes and the thymus. In Fundamental Immunology, ed. W. E. Paul, 2nd ed., pp. 69-93. New York: Raven Press. Thomas, P. S. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201-5205. Received October 1, 1990 Accepted March 8, 1991

Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression.

The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute...
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