Intestinal Absorption of Cadmium Is Associated with Divalent Metal Transporter 1 in Rats

Jung D. Park,1, Nathan J. Cherrington and Curtis D. Klaassen,2

Center for Environmental and Occupational Health, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 2018 Breidenthal Building, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7417

Received February 11, 2002; accepted April 8, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The intestinal absorption of cadmium (Cd) increases when the body iron (Fe) stores are depleted. The depletion of Fe upregulates the expression of divalent metal transporter 1 (DMT1), which is located at the apical membrane of enterocytes lining the small intestine. DMT1 has been shown to transport Fe and other divalent metal ions in vitro. However, it is not known whether DMT1 mediates the intestinal absorption of Cd. To investigate DMT1 involvement in Cd absorption, rats were fed a diet for 4 weeks either deficient in Fe (FeD diet, 2–6 mg Fe/kg) or supplemented with Fe (FeS diet, 120 mg Fe/kg), followed by a single oral administration of 109 CdCl2. Body Fe status, hemoglobin, and tissue Cd concentration were determined at 48 h after Cd administration. Also, DMT1 mRNA levels were quantified in duodenum, kidney, and liver by the branched DNA signal amplification method. Animals fed the FeD diet exhibited a reduced body weight gain, depletion of body Fe, and Fe deficiency anemia. Tissue Cd concentration was significantly higher in FeD than in FeS diet-fed rats, especially in the duodenum. The amount of Cd retained in the body was 10-fold higher in rats fed the FeD diet than in those fed the FeS diet. DMT1 mRNA was highly expressed in duodenum and was 15-fold higher in the FeD diet group. The levels of DMT1 mRNA were significantly lower in kidney and liver than in duodenum, but were 30 and 40% higher, respectively, in rats fed the FeD diet than in rats fed the FeS diet. These findings suggest that functional DMT1 protein is likely upregulated in the small intestine at the mRNA level by body iron depletion and increases Cd uptake from the gastrointestinal tract with subsequent transfer of Cd to the circulation and body tissues. Furthermore, the data from this study may indicate that DMT1 is a nonspecific metal transporter, which can transport not only Fe, but probably the toxic metal as well.

Key Words: absorption; cadmium; iron; divalent metal transporter 1 (DMT1); duodenum; tissue Cd concentration; total body Cd burden.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium (Cd), a toxic and nonessential metal, is a widespread environmental pollutant that causes damage to kidneys and bone in humans (Järup et al., 1998Go; Tsuchiya, 1969Go). Food is the major source of Cd exposure in the general population. In earlier experimental and epidemiological studies, the intestinal absorption of Cd was found to be increased whenever total body iron was depleted (Flanagan et al., 1978Go; Kowal, 1988Go; Ragan, 1977Go; Schümann et al., 1996Go). Iron deficiency is a worldwide health problem, especially for rapidly growing adolescents, pregnant women, and aged women. The mechanism of absorption of Cd in the gastrointestinal tract is unknown, and it is also not clear why and how iron deficiency increases Cd absorption in the intestine.

Iron plays an essential role in many biological processes, and it is important to maintain iron concentration within its narrow normal range because excess iron induces toxicity (Andrews, 1999aGo). Intestinal absorption of iron is regulated by the body’s store of iron and the rate of erythropoiesis in the bone marrow. Recently, a protein named divalent metal transporter 1 (DMT1), which is involved in transmembrane iron transport, was cloned, using 2 approaches: a functional study and a study using an animal model with a genetic defect, which resulted in a glycine-to-arginine missense mutation (G185R) in DMT1 (Fleming et al., 1997Go, 1998Go; Gunshin et al., 1997Go).

DMT1 encodes a protein with 12 putative transmembrane domains. DMT1 is the apical membrane iron transporter in intestinal epithelial cells, as well as the endosomal iron transporter in most mammalian cells. DMT1 expression in intestine is upregulated by iron deficiency states and in the paradoxical anemia of the genetic disorder hereditary hemochromatosis (Fleming et al., 1999Go; Gunshin et al., 1997Go). It was also shown that DMT1 has a broad substrate specificity favoring divalent metals, including Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+ in vitro (Gunshin et al., 1997Go).

During the last few years, studies have been performed on the localization and regulation of expression of DMT1 in laboratory animals (Canonne-Hergaux et al., 1999Go; Trinder et al., 2000Go; Ferguson et al., 2001Go). However, very little is known about the role of DMT1 in the intestinal absorption of toxic metals. Therefore, the purpose of the present study was to determine whether there is an increase in Cd absorption from the gastrointestinal tract in rats fed an iron-deficient diet, and whether this might be due to an increase in expression of DMT1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Young male Sprague-Dawley rats, aged 21 days, were purchased from Sasco (Omaha, NE). The animals were housed in our AAALAC-accredited facility at 22 ± 1°C with a 12-h light/dark cycle. Rats were provided standard rodent chow and tap water ad libitum for acclimatization to the environment.

Experimental design.
Iron deficiency was induced in the rats by feeding them a diet deficient in Fe. Animals were divided into 2 groups; one group was fed a Fe-deficient diet (FeD diet) and the other, an Fe-supplemented diet (FeS diet) (15 rats per group). The semisynthetic FeD and FeS diets, formulated by modification with ferrous sulfate and based on the recommendations of the American Institute of Nutrition (Bieri et al., 1977Go), were purchased from Harlan (Madison, WI). The FeD diet contained 2–6 mg of Fe per kg chow and the FeS diet, 120 mg of Fe per kg chow. These diets were fed to rats for 4 weeks and their drinking water was replaced with distilled-deionized water. The body weight of the animals was recorded twice weekly throughout the experimental period.

At the end of 4 weeks, the rats were fasted for 18 h followed by a single oral administration of Cd. Cadmium, labeled with a trace of 109CdCl2, was administered at a dose of 0.4 µmol CdCl2/kg in a volume of 4 ml/kg saline (Fischer Scientific, Fair Lawn, NJ). Diets were returned 4 h later.

Animals were decapitated 48 h after administration of Cd. Major organs (liver, kidney, lung, heart, brain, stomach, duodenum, jejunum, ileum, large intestine, testis, and bone) and blood were collected and weighed (luminal contents of the GI sections were removed). Parts of duodenum, liver, and kidney were immediately frozen in liquid nitrogen and stored at –80°C for DMT1 mRNA analysis. Two ml of blood was centrifuged to obtain serum.

Iron (Fe) and hemoglobin (Hb) assays.
Body iron status was analyzed by quantifying total iron, unsaturated iron binding capacity (UIBC), total iron binding capacity (TIBC), and transferrin saturation (TS, %) in serum. Total Fe and UIBC were measured colorimetrically, using a Sigma diagnostic kit (Sigma, St. Louis, MO). TIBC was calculated as the sum of total Fe, and UIBC and TS were determined as the fraction of TIBC in serum. Total Hb in blood was measured using a commercially available kit from Sigma.

Quantification of Cd.
Tissue 109Cd content was quantified with a Packard Model 5130 auto-{gamma} scintillation spectrometer (LaGrange, IL). To estimate the Cd content in bone and blood, they were assumed to constitute 6.7 and 7%, respectively, of body weight (Kreppel et al., 1994Go). Total body burden of Cd was calculated by summing up the Cd content in all the tissues analyzed. The relative body burden of Cd was normalized by body weight. The absorption index of Cd (%) was calculated from the following formula:

DMT1 mRNA analysis.
Analysis of DMT1 mRNA was performed by the branched DNA (bDNA) signal amplification method, using the Quantigene bDNA Signal Amplification Kit (Bayer Diagnostics, East Walpole, MA), as described previously (Hartley and Klaassen, 2000Go). Briefly, total RNA was isolated from duodenum, liver, and kidney using RNAzol B reagent (Tel-Test Inc., Friendswood, TX). Specific oligonucleotide probes to DMT1 mRNA (5 capture extenders, 10 label extenders, and 5 blocker probes, Table 1Go) were designed using Probe Designer Software Version 1.0 (Bayer Diagnostics). The oligonucleotides composing the probe set were synthesized by Operon Technologies (Alameda, CA). Total RNA (10 µg/10 µl) was added to each well of a 96-well plate containing 100 µl per well of premixed probe set, and incubated overnight at 53°C. Subsequent steps were carried out as per the manufacturers instructions. Luminescence was measured from the 96-well plate with the Quantiplex 320 bDNA Luminometer (Bayer Diagnostics).


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TABLE 1 Oligonucleotide Probes Generated for DMT1 mRNA Analysis in Rat by bDNA Assay
 
Statistics.
Data were expressed as mean ± standard error. The comparison of body weight change between the rats fed the FeD and the FeS diets was performed using repeated-measures analysis of variance. Other differences between both groups were analyzed using the unpaired Student’s t-test. The level of statistical significance was set at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The body weights of FeD- or FeS-fed animals increased gradually and progressively with age. The weight gain was less in FeD- than in FeS-fed rats (Fig. 1Go). Feeding the FeD diet for 4 weeks produced iron deficiency anemia in those rats. Compared with the FeS diet rats, the FeD-fed animals showed much lower levels of total Fe (361 vs. 25.4 µg/dl) and much higher levels of UIBC (123 vs. 431 µg/dl) in serum (Fig. 2Go, upper panels). The TIBC values were not different between FeD and FeS animals (456 vs. 484 µg/dl) (Fig. 2Go, left lower panel). The percentage of Fe bound to transferrin in serum (transferrin saturation index; 74.8 vs. 5.4%) was much less in FeD-fed rats than FeS rats (Fig. 2Go, lower right panel). The FeD diet decreased hemoglobin values (15.4 vs. 6.4 g/dl) to about 40% of the values with the FeS diet.



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FIG. 1. Body weight following feeding of FeS (iron sufficient) or FeD (iron deficient) diet to rats for 4 weeks. Data are represented as mean ± SE (n = 15). Asterisk indicates that body weight in FeD-fed rats is significantly different from FeS-fed rats (p < 0.05).

 


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FIG. 2. Serum Fe status in rats fed FeS or FeD diets for 4 weeks. Data are represented as mean ± SE (n = 15). UIBC, Unsaturated iron binding capacity; TIBC, total iron binding capacity; TS, transferrin saturation. Asterisk indicates FeD diet rats are significantly different from FeS diet rats (p < 0.05).

 
Tissue Cd concentrations were much greater in FeD-fed animals 48 h after oral Cd following 4 weeks of Fe-D or Fe-S diets. The orally administered Cd was distributed highly to the gastrointestinal tract in relation to other tissues. The duodenum retained the highest level of Cd in the body. In FeD diet rats, Cd concentration in the stomach (0.3 vs. 0.8 ng/g wet weight), duodenum (9.3 vs. 90.9 ng/g wet weight), jejunum (1.8 vs. 10.4 ng/g wet weight), ileum (0.7 vs. 3.1 ng/g wet weight), and colon (0.3 vs. 1.1 ng/g wet weight) were 2.7-, 9.8-, 5.8-, 4.4-, and 3.7-fold higher than in the corresponding tissues of FeS diet rats. The largest difference in Cd concentration was in the duodenum (Fig. 3AGo).



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FIG. 3. Cadmium concentration in (A) stomach, duodenum, jejunum, ileum, and colon; (B) liver, kidney, heart, and bone; and (C) lung, testis, brain, and blood of FeS- and FeD-fed rats. Data are represented as mean ± SE (n = 10). Asterisk indicates FeD-fed rats are significantly different from FeS-fed rats (p < 0.05).

 
Cd was highly distributed to liver and kidney and moderately distributed to heart and bone. The Cd concentrations in liver (0.52 vs. 3.78 ng/g wet weight), kidney (1.15 vs. 4.13 ng/g wet weight), heart (0.023 vs. 0.152 ng/g wet weight) and bone (0.015 vs. 0.149 ng/g wet weight) were 7.3-, 3.6-, 6.5-, and 9.8-fold higher in FeD-fed rats than in FeS-fed rats, respectively (Fig. 3BGo). Much less Cd was distributed to lung, testis, brain, and blood. In rats fed the FeD diet, the concentration of Cd in lung (0.009 vs. 0.051 ng/g wet weight), testis (0.008 vs. 0.054 ng/g wet weight), brain (0.002 vs. 0.009 ng/g wet weight), and blood (0.004 vs. 0.032 ng/g) showed significant increases, with FeD rats having 5.7-, 6.8-, 4.5-, and 7.7-fold more Cd in the respective tissues than in the FeS diet animals (Fig. 3CGo).

The total and relative body burdens of Cd at 48 h after oral Cd administration in FeD-fed rats (337 ng per rat and 136 ng/100 g body weight, respectively), were about 10-fold higher than the corresponding values in FeS diet rats (37 ng per rat and 13 ng/100 g body weight, respectively). In FeD diet rats, the proportion of administered Cd retained by the body was 1.85% at 48 h after Cd administration. This is about 10-fold higher than the 0.18% retained by rats fed the FeS diet (Fig. 4Go).



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FIG. 4. Total (top panel) and relative (middle panel) body burden of cadmium in rats fed an FeS or FeD diet. Absorption index of cadmium after oral administration in FeS- or FeD-fed rats (bottom panel). TBB, total body burden; RBB, relative body burden. Data are represented as mean ± SE (n = 10). Asterisk indicates FeD diet rats are significantly different from FeS diet rats (p < 0.05).

 
DMT1 mRNA levels were very high in the duodenums, and moderate in kidneys and livers. The level of DMT1 mRNA in duodenum was increased about 15-fold in FeD diet rats compared to FeS-fed rats (Fig. 5Go). DMT1 mRNA levels in liver and kidney was 40 and 30 percent higher, respectively, in FeD diet rats than in FeS diet rats.



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FIG. 5. Expression level of DMT1 mRNA in duodenum, kidney, and liver of FeS- or FeD-fed rats. Data are represented as mean ± SE (n = 15). Asterisk indicates FeD diet rats are significantly different from FeS diet rats (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The findings in the current study show that the increase of DMT1 mRNA in rats fed the FeD diet correlates with increased absorption of Cd from the gastrointestinal tract. Earlier reports suggested that the transport of Cd in mammals might be associated with the Fe transporter, which was recently identified as DMT1 (Fleming et al., 1997Go; Gunshin et al., 1997Go). For example, the intestinal absorption of Cd is increased or decreased depending on the body Fe status in animal and human models (Berglund et al., 1994Go; Flanagan et al., 1978Go; Hamilton and Valberg, 1974Go; Ko,wal, 1988; Leon and Johnson, 1987Go; Schafer and Forth, 1984Go; Schumann et al., 1996Go; Sullivan and Ruemmler, 1987Go). Second, DMT1 is expressed in the intestinal brush border and is up- and downregulated in Fe deficiency and Fe loading, respectively (Canonne-Hergaux et al., 1999Go; Gunshin et al., 1997Go; Tandy et al., 2000Go). Third, DMT1 transports not only Fe in vitro but also other divalent metals including Cd (Gunshin et al., 1997Go; Olivi et al., 2001Go; Picard et al., 2000Go). Fourth, DMT1 expression in Caco-2 cells exposed to iron is reduced with a corresponding decrease in Cd uptake (Tallkvist et al., 2001Go). Fifth, duodenal DMT1 mRNA expression is increased in patients with hereditary hemochromatosis (Zoller et al., 1999Go); increased intestinal absorption of cobalt and increased concentrations of manganese and lead were observed in these patients (Altstatt et al., 1967Go; Barton et al., 1994Go; Valberg et al., 1969Go). Finally, Cd concentration in blood is increased with phlebotomy in patients with hereditary hemochromatosis (Åkesson et al., 2000). To our knowledge, this is the first study to link the mechanism of intestinal Cd absorption in mammals with increased expression of DMT1 induced by Fe deficiency in vivo.

In the present study, the FeD diet caused a reduction in weight gain, depletion of body Fe stores, and Fe deficiency anemia. The expression of DMT1 mRNA in duodenum was markedly increased in the rats fed the FeD diet. DMT1 mRNA expression in duodenum was about 15-fold higher in FeD diet-fed animals than in FeS diet-fed animals. This is consistent with previous studies (Canonne-Hergaux et al., 1999Go; Gunshin et al., 1997Go; Trinder et al., 2000Go). Tissue Cd concentrations 48 h after a single oral administration of Cd in FeD diet-fed rats were higher than in FeS diet-fed rats. In FeD diet-fed rats, total body Cd burden and the proportion of administered Cd retained by the body 48 h after administration were about 10-fold higher than the corresponding values in FeS-fed rats. The tissue Cd concentration was highest in the small intestine, especially in the duodenum, and it displayed a decreasing Cd gradient from proximal to distal portions of the intestine.

Interestingly, the Cd distribution pattern (proximal to distal gradient) observed in intestine is similar to the DMT1 mRNA expression pattern in previous reports (Gunshin et al., 1997Go). The intestinal sites of highest Cd distribution and DMT1 mRNA expression coincide with the primary sites for intestinal absorption of most divalent cations (Gunshin et al., 1997Go; Han et al., 1995Go). The observed Cd distribution and DMT1 mRNA expression patterns between the FeD- and FeS-fed animals provide evidence that Fe deficiency upregulates de novo synthesis of DMT1 protein at the mRNA level in the duodenum (Andrews, 1999bGo). The upregulation of DMT1 in the small intestine, induced by Fe deficiency, may increase the uptake of Fe and other divalent metals, including Cd, from the gastrointestinal lumen and their transfer to other tissues. Therefore, it is important to have adequate nutritional intake of Fe; otherwise, the intestinal adaptive response to Fe deficiency (increased DMT1 expression) may lead to enhanced absorption of Cd and other toxic metals. While it is likely that the increased distribution of Cd to liver and kidney in FeD diet rats is due to the increased intestinal absorption, which correlates to the increase in DMT1 (15-fold), we cannot rule out that the minor increase (30–40%) of DMT1 expression in liver and kidney contributes to elevated Cd accumulation.

Iron transport in enterocytes involves the following 3 steps:(1) transport across the apical cell membrane, (2) movement within the enterocyte, and (3) transport across the basolateral membrane into the circulation (Powell et al., 1999Go). DMT1 is the transporter at the apical, microvillous membrane. Recently, the transporters ferroportin 1 and hephaestin have been postulated to export Fe across the basolateral membrane to the circulation (Vulpe et al., 1999Go; Abboud and Haile, 2000Go; Donovan et al., 2000Go; McKie et al., 2000Go). Our findings support the hypothesis that the uptake of Cd from intestinal lumen into enterocytes is mediated by the apical membrane transporter DMT1, because high concentrations of Cd were found in the duodenum.

DMT1 is ubiquitously distributed in mammals (Gunshin et al., 1997Go). In this study, DMT1 mRNA was found to be highly expressed in duodenum and moderately expressed in kidney and liver in FeS diet-fed animals. The DMT1 mRNA in kidney and liver were upregulated by the FeD diet but to a much lower extent than in duodenum. This finding is different from the situation in hereditary hemochromatosis, where the increased expression of DMT1 is restricted to duodenum and does not occur in kidney and liver (Fleming et al., 1999Go). However, our results are consistent with previous reports that DMT1 expression is increased in multiple tissues by the depletion of body Fe (Gunshin et al., 1997Go). In FeD diet rats, the expression of DMT1 mRNA in kidney and liver was higher than in the corresponding tissues in FeS diet rats. The expression level of a metal transporter in a tissue presumably is related to its biological role in the tissue. In a recent study, DMT1 was found to be highly expressed in the loop of Henle and the collecting duct of the kidney. This finding suggests that DMT1 plays an important role in Fe reabsorption in these portions of the kidney (Ferguson et al., 2001Go; Wareing et al., 2000Go).

In summary, the present FeD diet causes a reduction in body weight gain and results in the depletion of body Fe stores. Iron deficiency markedly upregulates DMT1 at the mRNA level in the duodenum. This high expression of DMT1 in duodenum coincides with the increased uptake of Cd from the gastrointestinal tract into the enterocyte and the subsequent transfer of Cd to the circulation and body tissues. This study also strongly supports a role for DMT1 in mammalian small intestine transport, not only of Fe but also other metals, including the toxic metal Cd.


    ACKNOWLEDGMENTS
 
The authors would like to thank Sultan Habeebu, Dylan Hartley, Eric Harstad, David Johnson, Ning Li, and Yaping Liu for their technical assistance. This research was supported by NIH grant ES-01142.


    NOTES
 
1 Present address: Department of Preventive Medicine, College of Medicine, Chung-Ang University, Seoul, Korea 156–756. Back

2 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu. Back


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