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Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Cadmium‐induced enteropathy: Comparative toxicity of cadmium chloride and cadmium‐thionein b
a
a
Leslie S. Valberg , James Haist , M. George Cherian , a
Liliane Delaquerriere‐Richardson & Robert A. Goyer
a
a
Departments of Medicine and Pathology , University of Western Ontario and University Hospital , London, Ontario, Canada b
Departments of Medicine and Pathology , University of Western Ontario and University Hospital , London, Ontario, N6A 5C1, Canada Published online: 20 Oct 2009.
To cite this article: Leslie S. Valberg , James Haist , M. George Cherian , Liliane Delaquerriere‐Richardson & Robert A. Goyer (1977) Cadmium‐induced enteropathy: Comparative toxicity of cadmium chloride and cadmium‐thionein, Journal of Toxicology and Environmental Health: Current Issues, 2:4, 963-975, DOI: 10.1080/15287397709529495 To link to this article: http://dx.doi.org/10.1080/15287397709529495
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms
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CADMIUM-INDUCED ENTEROPATHY: COMPARATIVE TOXICITY OF CADMIUM CHLORIDE AND CADMiUM-THIONEIN
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Leslie S. Valberg, James Haist, M. George Cherian, Liliane Delaquerriere-Richardson, Robert A. Goyer Departments of Medicine and Pathology, University of Western Ontario and University Hospital, London, Ontario, Canada
In protecting the body against the noxious effects of dietary cadmium ions, cadmium is bound to metallothionein in the proximal intestine, and subsequently excreted into the lumen with desquamation of the epithelium. The purpose of this study was to determine the extent to which cadmium in the form of intestinal cadmium-thionein is absorbed from the intestinal lumen and to appraise the toxicity of cadmium-thionein on the intestinal mucosa. With open-ended duodenal perfusion, equivalent amounts of cadmium administered as CdCI2 or cadmium-thionein entered the mucosa, but significantly less cadmium from the perfusate of cadmium-thionein passed into the body. Exposure of the mucosa to CdCI2 for 1 hr led to minor abnormalities in the form of broadening of villi with pseudostratification of epithelium, and swelling of mitochondria, whereas cadmium-thionein produced extensive necrosis of absorptive cells. The results suggest that cadmium-thionein may play a paradoxical role, providing protection against the cadmium ion in the intracellular milieu, but promoting cadmium toxicity when it is present in sufficient amounts in the lumen of the intestine.
INTRODUCTION The gastrointestinal tract plays a key role in protecting the body against the noxious effects of dietary cadmium. In experimental animals only a small portion of dietary cadmium is taken up by the intestinal mucosa and less than 5% of the cadmium that enters the mucosa actually passes into the body (Hamilton and Valberg, 1974). The remainder is bound in the mucosal cells to metallothionein and subsequently lost into the lumen with desquamation of the epithelium. Intracellular binding of cadmium to metallothionein is considered to provide protection against the toxic cadmium ion (Friberg et al., 1974; Rugstad and Norseth, 1975; Sugawara and Sugawara, 1975; Webb and Versehoyle, 1976). Whether cadmium-thionein actually serves this function in the intestine, or continues to serve it once it is discharged into the lumen of the intestine, has not been established. This research was supported by a grant from the Medical Research Council of Canada. Requests for reprints should be sent to Leslie S. Valberg, Departments of Medicine and Pathology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
963 Journal of Toxicology and Environmental Health, 2:963-975,1977 Copyright© 1977 by Hemisphere Publishing Corporation
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The intestinal mucosa, while serving to protect the body against dietary cadmium toxicity, is vulnerable to injury. Cadmium given to Japanese quail in a concentration of 75 mg per kilogram of diet for 4 wk produced shortening and thickening of the villi of the duodenum with stratification of surface epithelium (Richardson and Spivey-Fox, 1974). Similar lesions have been reported in Japanese women eating cadmium-contaminated food (Murataetal., 1969). This study was undertaken to determine the extent to which cadmiumthionein is absorbed from the lumen of the intestine and to appraise the toxic effect of cadmium-thionein on the intestinal mucosa in comparison to an equivalent amount of cadmium in the form of CdCI2. The experiments were carried out on mice fed a low-iron diet, which enhances the intestinal absorption of cadmium (Valberg et al., 1976). METHODS Animals and Diets Mice, 12 wk of age, of the C57BL/6 strain were obtained from Jackson Laboratories, Bar Harbor, Maine. On arrival from the laboratory they were allocated in a random manner to the various experiments. The animals were housed in plastic cages and provided with distilleddeionized water. The diet consisted of a low-iron semisynthetic powdered diet (< 2 mg/kg iron), prepared as described previously (McCall et al., 1962). The mice were fed this diet for 14 days before the experiment. Preparation of Cadmium-Thionein The synthesis of cadmium-thionein was induced in 12 Sprague-Dawley male rats weighing 150-200 g by intraperitoneal injection with 0.6 mg CdCI2 per kilogram of body weight for five consecutive days. At the end of 3 wk the rats were sacrificed and the proximal two-thirds of the small intestine was removed. The intestinal mucosa was scraped off using a glass slide, pooled, and a 40% homogenate was prepared in 0.25 M sucrose and 0.01 M Tris-HCI at pH 8.6. The homogenate was frozen overnight, thawed, and centrifuged at 10,000 g for 10 min. The postmitochondrial supernatant, which contained most of the cadmiumthionein, was spiked with 5 juCi of carrier-free 109CdCI2. The cadmiumthionein enriched fraction was isolated as described elsewhere (Cherian, 1974). Since the final sample contained only a few milligrams of protein, it was freeze-dried in the presence of Tris buffer for complete recovery of the protein. Thus the purified sample contained Tris buffer together with cadmium-thionein. Ten milligrams of the freeze-dried powder contained 11.69 jug of cadmium, as determined by atomic absorption spectroscopy (Pulido et al., 1966).
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Mice were given 400 nmol CdCI2 labeled with 1 nC'\ of 109CdCI2 by intragastric injection. Twenty-four hours later the animals were killed and the proximal one-half of the small intestine removed; the mucosa was scraped off and pooled, and cadmium-thionein was prepared as described above.
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Electrophoresis of Cadmium-Thionein Polyacrylamide gel electrophoresis of cadmium-thionein isolated from rat intestine and mouse intestine was carried out as described by Davis (1964). The gel was stained for protein with 0.2% Commassie Blue. Two-millimeter slices of polyacrylamide gel were counted for radioactivity in a scintillation spectrometer after adjusting the volume with water to that in radioactive standards. Cadmium Absorption After the animals were fasted overnight an open-ended loop of duodenum and proximal jejunum (8 cm) was perfused for 1 hr, employing a technique described previously (Hamilton and Valberg, 1974). Test solutions of either 100 \iM CdCI2 or cadmium-thionein with an equivalent amount of cadmium labeled with carrier-free 109CdCI2 were prepared in 0.15 M NaCI adjusted to pH 8.5 with NaOH. To prepare the perfusate of cadmium-thionein with 100 nM cadmium, 10 mg of the powder from rat intestine were added to 1 ml of 0.15 M NaCI adjusted to pH 8.5 with dilute NaOH. Each animal was perfused with a total of 2 ml of the test solution over a period of 60 min. Immediately following the perfusion, the lumen was flushed three times with 10 ml of ice-cold 0.15 M NaCI followed by 10 ml of air, and then the loop was excised. It was split open and placed in 3% glutaraldehyde in a counting tube. After counting, samples of the tissue were prepared for microscopic examination. To ascertain whether the perfused loop leaked during the perfusion, the peritoneal cavity was injected with 3 ml of 0.15 M NaCI at the end of the experiment. The solution was aspirated from the cavity and counted for radioactivity. Negligible amounts of radioactivity were found. Methods for measuring radioactivity in the intestinal mucosa and carcass and for calculating intestinal uptake and transfer from the lumen of the intestine to the body have been described previously (Hamilton and Valberg, 1974). After the carcass was counted, the liver and kidneys were removed and counted against standards of equivalent volume. The radioactivity was converted to micrograms of cadmium on the basis of the specific activity of the perfusate.
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Electron Microscopy Samples of tissue were taken from multiple areas of the intestinal loops obtained at the end of the various experiments. Samples were dehydrated and embedded, and multiple thick (0.75 /xm) and thin (0.05 fim) sections were prepared for light and electron microscopy. The slides were coded and evaluated by two of us (L.D.-R and R.A.G), without knowledge of the experimental conditions.
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Experimental Design Three groups of nine animals each were perfused respectively with (1) 0.15 M NaCI adjusted to pH 8.5, (2) CdCI2 in 0.15 M NacL adjusted to pH 8.5, and (3) cadmium-thionein in 0.15 M NaCI adjusted to pH 8.5. RESULTS Intestinal Absorption of Cadmium Out of the total of 22.4 jug perfused in the form of CdCI2, an average of 6.6 fig of cadmium entered the intestinal mucosa and 0.26 ng passed into the body (Table 1). A similar amount of cadmium was taken up from the perfusate of cadmium-thionein, but an average of only 0.10 jug was transferred from the intestinal mucosa to the body. About 40-50% of the radiocadmium that was absorbed was found in the liver, and less than 1% was detected in the kidneys (Table 1).
TABLE 1. Intestinal Absorption and Distribution of Cadmium Supplied as Cadmium Chloride or Cadmium-Thionein in Iron-Deficient Mice Transfer /uptake X 100 (%)
Intestinal mucosa (Mg)
0.26 + 0.03 c
3.9
6.3 + 0.37 0.09 ± 0.02 c '° f
0.005 ± 0.00
0.10 + 0.02
1.7
5.7 ± 0.51 0.05 ± 0.01°'
0.004 + 0.00
Perfusate
No. of mice
Intestinal uptake (Kg)0
Transfer to body (Mg)
Cadmium chloride
9
6.6 ± 0.39 6
Cadmiumthionein
9
5.8 + 0.50
Liver
Kidney (Mg)
"Amount of cadmium taken up from the intestinal perfusate. Mean ± SEM. c The difference between the mean of this group and the mean of the group given cadmium-thionein is statistically significant, p < 0.01. The difference between the mean cadmium level in the liver and kidney in this group of animals is statistically significant, p < 0.01. 6
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GEL SLICE NUMBER FIGURE 1. Polyacrylamide gel electrophoresis of cadmium-thionein labeled with ""Cd isolated from homogenate of proximal intestinal mucosa of the mouse (see text for details). The anode is on the right.
Polyacrylamide Gel Electrophoresis A single homogeneous peak of radioactivity was obtained with the cadmium-thionein preparation isolated from the small intestine of the mouse (Fig. 1). An identical radioactive peak was observed with the preparation of cadmium-thionein from rat small intestine. A partial characterization of the isolated intestinal protein as metallothionein was done by heat treatment and absorption spectra. The cadmium-binding proteins (molecular weight 10,000) isolated from both rat and mice intestine showed a characteristic absorption maximum at 254 nm (the ratio of absorption at 254 nm to that at 280 nm was 12.5), consistent with the absorption spectra of metallothionein (Kagi and Vallee, 1961). These proteins were also stable to heat treatment (80°C for 2 min) as observed by identical elution profiles from Sephadex G-75 columns before and after heating. These results are similar to those obtained for rat liver cadmium-thionein (Cherian, 1974). HISTOLOG1CAL EFFECTS Saline Perfusion (Controls) The segments of duodenum and proximal jejunum perfused with saline were lined with tall slender villi covered with a single layer of
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FIGURE 2. Villus from small intestine of control mouse, showing a single layer of columnar epithelium. The brush border is thin and regular. The lamina propria is narrow, containing vascular channels and a few macrophages and other mononuclear cells. (Light microscopy, toluidine blue stain, X 1,280.)
mv,
9-"
FIGURE 3. Electron photomicrograph of brush border and luminal portions of adjacent absorptive cells from control mouse, showing microvilli (mv), mitochondria (mt), intercellular plasmalemmal membrane (pm) and junctional complexes (jc). (X 12,000)
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columnar epithelium (Fig. 2). The ultrastructurai details of the absorptive cells are shown in Fig. 3. Cadmium Chloride Perfusion The viIIi of the duodenum and proximal jejunum perfused with CdCI2 solution were shortened and the epithelium contained multiple vacuoles (Fig. 4). Electron micrographs revealed that these vacuoles were greatly enlarged intercellular spaces (Fig. 5). Many of the vacuoles contained myelin figures that appeared to be continuous with plasmalemmal membranes. The microvilli were intact but matrical swelling of mitochondria was prominent, particularly in absorptive cells of the lower two-thirds of the villi (Fig. 6). Goblet cells and Paneth cells were unaffected by the cadmium perfusion. Cadmium-Thionein Perfusion Perfusion of the intestine with cadmium-thionein produced severe necrosis of the absorptive cells, especially in the outer portion of the villi. The result was shortening of villi with the appearance in some areas of fusion of adjacent villi (Fig. 7). Figure 8 shows two adjacent absorptive cells with changes of early necrosis. The microvilli are disrupted and cytoplasmic contents swollen. The mitochondria have lost their cristae and the endoplasmic reticulum is greatly dilated. Cell sap is diluted, ',?*
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FIGURE 4. Villus from small intestine of CdCI2-treated mouse, showing pseudostratification of absorptive cells and diffuse vacuolation. (Light microscopy, toluidine blue stain, X 1,280.)
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FIGURE 5. Electron photomicrograph of absorptive cells of small intestine of CdCI2-treated mouse. Vacuoles appear to be expanded intercellular spaces. (X 12,000) Insert: myelin figures within these spaces may be continuous with plasmalemmal membranes (X 21,600).
FIGURE 6. Electron photomicrograph of absorptive cell in lower one-third of villus of small intestine of mouse treated with CdCI,, showing mitochondrial swelling (arrows). (X 21,600)
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ac FIGURE 7. Epithelium of small intestine of mouse treated with cadmium-thionein, showing necrosis of absorptive cells (ac) of most of the luminal portions of villi. The crypts are relatively less damaged and contain morphologically intact cells. (X 625)
FIGURE 8. Higher magnification of necrotic absorptive cells of intestine of mouse treated with cadmium-thionein. There is marked swelling and degeneration of all cytoplasmic organelles and clumping of chromatin in nuclei, (x 21,600) 971
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ac
FIGURE 9. Base of crypt of small intestine of mouse treated with cadmium-thionein, necrosis of absorptive cells (ac) but preservation of Paneth cells (pc). (X 3,600)
leaving focal clear spaces, particularly in the basilar portions of the cells. Deep in the crypts morphologically intact and apparently uninjured Paneth cells were present between damaged absorptive cells (Fig. 9). DISCUSSION In comparison to the innocuous effects of 150 xnM NaCI, exposure of the proximal intestinal mucosa to 100 \iM CdCI2 for 1 hr produced broadening and shortening of villi with pseudostratification of epithelium. At the cell level, there was widening of intercellular spaces between the lateral margin of absorptive cells, and swelling of mitochondria. Similar changes, including separation of cell margins and enlarged pale mitochondria, were found by Richardson and Spivey-Fox (1974) in Japanese quail fed CdCI2, 75 mg/kg of diet, for 4 wk. Other abnormalities found in the quail—goblet cell hyperplasia and inflammatory cell infiltration of the lamina propria—were not observed in this experiment, probably due to the short exposure to a lower concentration of CdCI2. Experimentally, intercellular adhesions may be altered by calcium depletion of the intact mucosa (Weisberg and Rhoden, 1970; Imbar et al., 1971). Hamilton and Smith (1975) found that cadmium competes with calcium for binding sites in the intestinal mucosa, and it is possible that the
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separation of cell margins observed in our experiment was due to cadmiuminduced calcium loss from adjacent cell membranes. Our results indicate that alterations in cellular organelles, including disruption of intercellular junctions and swelling of mitochondria, are early manifestations of acute cadmium toxicity, and these precede any apparent structural change in the microvilli. It should be emphasized that the dose of cadmium administered, 22.4 ng, was large in relation to the 50-70 jug of cadmium ingested daily by humans. In a previous study (Hamilton and Valberg, 1974) of cadmium absorption in mice fed an iron-supplemented diet, no histological abnormalities of the intestinal mucosa were apparent with a cadmium concentration equivalent to that used in this experiment. The presence of histological changes in this experiment may be explained by several factors: (1) the use of a low-iron diet to enhance intestinal cadmium intake; (2) the longer exposure of the mucosa to cadmium (60 min compared to 30 min), which provided more time for structural alterations to occur; (3) the use of a more sensitive histological technique, electron microscopy; and (4) the elimination of ascorbic acid from the perfusate, which may have increased the cadmium toxicity. Richardson et al. (1974) observed that ascorbic acid added to a diet containing cadmium alleviated or prevented cadmium toxicity and decreased the incidence of intestinal mucosal damage. The finding of structural changes in the intestinal mucosa with a cadmium level equivalent to the minimum concentration required to inhibit iron absorption in mice on a low-iron diet (Hamilton and Valberg, 1974) implies that the effect of cadmium on iron transport may be a nonspecific consequence of cell injury. The degree of mitochondrial injury observed with electron microscopy (Fig. 6) would appear to be sufficient to deprive the cell of adequate energy for normal transport functions. However, further investigation is required before definitive conclusions can be drawn about the specificity of cadmium inhibition of iron absorption. The histological changes produced by CdCI2 were mild compared to those induced by an equivalent dose of cadmium in the form of cadmium-thionein (Figs. 7 and 8), even though equivalent amounts of cadmium were present in the intestinal mucosa (Table 1). There was such severe mucosal damage with cadmium-thionein, however, that it is not feasible to reconstruct the sequence of intracellular events leading to injury, nor to ascertain whether cadmium bound to metallothionein entered the intact mucosa. It is of interest that the absorptive cells on the villi were very susceptible to the toxic effects of cadmium, whereas the Paneth cells were relatively resistant (Fig. 9). This is in contrast to the results obtained with chronic low-dose feeding of methyl mercury to primates, which selectively altered Paneth cells, leaving the intestinal absorptive cells intact (Mottet and Body, 1976).
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It is unlikely that the toxic effects of cadmium-thionein were due to species differences in metallothionein. The cadmium-thionein was prepared from rat intestine because it was not technically feasible to make sufficient amounts from mice. No chemical difference was found between mouse and rat metallothionein on polyacrylamide electrophoresis (Fig. 1). Another unlikely possibility is that the toxic effects were due to thionein perse. Further investigation is required to exclude this. The toxic effect of cadmium on the intestinal mucosa resembles its effect on the renal tubules. Cherian et al. (1976) found extensive degerative changes in the proximal renal tubular lining cells 24 hr after the intraperitoneal administration of cadmium-thionein, whereas no structural alteration was observed after a similar dose of CdCI2. Hence, cadmium-thionein may play a paradoxical role in the pathogenesis of cadmium toxicity (Nordberg et al., 1975). In certain organs, such as the intestinal tract and liver, metallothionein may provide protection against cadmium toxicity by binding cadmium in the cells, but once released from these organs into the extracellular environment or the lumen of the intestine, cadmium-thionein appears to be highly toxic to tissues, such as the renal tubules or the intestinal mucosa. REFERENCES Cherian, M. G., Goyer, R. A. and Delaquerriere-Richardson, L. 1976. Cadmium-metallothionein induced nephropathy. Toxicol. Appl. Pharmacol. 38: in press. Cherian, M. G. 1974. Isolation and purification of cadmium binding proteins from rat liver. Biochem. Biophys. Res. Commun. 61:923-926. Davis, C. J. 1964. Disc electrophoresis. I I . Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. Friberg, L., Piscator, M., Nordberg, G. F, and Kjellstrom, T. 1974. Cadmium in the environment, 2nd ed., pp. 126-127. Cleveland: CRC Press. Hamilton, D. L. and Smith, M. W. 1975. Effect of cadmium on calcium uptake by rat duodenum. Clin. Res. 23: 635A. Hamilton, D. L. and Valberg, L. S. 1974. Relationship between cadmium and iron absorption. Am. j. Physiol. 227:1033-1037. Imbar, M. Ben-Basset, H. and Sachs, L. 1971. Location of amino acid and carbohydrate transport sites in the surface membrane of normal and transformed mammalian cells. J. Membr. Biol. 6:195-209. Kägi, J. H. R. and Vallee, B. L. 1961. Metallothionein: A cadmium and zinc-containing protein from equine renal cortex. J. Biol. Chem. 236:2435-2442. McCall, M. G., Newman, G. E., O'Brien, J. R. P., Valberg, L. S. and Witts, L. J. 1962. Studies on iron metabolism. I. The experimental production of iron deficiency in the growing rat. Br. J. Nutr. 16:297-304. Mottet, K. N. and Body, R. L. 1976. Primate Paneth cell degeneration following methylmercury hydroxide ingestion. Am. J. Pathol. 84:93-102. Murata, I., Hirono, T., Saeki, Y. and Nakashima, S. 1969. Cadmium enteropathy, renal osteomalacia ("Itai-ltai" disease) in Japan. Bull. Soc. Int. Chir. 29:34. Nordberg, G. F., Goyer, R. and Nordberg, M. 1975. Comparative toxicity of cadmium-metallothionein and cadmium chloride on mouse kidney. Arch. Pathol. 99:192-197.
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Pulido, P., Fuwa, K. and Vallee, B. L. 1966. Determination of cadmium in biological material by atomic absorption spectrophotometry. Anal. Biochem. 14:393-404. Richardson, M. E. and Spivey-Fox, M. R. 1974. Dietary cadmium and enteropathy in the Japanese quail. Lab. Invest. 31:722-731. Richardson, M. E., Spivey-Fox, M. R. and Fry, B. E. 1974. Pathological changes produced in Japanese quail by ingestion of cadmium. J. Nutr. 104:323-338. Rugstad, H. E. and Norseth, T. 1975. Cadmium resistance and content of cadmium binding protein in cultured human cells. Nature (Lond.) 257:136-137. Sugawara, C. and Sugawara, N. 1975. The inductive effect of cadmium on protein synthesis of rat intestine. Bull. Environ. Contam. Toxicol. 14:159-162. Valberg, L. S., Sorbie, J. and Hamilton, D. L. 1976. Gastro-intestinal metabolism of cadmium in experimental iron deficiency. Am. J. Physiol., in press. Webb, M. and Versehoyle, R. D. 1976. An investigation of the role of metallothionein in protection against the acute toxicity of the cadmium ion. Biochem. Pharmacol. 25:673-679. Weisberg, H. and Rhodin, J. 1970. Relation of calcium to mucosal structure and vitamin B12 absorption in the canine intestine. Am. J. Pathol. 61:145-150. Received September 10, 1976 Accepted December 14, 1976
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Cadmium‐induced enteropathy: Comparative toxicity of cadmium chloride and cadmium‐thionein b
a
a
Leslie S. Valberg , James Haist , M. George Cherian , a
Liliane Delaquerriere‐Richardson & Robert A. Goyer
a
a
Departments of Medicine and Pathology , University of Western Ontario and University Hospital , London, Ontario, Canada b
Departments of Medicine and Pathology , University of Western Ontario and University Hospital , London, Ontario, N6A 5C1, Canada Published online: 20 Oct 2009.
To cite this article: Leslie S. Valberg , James Haist , M. George Cherian , Liliane Delaquerriere‐Richardson & Robert A. Goyer (1977) Cadmium‐induced enteropathy: Comparative toxicity of cadmium chloride and cadmium‐thionein, Journal of Toxicology and Environmental Health: Current Issues, 2:4, 963-975, DOI: 10.1080/15287397709529495 To link to this article: http://dx.doi.org/10.1080/15287397709529495
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms
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& Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
CADMIUM-INDUCED ENTEROPATHY: COMPARATIVE TOXICITY OF CADMIUM CHLORIDE AND CADMiUM-THIONEIN
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Leslie S. Valberg, James Haist, M. George Cherian, Liliane Delaquerriere-Richardson, Robert A. Goyer Departments of Medicine and Pathology, University of Western Ontario and University Hospital, London, Ontario, Canada
In protecting the body against the noxious effects of dietary cadmium ions, cadmium is bound to metallothionein in the proximal intestine, and subsequently excreted into the lumen with desquamation of the epithelium. The purpose of this study was to determine the extent to which cadmium in the form of intestinal cadmium-thionein is absorbed from the intestinal lumen and to appraise the toxicity of cadmium-thionein on the intestinal mucosa. With open-ended duodenal perfusion, equivalent amounts of cadmium administered as CdCI2 or cadmium-thionein entered the mucosa, but significantly less cadmium from the perfusate of cadmium-thionein passed into the body. Exposure of the mucosa to CdCI2 for 1 hr led to minor abnormalities in the form of broadening of villi with pseudostratification of epithelium, and swelling of mitochondria, whereas cadmium-thionein produced extensive necrosis of absorptive cells. The results suggest that cadmium-thionein may play a paradoxical role, providing protection against the cadmium ion in the intracellular milieu, but promoting cadmium toxicity when it is present in sufficient amounts in the lumen of the intestine.
INTRODUCTION The gastrointestinal tract plays a key role in protecting the body against the noxious effects of dietary cadmium. In experimental animals only a small portion of dietary cadmium is taken up by the intestinal mucosa and less than 5% of the cadmium that enters the mucosa actually passes into the body (Hamilton and Valberg, 1974). The remainder is bound in the mucosal cells to metallothionein and subsequently lost into the lumen with desquamation of the epithelium. Intracellular binding of cadmium to metallothionein is considered to provide protection against the toxic cadmium ion (Friberg et al., 1974; Rugstad and Norseth, 1975; Sugawara and Sugawara, 1975; Webb and Versehoyle, 1976). Whether cadmium-thionein actually serves this function in the intestine, or continues to serve it once it is discharged into the lumen of the intestine, has not been established. This research was supported by a grant from the Medical Research Council of Canada. Requests for reprints should be sent to Leslie S. Valberg, Departments of Medicine and Pathology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
963 Journal of Toxicology and Environmental Health, 2:963-975,1977 Copyright© 1977 by Hemisphere Publishing Corporation
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L. S. VALBERG ET AL.
The intestinal mucosa, while serving to protect the body against dietary cadmium toxicity, is vulnerable to injury. Cadmium given to Japanese quail in a concentration of 75 mg per kilogram of diet for 4 wk produced shortening and thickening of the villi of the duodenum with stratification of surface epithelium (Richardson and Spivey-Fox, 1974). Similar lesions have been reported in Japanese women eating cadmium-contaminated food (Murataetal., 1969). This study was undertaken to determine the extent to which cadmiumthionein is absorbed from the lumen of the intestine and to appraise the toxic effect of cadmium-thionein on the intestinal mucosa in comparison to an equivalent amount of cadmium in the form of CdCI2. The experiments were carried out on mice fed a low-iron diet, which enhances the intestinal absorption of cadmium (Valberg et al., 1976). METHODS Animals and Diets Mice, 12 wk of age, of the C57BL/6 strain were obtained from Jackson Laboratories, Bar Harbor, Maine. On arrival from the laboratory they were allocated in a random manner to the various experiments. The animals were housed in plastic cages and provided with distilleddeionized water. The diet consisted of a low-iron semisynthetic powdered diet (< 2 mg/kg iron), prepared as described previously (McCall et al., 1962). The mice were fed this diet for 14 days before the experiment. Preparation of Cadmium-Thionein The synthesis of cadmium-thionein was induced in 12 Sprague-Dawley male rats weighing 150-200 g by intraperitoneal injection with 0.6 mg CdCI2 per kilogram of body weight for five consecutive days. At the end of 3 wk the rats were sacrificed and the proximal two-thirds of the small intestine was removed. The intestinal mucosa was scraped off using a glass slide, pooled, and a 40% homogenate was prepared in 0.25 M sucrose and 0.01 M Tris-HCI at pH 8.6. The homogenate was frozen overnight, thawed, and centrifuged at 10,000 g for 10 min. The postmitochondrial supernatant, which contained most of the cadmiumthionein, was spiked with 5 juCi of carrier-free 109CdCI2. The cadmiumthionein enriched fraction was isolated as described elsewhere (Cherian, 1974). Since the final sample contained only a few milligrams of protein, it was freeze-dried in the presence of Tris buffer for complete recovery of the protein. Thus the purified sample contained Tris buffer together with cadmium-thionein. Ten milligrams of the freeze-dried powder contained 11.69 jug of cadmium, as determined by atomic absorption spectroscopy (Pulido et al., 1966).
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Mice were given 400 nmol CdCI2 labeled with 1 nC'\ of 109CdCI2 by intragastric injection. Twenty-four hours later the animals were killed and the proximal one-half of the small intestine removed; the mucosa was scraped off and pooled, and cadmium-thionein was prepared as described above.
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Electrophoresis of Cadmium-Thionein Polyacrylamide gel electrophoresis of cadmium-thionein isolated from rat intestine and mouse intestine was carried out as described by Davis (1964). The gel was stained for protein with 0.2% Commassie Blue. Two-millimeter slices of polyacrylamide gel were counted for radioactivity in a scintillation spectrometer after adjusting the volume with water to that in radioactive standards. Cadmium Absorption After the animals were fasted overnight an open-ended loop of duodenum and proximal jejunum (8 cm) was perfused for 1 hr, employing a technique described previously (Hamilton and Valberg, 1974). Test solutions of either 100 \iM CdCI2 or cadmium-thionein with an equivalent amount of cadmium labeled with carrier-free 109CdCI2 were prepared in 0.15 M NaCI adjusted to pH 8.5 with NaOH. To prepare the perfusate of cadmium-thionein with 100 nM cadmium, 10 mg of the powder from rat intestine were added to 1 ml of 0.15 M NaCI adjusted to pH 8.5 with dilute NaOH. Each animal was perfused with a total of 2 ml of the test solution over a period of 60 min. Immediately following the perfusion, the lumen was flushed three times with 10 ml of ice-cold 0.15 M NaCI followed by 10 ml of air, and then the loop was excised. It was split open and placed in 3% glutaraldehyde in a counting tube. After counting, samples of the tissue were prepared for microscopic examination. To ascertain whether the perfused loop leaked during the perfusion, the peritoneal cavity was injected with 3 ml of 0.15 M NaCI at the end of the experiment. The solution was aspirated from the cavity and counted for radioactivity. Negligible amounts of radioactivity were found. Methods for measuring radioactivity in the intestinal mucosa and carcass and for calculating intestinal uptake and transfer from the lumen of the intestine to the body have been described previously (Hamilton and Valberg, 1974). After the carcass was counted, the liver and kidneys were removed and counted against standards of equivalent volume. The radioactivity was converted to micrograms of cadmium on the basis of the specific activity of the perfusate.
L. S. VALBERG ET AL.
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Electron Microscopy Samples of tissue were taken from multiple areas of the intestinal loops obtained at the end of the various experiments. Samples were dehydrated and embedded, and multiple thick (0.75 /xm) and thin (0.05 fim) sections were prepared for light and electron microscopy. The slides were coded and evaluated by two of us (L.D.-R and R.A.G), without knowledge of the experimental conditions.
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Experimental Design Three groups of nine animals each were perfused respectively with (1) 0.15 M NaCI adjusted to pH 8.5, (2) CdCI2 in 0.15 M NacL adjusted to pH 8.5, and (3) cadmium-thionein in 0.15 M NaCI adjusted to pH 8.5. RESULTS Intestinal Absorption of Cadmium Out of the total of 22.4 jug perfused in the form of CdCI2, an average of 6.6 fig of cadmium entered the intestinal mucosa and 0.26 ng passed into the body (Table 1). A similar amount of cadmium was taken up from the perfusate of cadmium-thionein, but an average of only 0.10 jug was transferred from the intestinal mucosa to the body. About 40-50% of the radiocadmium that was absorbed was found in the liver, and less than 1% was detected in the kidneys (Table 1).
TABLE 1. Intestinal Absorption and Distribution of Cadmium Supplied as Cadmium Chloride or Cadmium-Thionein in Iron-Deficient Mice Transfer /uptake X 100 (%)
Intestinal mucosa (Mg)
0.26 + 0.03 c
3.9
6.3 + 0.37 0.09 ± 0.02 c '° f
0.005 ± 0.00
0.10 + 0.02
1.7
5.7 ± 0.51 0.05 ± 0.01°'
0.004 + 0.00
Perfusate
No. of mice
Intestinal uptake (Kg)0
Transfer to body (Mg)
Cadmium chloride
9
6.6 ± 0.39 6
Cadmiumthionein
9
5.8 + 0.50
Liver
Kidney (Mg)
"Amount of cadmium taken up from the intestinal perfusate. Mean ± SEM. c The difference between the mean of this group and the mean of the group given cadmium-thionein is statistically significant, p < 0.01. The difference between the mean cadmium level in the liver and kidney in this group of animals is statistically significant, p < 0.01. 6
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GEL SLICE NUMBER FIGURE 1. Polyacrylamide gel electrophoresis of cadmium-thionein labeled with ""Cd isolated from homogenate of proximal intestinal mucosa of the mouse (see text for details). The anode is on the right.
Polyacrylamide Gel Electrophoresis A single homogeneous peak of radioactivity was obtained with the cadmium-thionein preparation isolated from the small intestine of the mouse (Fig. 1). An identical radioactive peak was observed with the preparation of cadmium-thionein from rat small intestine. A partial characterization of the isolated intestinal protein as metallothionein was done by heat treatment and absorption spectra. The cadmium-binding proteins (molecular weight 10,000) isolated from both rat and mice intestine showed a characteristic absorption maximum at 254 nm (the ratio of absorption at 254 nm to that at 280 nm was 12.5), consistent with the absorption spectra of metallothionein (Kagi and Vallee, 1961). These proteins were also stable to heat treatment (80°C for 2 min) as observed by identical elution profiles from Sephadex G-75 columns before and after heating. These results are similar to those obtained for rat liver cadmium-thionein (Cherian, 1974). HISTOLOG1CAL EFFECTS Saline Perfusion (Controls) The segments of duodenum and proximal jejunum perfused with saline were lined with tall slender villi covered with a single layer of
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FIGURE 2. Villus from small intestine of control mouse, showing a single layer of columnar epithelium. The brush border is thin and regular. The lamina propria is narrow, containing vascular channels and a few macrophages and other mononuclear cells. (Light microscopy, toluidine blue stain, X 1,280.)
mv,
9-"
FIGURE 3. Electron photomicrograph of brush border and luminal portions of adjacent absorptive cells from control mouse, showing microvilli (mv), mitochondria (mt), intercellular plasmalemmal membrane (pm) and junctional complexes (jc). (X 12,000)
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CADMIUM ENTEROPATHY
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columnar epithelium (Fig. 2). The ultrastructurai details of the absorptive cells are shown in Fig. 3. Cadmium Chloride Perfusion The viIIi of the duodenum and proximal jejunum perfused with CdCI2 solution were shortened and the epithelium contained multiple vacuoles (Fig. 4). Electron micrographs revealed that these vacuoles were greatly enlarged intercellular spaces (Fig. 5). Many of the vacuoles contained myelin figures that appeared to be continuous with plasmalemmal membranes. The microvilli were intact but matrical swelling of mitochondria was prominent, particularly in absorptive cells of the lower two-thirds of the villi (Fig. 6). Goblet cells and Paneth cells were unaffected by the cadmium perfusion. Cadmium-Thionein Perfusion Perfusion of the intestine with cadmium-thionein produced severe necrosis of the absorptive cells, especially in the outer portion of the villi. The result was shortening of villi with the appearance in some areas of fusion of adjacent villi (Fig. 7). Figure 8 shows two adjacent absorptive cells with changes of early necrosis. The microvilli are disrupted and cytoplasmic contents swollen. The mitochondria have lost their cristae and the endoplasmic reticulum is greatly dilated. Cell sap is diluted, ',?*
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FIGURE 4. Villus from small intestine of CdCI2-treated mouse, showing pseudostratification of absorptive cells and diffuse vacuolation. (Light microscopy, toluidine blue stain, X 1,280.)
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. * * •
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FIGURE 5. Electron photomicrograph of absorptive cells of small intestine of CdCI2-treated mouse. Vacuoles appear to be expanded intercellular spaces. (X 12,000) Insert: myelin figures within these spaces may be continuous with plasmalemmal membranes (X 21,600).
FIGURE 6. Electron photomicrograph of absorptive cell in lower one-third of villus of small intestine of mouse treated with CdCI,, showing mitochondrial swelling (arrows). (X 21,600)
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ac FIGURE 7. Epithelium of small intestine of mouse treated with cadmium-thionein, showing necrosis of absorptive cells (ac) of most of the luminal portions of villi. The crypts are relatively less damaged and contain morphologically intact cells. (X 625)
FIGURE 8. Higher magnification of necrotic absorptive cells of intestine of mouse treated with cadmium-thionein. There is marked swelling and degeneration of all cytoplasmic organelles and clumping of chromatin in nuclei, (x 21,600) 971
L. S. VALBERGETAL.
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ac
FIGURE 9. Base of crypt of small intestine of mouse treated with cadmium-thionein, necrosis of absorptive cells (ac) but preservation of Paneth cells (pc). (X 3,600)
leaving focal clear spaces, particularly in the basilar portions of the cells. Deep in the crypts morphologically intact and apparently uninjured Paneth cells were present between damaged absorptive cells (Fig. 9). DISCUSSION In comparison to the innocuous effects of 150 xnM NaCI, exposure of the proximal intestinal mucosa to 100 \iM CdCI2 for 1 hr produced broadening and shortening of villi with pseudostratification of epithelium. At the cell level, there was widening of intercellular spaces between the lateral margin of absorptive cells, and swelling of mitochondria. Similar changes, including separation of cell margins and enlarged pale mitochondria, were found by Richardson and Spivey-Fox (1974) in Japanese quail fed CdCI2, 75 mg/kg of diet, for 4 wk. Other abnormalities found in the quail—goblet cell hyperplasia and inflammatory cell infiltration of the lamina propria—were not observed in this experiment, probably due to the short exposure to a lower concentration of CdCI2. Experimentally, intercellular adhesions may be altered by calcium depletion of the intact mucosa (Weisberg and Rhoden, 1970; Imbar et al., 1971). Hamilton and Smith (1975) found that cadmium competes with calcium for binding sites in the intestinal mucosa, and it is possible that the
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separation of cell margins observed in our experiment was due to cadmiuminduced calcium loss from adjacent cell membranes. Our results indicate that alterations in cellular organelles, including disruption of intercellular junctions and swelling of mitochondria, are early manifestations of acute cadmium toxicity, and these precede any apparent structural change in the microvilli. It should be emphasized that the dose of cadmium administered, 22.4 ng, was large in relation to the 50-70 jug of cadmium ingested daily by humans. In a previous study (Hamilton and Valberg, 1974) of cadmium absorption in mice fed an iron-supplemented diet, no histological abnormalities of the intestinal mucosa were apparent with a cadmium concentration equivalent to that used in this experiment. The presence of histological changes in this experiment may be explained by several factors: (1) the use of a low-iron diet to enhance intestinal cadmium intake; (2) the longer exposure of the mucosa to cadmium (60 min compared to 30 min), which provided more time for structural alterations to occur; (3) the use of a more sensitive histological technique, electron microscopy; and (4) the elimination of ascorbic acid from the perfusate, which may have increased the cadmium toxicity. Richardson et al. (1974) observed that ascorbic acid added to a diet containing cadmium alleviated or prevented cadmium toxicity and decreased the incidence of intestinal mucosal damage. The finding of structural changes in the intestinal mucosa with a cadmium level equivalent to the minimum concentration required to inhibit iron absorption in mice on a low-iron diet (Hamilton and Valberg, 1974) implies that the effect of cadmium on iron transport may be a nonspecific consequence of cell injury. The degree of mitochondrial injury observed with electron microscopy (Fig. 6) would appear to be sufficient to deprive the cell of adequate energy for normal transport functions. However, further investigation is required before definitive conclusions can be drawn about the specificity of cadmium inhibition of iron absorption. The histological changes produced by CdCI2 were mild compared to those induced by an equivalent dose of cadmium in the form of cadmium-thionein (Figs. 7 and 8), even though equivalent amounts of cadmium were present in the intestinal mucosa (Table 1). There was such severe mucosal damage with cadmium-thionein, however, that it is not feasible to reconstruct the sequence of intracellular events leading to injury, nor to ascertain whether cadmium bound to metallothionein entered the intact mucosa. It is of interest that the absorptive cells on the villi were very susceptible to the toxic effects of cadmium, whereas the Paneth cells were relatively resistant (Fig. 9). This is in contrast to the results obtained with chronic low-dose feeding of methyl mercury to primates, which selectively altered Paneth cells, leaving the intestinal absorptive cells intact (Mottet and Body, 1976).
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974
L.S. VALBERGETAL.
It is unlikely that the toxic effects of cadmium-thionein were due to species differences in metallothionein. The cadmium-thionein was prepared from rat intestine because it was not technically feasible to make sufficient amounts from mice. No chemical difference was found between mouse and rat metallothionein on polyacrylamide electrophoresis (Fig. 1). Another unlikely possibility is that the toxic effects were due to thionein perse. Further investigation is required to exclude this. The toxic effect of cadmium on the intestinal mucosa resembles its effect on the renal tubules. Cherian et al. (1976) found extensive degerative changes in the proximal renal tubular lining cells 24 hr after the intraperitoneal administration of cadmium-thionein, whereas no structural alteration was observed after a similar dose of CdCI2. Hence, cadmium-thionein may play a paradoxical role in the pathogenesis of cadmium toxicity (Nordberg et al., 1975). In certain organs, such as the intestinal tract and liver, metallothionein may provide protection against cadmium toxicity by binding cadmium in the cells, but once released from these organs into the extracellular environment or the lumen of the intestine, cadmium-thionein appears to be highly toxic to tissues, such as the renal tubules or the intestinal mucosa. REFERENCES Cherian, M. G., Goyer, R. A. and Delaquerriere-Richardson, L. 1976. Cadmium-metallothionein induced nephropathy. Toxicol. Appl. Pharmacol. 38: in press. Cherian, M. G. 1974. Isolation and purification of cadmium binding proteins from rat liver. Biochem. Biophys. Res. Commun. 61:923-926. Davis, C. J. 1964. Disc electrophoresis. I I . Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. Friberg, L., Piscator, M., Nordberg, G. F, and Kjellstrom, T. 1974. Cadmium in the environment, 2nd ed., pp. 126-127. Cleveland: CRC Press. Hamilton, D. L. and Smith, M. W. 1975. Effect of cadmium on calcium uptake by rat duodenum. Clin. Res. 23: 635A. Hamilton, D. L. and Valberg, L. S. 1974. Relationship between cadmium and iron absorption. Am. j. Physiol. 227:1033-1037. Imbar, M. Ben-Basset, H. and Sachs, L. 1971. Location of amino acid and carbohydrate transport sites in the surface membrane of normal and transformed mammalian cells. J. Membr. Biol. 6:195-209. Kägi, J. H. R. and Vallee, B. L. 1961. Metallothionein: A cadmium and zinc-containing protein from equine renal cortex. J. Biol. Chem. 236:2435-2442. McCall, M. G., Newman, G. E., O'Brien, J. R. P., Valberg, L. S. and Witts, L. J. 1962. Studies on iron metabolism. I. The experimental production of iron deficiency in the growing rat. Br. J. Nutr. 16:297-304. Mottet, K. N. and Body, R. L. 1976. Primate Paneth cell degeneration following methylmercury hydroxide ingestion. Am. J. Pathol. 84:93-102. Murata, I., Hirono, T., Saeki, Y. and Nakashima, S. 1969. Cadmium enteropathy, renal osteomalacia ("Itai-ltai" disease) in Japan. Bull. Soc. Int. Chir. 29:34. Nordberg, G. F., Goyer, R. and Nordberg, M. 1975. Comparative toxicity of cadmium-metallothionein and cadmium chloride on mouse kidney. Arch. Pathol. 99:192-197.
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Pulido, P., Fuwa, K. and Vallee, B. L. 1966. Determination of cadmium in biological material by atomic absorption spectrophotometry. Anal. Biochem. 14:393-404. Richardson, M. E. and Spivey-Fox, M. R. 1974. Dietary cadmium and enteropathy in the Japanese quail. Lab. Invest. 31:722-731. Richardson, M. E., Spivey-Fox, M. R. and Fry, B. E. 1974. Pathological changes produced in Japanese quail by ingestion of cadmium. J. Nutr. 104:323-338. Rugstad, H. E. and Norseth, T. 1975. Cadmium resistance and content of cadmium binding protein in cultured human cells. Nature (Lond.) 257:136-137. Sugawara, C. and Sugawara, N. 1975. The inductive effect of cadmium on protein synthesis of rat intestine. Bull. Environ. Contam. Toxicol. 14:159-162. Valberg, L. S., Sorbie, J. and Hamilton, D. L. 1976. Gastro-intestinal metabolism of cadmium in experimental iron deficiency. Am. J. Physiol., in press. Webb, M. and Versehoyle, R. D. 1976. An investigation of the role of metallothionein in protection against the acute toxicity of the cadmium ion. Biochem. Pharmacol. 25:673-679. Weisberg, H. and Rhodin, J. 1970. Relation of calcium to mucosal structure and vitamin B12 absorption in the canine intestine. Am. J. Pathol. 61:145-150. Received September 10, 1976 Accepted December 14, 1976
