the influence of nutritional deficiencies on gastrointestinal uptake of cadmium and...

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ELSEVIER Toxicology 97 (1995) 71-80 The influence of nutritional deficiencies on gastrointestinal uptake of cadmium and cadmium-metallothionein in rats Hisayoshi Ohta *, M. George Cherian Department of Pathology, The University of Western Ontario, London, Ontario, Canada N6A SC1 Received 19 March 1994; accepted 28 July 1994 Abstract The intestinal uptake and tissue distribution of cadmium (Cd) were studied in control rats and those deficient in zinc (Zn), iron (Fe) or cysteine (SH) using an in situ model where an intestinal loop of 5 cm was incubated with CdCl, or Cd-MT (MT, metallothionein) for 30 and 60 min. The intestinal content of Cd after incubation with CdCl, or Cd- MT was not affected by nutritional deficiencies, but the Cd uptake from CdCl* was always higher than that from Cd- MT. However, both Fe and Zn deficiencies had a marked effect on distribution of Cd in liver, kidney and pancreas. After 30 min incubation in situ with CdCl,, Cd was deposited only in liver in control and SH deficient rats, while Cd was detected also in kidney and pancreas of both Fe and Zn deficient rats. After 60 min incubation with CdCl,, the deposition of Cd in the liver, kidney and pancreas of Fe deficient rat was significantly higher than that in the control. The deposition of Cd after Cd-MT incubation in situ was mainly found in kidney, and nutritional deficiencies increased the tissue deposition of Cd from Cd-MT. Similarly, the renal deposition of Cd absorbed from CdCl, was markedly increased in Fe deficient rats. These results suggest that the intestinal uptake mechanisms of Cd from CdCl, and Cd- MT are different and nutritional deficiencies can markedly increase the deposition of Cd in the kidney, the critical organ in chronic cadmium exposure. Keywords: Metallothionein; Metal absorption; Nutritional deficiency 1. IntradWtion The major source of cadmium (Cd) intake in the general population is food and water, and normal- ly there is little detectable Cd in the air. In experi- mental animals and humans, the absorption of Cd from the gastrointestinal tract is estimated to be about l-6% of total intake (Hamilton and Valberg, 1974; Neathery and Miller, 1975; Yamagata et al., 1975; Flanagan et al., 1978). After absorption, Cd is mainly accumulated in liver and kidney and bound to metallothionein l Corresponding author, Department of Occupational Health and Toxi~logy, School of Allied Health Sciences, Kitasato University, 1-15-l Kitasato, Sagamihara, Kanagawa 228, Japan. (MT), a low molecular weight, sulphur-rich, metalloprotein (Kagi and Vallee, 1961; Cherian and Goyer, 1978). Intracellular metal binding pro- teins, including MT or MT-like Cd binding pro- 0300-483x/95/$09.50 @ 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(94)02925-K

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ELSEVIER Toxicology 97 (1995) 71-80

The influence of nutritional deficiencies on gastrointestinal uptake of cadmium and cadmium-metallothionein in rats

Hisayoshi Ohta *, M. George Cherian

Department of Pathology, The University of Western Ontario, London, Ontario, Canada N6A SC1

Received 19 March 1994; accepted 28 July 1994

Abstract

The intestinal uptake and tissue distribution of cadmium (Cd) were studied in control rats and those deficient in zinc (Zn), iron (Fe) or cysteine (SH) using an in situ model where an intestinal loop of 5 cm was incubated with CdCl, or Cd-MT (MT, metallothionein) for 30 and 60 min. The intestinal content of Cd after incubation with CdCl, or Cd- MT was not affected by nutritional deficiencies, but the Cd uptake from CdCl* was always higher than that from Cd- MT. However, both Fe and Zn deficiencies had a marked effect on distribution of Cd in liver, kidney and pancreas. After 30 min incubation in situ with CdCl,, Cd was deposited only in liver in control and SH deficient rats, while Cd was detected also in kidney and pancreas of both Fe and Zn deficient rats. After 60 min incubation with CdCl,, the deposition of Cd in the liver, kidney and pancreas of Fe deficient rat was significantly higher than that in the control. The deposition of Cd after Cd-MT incubation in situ was mainly found in kidney, and nutritional deficiencies increased the tissue deposition of Cd from Cd-MT. Similarly, the renal deposition of Cd absorbed from CdCl, was markedly increased in Fe deficient rats. These results suggest that the intestinal uptake mechanisms of Cd from CdCl, and Cd- MT are different and nutritional deficiencies can markedly increase the deposition of Cd in the kidney, the critical organ in chronic cadmium exposure.

Keywords: Metallothionein; Metal absorption; Nutritional deficiency

1. IntradWtion

The major source of cadmium (Cd) intake in the general population is food and water, and normal- ly there is little detectable Cd in the air. In experi- mental animals and humans, the absorption of Cd

from the gastrointestinal tract is estimated to be about l-6% of total intake (Hamilton and Valberg, 1974; Neathery and Miller, 1975; Yamagata et al., 1975; Flanagan et al., 1978). After absorption, Cd is mainly accumulated in liver and kidney and bound to metallothionein

l Corresponding author, Department of Occupational Health and Toxi~logy, School of Allied Health Sciences, Kitasato University, 1-15-l Kitasato, Sagamihara, Kanagawa 228, Japan.

(MT), a low molecular weight, sulphur-rich, metalloprotein (Kagi and Vallee, 1961; Cherian and Goyer, 1978). Intracellular metal binding pro- teins, including MT or MT-like Cd binding pro-

0300-483x/95/$09.50 @ 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(94)02925-K

72 H. Ohra. M.G. Cherian / Toxicology 97 (1995) 71-80

teins have been purified from beef liver and kidney, shellfish, and also from plants, especially leafy vegetables (Wagner and Trotter, 1982; Stone and Overnell, 1985).

It is well known that Cd absorption from the gastrointestinal tract and its body retention are affected by various nutritional factors, such as deficiencies in calcium (Ca), iron (Fe), zinc (Zn) (Bremner, 1974; Washko and Cousins, 1974; Valberg, et al., 1976), protein, and also difference in tibre content of diet (Suzuki et al., 1969; Omori and Muto, 1977). In addition, the chemical form can markedly affect the organ distribution of Cd when administered parenterally (Cherian and Shaikh, 1975; Nordberg et al., 1975; Tanaka et al., 1975). The oral administration of Cd in the form of Cd-MT resulted in increased accumulation of Cd in the kidney as compared to feeding a similar dose of CdC12 (Cherian, 1983). Although there are several reports on intestinal absorption of Cd from CdC12, the factors affecting absorption of Cd from Cd-MT, the form normally found in food, are not well understood.

In our previous study on gastrointestinal uptake of Cd, we reported that the intestinal uptake of Cd-MT was a slow process, compared with CdClz, using an everted rat intestinal sac in in situ experiments, and that the absorbed exogenous Cd- MT was mainly deposited in the kidney (Ohta et al., 1989; Ohta and Cherian, 1991). We also reported that the endogenous MT levels in intes- tine did not affect the uptake of Cd from the lumen, but high intestinal MT level can decrease both the release of Cd from the intestine and its deposition in tissues. The differences in the absorption of Cd from CdCl, and Cd-MT, and its tissue distribution, have been reported in mice (Sugawara and Sugawara, 199 1).

The present study was carried out using an in situ model of rat intestine to investigate the effects of nutritional deficiencies, such as methionine, cys- teine (SH), Zn and Fe, on the uptake of Cd or Cd- MT from gastrointestinal tract and its distribution to various organs. The hypotheses tested in these experiments are whether nutritional changes can alter the uptake of Cd from two different forms, CdClz and Cd-MT, in the intestine, and also whether they have any effect on the deposition of Cd in kidney, the critical organ in Cd toxicity.

2. Methods

2.1. Animals Male Sprague-Dawley rats (Canadian Breeding

Farm and Laboratory, Saint-Constant, Quebec, Canada) weighing 250-300 g were housed in a temperature-controlled room on an alternating 12 h light/dark cycle. Animals were fed with a control rodent diet or three separate diets deficient in sulphur amino acids, i.e. no methionine, cysteine (SH) or Zn (< 1 mg/kg), or Fe (<2 mg/kg), and water ad libitum for 6 days. They were fasted for -20 h before experimentation. The control and three diets deficient in Zn, Fe and SH were pur- chased from Zeigler Bros. Inc. (Gardners, PA). These diets were selected for this study because of the potential interaction of Cd in these nutritional deficiencies.

2.2. Sample preparation The ‘%d-labelled MT-II (Cd-MT) was isolal

ted from livers of rats subcutaneously injected daily with ‘%dCl, (1 mgCd/kg, 1 &i) for 2 weeks. The liver homogenate was heated in a water bath at 80°C for 2 min and then centrifuged at 105 000 x g for 60 min at 4°C to obtain the heated supematant. Samples (30 ml) were frac- tionated on a calibrated Sephadex G-75 column (15 x 90 cm) to separate ‘%d containing 10 000 molecular weight fraction. The two major forms of Cd-MT (MT-I and II) were separated on DEAE Sephacel ion-exchange chromatography (Templeton and Cherian, 1984). In this study, Cd-MT II was used because it is present in high concentration in rat liver after induction and can be isolated in large amounts. The contents of Cd, Zn, MT and the specific activity of ‘@Cd-MT were estimated for the purified Cd-MT II samples. The CdCL solution was spiked with ‘%d (carrier free, New England Nuclear Corp., Boston, MA) and the spe- cific radioactivity of ‘09Cd in both CdCl, and Cd- MT were adjusted to comparable values.

Preparation of intestinal loop and in situ in- cubation conditions were similar to our previous publication (Ohta and Cherian, 1991). Briefly, rats were anaesthetized with 60 mg/kg of sodium pen- tobarbital (Ohta et al., 1989). The abdomen was opened and the jejunum (5-6 cm) was cannulated with a polyethylene tubing (PE-50). The intestinal

H. Ohta. M.G. Cherian / Toxicology 97 (1995) 71-80 73

content was flushed out with 10 ml of saline solu- tion. About 0.5 ml incubation solution (Stacey and Klaassen, 1980; Ohta et al., 1989; Ohta and Cherian, 1991) containing rWCdC12 or lWCd-MT (- 50 PgCd) was injected through the cannula into the jejunum segment and the isolated segment clamped at both ends. The jejunum segment was put back into the peritoneal cavity and covered with a gauze to keep in a moist condition, The ani- mal was maintained under anaesthesia at about 37°C during the experiment on a heated pad (Doluisio et al., 1969; Barr and Riegelman, 1970). At the end of the experiment period (30 or 60 min), the jejunum segment was removed and rats were killed by exsanguination. Blood samples were col- lected in heparinized tubes from abdominal aorta.

2.3. Analysis The removed jejunum segment was washed out

immediately with 20 ml fresh incubation buffer, after collecting the incubation solution of ‘WCdC12 or lWCd-MT. The intestinal segment was blotted on absorbent filter paper and weighed. The amount of lWCd in the intestine, various or- gans, blood and incubation medium was determin- ed by radioactive counting using a LKB rack

gamma scintillation spectrometer with a counting eff’ciency of 85%. Plasma samples were analyzed for Zn and Fe by atomic absorption spec- trophotometry (Varian Spectra 30) with an air- acetylene flame, directly or after dilution with distilled water. The intestinal tissue samples, after incubation, were homogenized in ice-cold 0.25 M sucrose/O. 1 M Tris-HCL buffer (pH 7.4) and 20% homogenate was centrifuged at 15 000 x g for 15 min at 4” to separate the particulate and cytosol fractions. The Cd content of these fractions was determined by ‘@Cd radioactivity. A portion (0.3 ml) of the cytosol fraction was applied on a calibrated Sephadex G-75 column (0.9 x 60 cm at 4°C) to determine the distribution of Cd and Zn in various binding ligands. MT levels were determin- ed by modified Ag-Heme method without radio- active silver, as described by Scheuhammer and Cherian (1991). The content of silver in samples was estimated by atomic absorption spectrometry.

2.4. Statistics All results are expressed as the arithmetic mean

with the standard deviation. The data was statistically analyzed by Student’s t-test and a one- way ANOVA, with a Neuman-Keuls follow-up

Table 1 The distribution of Cd in cytosolic and particulate fractions of rat intestine incubated with ‘WCdCI, for 30 and 60 min

Experimental

group

Control

SH-Def.

Zn-Def.

Fe-Def.

Incubation time

(mm)

30

60

30

60

30

60

30

60

Cd concentration (pglg and %)”

Cytosol Particulate

16.0 f 6.4 11.0 zt 2.7 (58.2 zt 6.5) (41.8 f 6.5) 16.9 f 5.0 16.7 f 3.6 (50.1 f 6.8) (49.9 f 6.8) 15.6 f 1.5 13.5 f 3.6 (54.1 f 6.0) (46.0 f 6.0) 23.5 f 3.8 19.4 zt 2.5 (54.6 f 4.9) (45.4 * 4.9) 21.5 f 4.3 17.5 f 3.2 (54.9 f 2.3) (45.1 f 2.3) 26.5 f 9.2 19.9 f 3.7 (56.3 f 4.7) (43.8 f 4.7) 19.1 l 3.2 14.3 f 3.0 (57.2 f 5.0) (42.8 f 5.0) 22.8 f 3.7 19.1 f 5.4 (54.9 f 6.6) (45.1 f 6.6)

Total

27.0 f 8.6

33.7 * 7.2

29.1 f 4.4

42.9 f 5.0

39.0 l 7.3

46.4 zt 1.28

33.4 f 5.4

42.0 -f 7.8

‘Data @g/g) are expressed as mean f SD. The distribution of Cd in each fraction is shown in parentheses as percent.

14 H. Ohra, M.G. Cherian / Toxicology 97 (1995) 71-80

test, was used for all comparisons involving more than two groups. In all statistical comparisons, a P s 0.05 was considered a significant difference.

3. Results

When compared to age-matched controls, the concentrations of Zn and Fe in plasma (&ml) were significantly decreased in rats fed with the de- ficient diet of Zn (0.90 f 0.1 vs. 1.22 f 0.2) and Fe (0.98 f 0.3 vs. 1.33 i 0.2) for 6 days. How- ever, the SH deficiency was difficult to demonstrate after feeding of SH deficient diet for 6 days. There was no change in the plasma glutathione levels, but these rats did not gain any weight. The intestinal uptake of Cd from CdCl, was not significantly affected by nutritional deti- ciencies of Fe, Zn or SH (Table 1). At both 30 and 60 min incubation periods, the total Cd uptake, and that associated with cytosol and particulate fractions in intestine, did not change in any of the nutritional deficiencies. However, in all experi- ments, the total Cd concentration in intestinal tis- sue was higher at 60 min incubation period than at the 30 min. Intestinal MT content remained un- changed in all experiments following incubation with Cd (Table 2). The MT concentration in intes- tine of SH and Zn deficient rats was slightly lower than control values, but was not statistically signi-

Table 2 Metallothionein concentration in the intestine after in situ incubation with CdCl,

Experimental

group

n Incubation Metallothionein time (mitt) concentrationa

(fig/g tissue)

Control 5 30/60 20.45 f 5.56 CdCl, 8 30 19.19 f 3.20

4 60 16.42 zt 1.93

SH-Defb 3 30 17.12 f 3.92 3 60 13.78 f 2.05

Zn-Defb 4 30 13.11 f 3.69 4 60 17.02 +z 3.29

Fe-Defb 4 30 28.74 f 1.73 4 60 24.59 +z 6.19

aValues are expressed as mean f S.D.in &g tissue. bAnimals were fed the deficient diet for 6 days prior to the experiment.

E

‘$ ,

0.06 7 Pancreas ( * - * m 0” 0.04 -

0.02 -

0 I I I Control SH-D Zn-D Fe-D

Experimental Groups

Fig. I. Cadmium concentration in liver, kidney and pancreas of control and nutritional deficient rats after incubation with CdCl,. Rats were fed a control diet, or a diet deficient in methionine and cysteine (SH), zinc (Zn) or iron (Fe), for 6 days. A 5 cm intestinal segment was incubated with ‘09CdC12 in situ for 30 (0) min or 60 min @) and the deposition of Cd in tissues (liver, kidney and pancreas) were determined by measuring radioactivity. The results are from four different experiments. Control: Control rat incubated with ‘WCdC12. SH-D: Methinine and cyteine deficient rat incubated with ‘WCdCI,. Zn-D: Zn deficient rat incubated with ‘%dC12. Fe-D: Fe de- ficient rat incubated with ‘@‘CdCl,. *Significantly different from the control group at P < 0.05. *Significantly different from the experimental groups (Zn, Fe or SH deficient groups) at P < 0.05.

ficant. The concentration of Cd in liver, kidney and pancreas after CdCl, incubation was increas- ed in both the deficiencies of Fe and Zn (Fig. 1). After 30 min incubation with CdCl,, while Cd was detected in the liver of all groups of rats, it was not detectable in kidney or pancreas of control or SH deficient rats. However, Cd was detected in the kidney and pancreas, in both Fe and Zn deficient

H. Ohra. M.G. Cherian / Toxicology 97 (1995) 71-80 15

Experimental Groups

Fig. 2. Intestinal uptake of Cd in the control rats incubated with CdClz or Cd-MT and in Zn and Fe deficient rats incubated with Cd-MT. Experimental details are as described in Fig. 1. Incubation time: 30 min 0, 60 min 0. *Significantly different from the control group at P < 0.05.

rats, after 30 min incubation. Furthermore, at 60 min after CdClz incubation, the Cd deposition in the liver, kidney and pancreas of Fe deficient rats was significantly higher than those of control rats. The increased tissue deposition of Cd was more

Table 3 Metalfothionein concentration in the intestine after in situ incubation with ‘@%d-metailothionein

Experimental n

group

Incubation 5 mediumb

ControP 4 4

Zn-Defd 3 3

Fe-DeP 4 4

Incubation time (min)

30/60 20.45 f 5.56

30 50.19 + 18.15* 60 76.11 f 39.66* 30 41.79 l 10.47* 60 87.97 f 25.56* 30 40.69 f 14.50’ 60 66.37 zt 32.70’

Metallothionein concentrationa (j&g tissue)

‘Values are expressed as mean f S.D. in &g tissue. Metallo- thionein was detetined by the Ag-heme method. bIncubation medium: incubated with incubation medium alone. FControl: control rat incubated with lWCd-MT. dZn-Def: zinc deficient rat incubated with ‘09Cd-MT, Fe-D& iron deficient rat incubated with ‘@Cd-MT. Animals were fed a diet deficient in Zn or Fe for 6 days prior to the experiment. *Significantly different from the group incubated with incuba- tion medium alone at P < 0.05.

prominent in Fe deficiency than in Zn deficiency (Fig. 1).

To evaluate the effect of chemical form on the uptake of Cd from intestine, “%d-MT was in- cubated under the same experimental condition. However, no experiments with Cd-MT were per- formed in SH deficient rats because of the insufli- cient amount of isolated Cd-MT. The intestinal Cd accumulation was - 6 pglg and 15 pglg after 30 min and 60 min incubations with Cd-MT, respec- tively, and these values were significantly lower than that from CdCl, incubation in control rats (Fig. 2 and Table 1). Similarly, the intestinal Cd accumulation in both Fe and Zn deficient rats from Cd-MT incubation at both 30 min and 60 min was significantly lower than that of rats in- cubated with CdC12. No significant difference in Cd accumulation in intestine was observed be- tween Zn deficient rats and the control after in- cubation with Cd-MT, while there was an increase in intestinal Cd content in Fe deficiency. When in- testinal loops were incubated with incubation me- dium alone, there was no change in MT levels at either 30 or 60 min incubation periods. However, there was a time-dependant increase in intestinal MT levels in control, as well as in Fe and Zn defici- ent rats, incubated with Cd-MT (Table 3). The amount of MT at 60 min incubation was about twice that after 30 min incubation period when in- cubated with Cd-MT. These results suggest that Cd in the form of Cd-MT can be taken up intact by intestinal mucosal cells.

The absorbed Cd from Cd-MT incubation was mostly recovered in kidney. The deficiencies of Fe and Zn had little effect on the tissue distribution of Cd from Cd-MT incubation. However, at 60 min after Cd-MT incubation, Cd deposition in the kid- ney of Fe deficient rat was markedly increased, compared with that of control and of the other experimental groups (Fig. 3).

Cadmium in control rat intestine, incubated with CdC12, was distributed almost equally be- tween cytosol and particulate fractions (Fig, 4). On the other hand, Cd in the intestine was recovered mainiy from the cytosol fraction (75-X%), after incubation with Cd-MT, in rats fed with control diet or nutritional deficient diets of Zn and Fe. Unlike in CdC12 incubation, only a

16 H. Ohta. M.G. Cherian / Toxicology 97 (1995) 71-80

0.03

0.02

0.01

” 0 g 0.05 I-

s _ 0.04 1 Zn-D

*z 0.03 r!

5 0.02

g 0.01 E: s 0 I-I4

._

5 0.21 Fe-D

o 0.15

0.1

0.05

0 l-4 Liver Kidney

Fig. 3. Tissue distribution of Cd from control (top panel) or nutritionally deficient rats (bottom two panels) at 30 min 0 and 60 min El after incubation with t’%d-MT. Control: Control diet rat incubated with ‘@kd-MT. Zn-D: Zn deficient rat in- cubated with ‘%d-MT. Fe-D: Fe deficient rat incubated with ‘c%d-MT. *Significantly different from the control group at P < 0.05.

small fraction was associated with the particulate fraction in Cd-MT incubated rats. Thus, there was a significant difference in cellular distribution of Cd in the intestine when incubated with CdC12 or Cd-MT. Compared to the control and Zn-deficient rats, the Fe-deficient rats sequestered significantly more Cd in the particulate fraction when in- cubated with Cd-MT. In addition, the cytosolic Cd, after incubation with Cd-MT, was mainly bound to MT, as shown by Sephadex G-75 gel Iil- tration (Fig. 5). More Cd was bound to MT in Fe deficient rats than the control. These results are

VW 100,

v*-- E 30min * * ’ I

80 n * 60

40

20

0

(%) ’ II

100 E 60 min * -*

* --! 80

60

1

Experimental Groups

Fig. 4. The distribution of Cd in cytosol and particulate frac- tions of the intestine of control and nutritional deficient rats incubated with tWCdC12 or ‘@kd-MT. Intestinal segments were washed after incubation, homogenized and centrifuged at 15 000 x g for I5 min to separate the cytosol Cl and particulate q fractions. I - control rat incubated with CdCI,; II - con-

trol rat incubated with Cd-MT; III - Zn-deficient rat in- cubated with Cd-MT; IV - Fe-deficient rat incubated with Cd-MT. *Significantly different from the control group P < 0.05.

different from the fractionation of the intestinal cytosol from rats incubated with CdClr, where most of the Cd was bound to high molecular weight proteins (>60 000) and there was little binding to MT-like proteins ( data not shown).

4. Di!?eusaloa

Several lines of evidence in experimental ani- mals suggest that interactions of essential metals (e.g. zinc, iron and calcium) and a toxic metal, like cadmium, can occur at the intestinal-absorption

H. Ohta. M.G. Cherian / Toxicology 97 (1995) 71-80 7-l

81200 g,ooo 0.4 i m

g 600 0.3 2

3 600 0.2

: 400

g

0.1 5 E

0 00

10 20 30 0.5 .E G

MT A"

BlOOO ".L(

800 0.3

600 0.2

400

200 0.1

0 0 0 10 20 30

Fraction Numbers

Fig. 5. Sephadex G-75 fractionation of cytosol fractions from intestine of control (top panel) and nutritionally deficient rats (bottom two panels) incubated with “%d-MT for 60 min. MT I indicates the elution position of rat liver metallothionein. Control: Control rat incubated with “%d-MT. Zn-D: Zn de- ficient rat incubated with tWCd-MT. Fe-D: Fe deficient rat in- cubated with tWCd-MT.

step (Jacobs et al. 1969; Hamilton and Valberg, 1974; Flanagan et al. 1978; Sugawara et al., 1984). It is unclear whether these interactions are directly related to competition for similar absorption mechanisms, structural changes in the intestinal cells affecting metal transport or due to alterations in metal binding ligands. Both mineral and sulfur amino acid deficiencies can affect all these factors and, thereby, influence the uptake and tissue dis- tribution of cadmium. The present study is an extension of our previous report (Ohta and Cherian, 1991) on the differences in intestinal up-

take of cadmium salts and Cd-MT in situ from rat jejunum, but focuses mainly on the effects of nutri- tional deficiencies, especially zinc, iron and sulfur amino acid deficiencies, on the uptake and tissue distribution of Cd from Cd-MT.

Mineral (zinc and iron) and sulfhydryl deticien- ties had little effect on the intestinal content of MT or cadmium after in situ incubation with CdCl,. However, the deposition of cadmium in liver, kid- ney and pancreas was increased significantly in iron and zinc deficient rats, suggesting an increas- ed uptake of cadmium in iron deficiency. Since these effects are not observed in cysteine deticien- cy, they cannot be considered as a general effect on membrane transport systems in nutritional deti- ciencies. Although the increased cadmium absorp- tion in iron deficiency has been reported both in animals (Valberg et al., 1976) and in human (Flanagan et al., 1978), the mechanisms involved are not yet understood. In the major pathways of intestinal iron absorption, iron binding proteins, such as ferritin and transferrin, or transferrin receptors, may be involved (Moore, 1961; Finch and Huebers, 1982; Schafer and Forth, 1984). In iron deficiency, the intestinal iron uptake mecha- nisms are activated and cadmium may compete with iron for absorptive sites at the mucosal brush- border membrane (Sugawara and Sugawara, 1987). In addition, chronic feeding of cadmium salts can cause anaemia in Japanese quail (Jacobs et al., 1969), showing the interaction between cadmium and iron in intestinal absorption mechanisms.

The steps involved in the intestinal absorption of cadmium have been studied in detail. In a series of experiments, Foulkes and co-workers have demonstrated the characteristics of cadmium bind- ing to intestinal epithelium and the mechanism of cadmium uptake by intestinal mucosal cells (Foulkes, 1980, 1985, 1988, 1991; Foulkes and McMullen 1987; Bevan and Foulkes, 1989). These studies show the electrostatic binding of cadmium to anionic membrane charges, which can be non- competitively and non-specifically inhibited by polyvalent cations. In a subsequent temperature- sensitive step, Cd was internalized into the cell. The membrane fluidity may also be a critical factor in internalization. There is no evidence for the ex- istence of specific carriers for cadmium uptake. It

78 H. Ohta. M.G. Cherian / Toxicology 97 (1995) 71-80

appears that this process is not an energy-dependent process, nor is the passage unidirectional. The lack of effect of sulfhydryl deficiency on uptake of cad- mium in the present study is not surprising because of the demonstrated non-involvement of reactive sulthydryl groups in cadmium binding on the intes- tinal brush-border membrane (Foulkes, 1991). A re- cent study has shown a lack of effect of glutathione on the uptake of Cd from the jejunal mucosa, sug- gesting a stronger binding of Cd to intestinal mem- brane than mercury (Foulkes, 1993). Thus, while there is some information on the uptake mechanisms of CdCl*, there are only a few studies on the intestinal uptake mechanism of Cd-MT, the major form of Cd found in food. The uptake of cad- mium, bound to metallothionein, by isolated intes- tinal brush-border membrane was shown to be small compared to cadmium salts, in in vitro studies (Sugawara et al., 1988), suggesting a different mech- anism for the uptake of Cd-MT.

Similar to our previous reports (Ohm et al., 1989; Ohta and Cherian, 1991), the uptake of exogenous Cd-MT from gastrointestinal tract was much slower than that of CdC12 in this study. Moreover, the cadmium absorbed from Cd-MT was mainly transported to the kidney, in contrast to the hepa- tic deposition of cadmium from CdCl, (Cherian, 1983). These results are supported by a study in mice (Sugawara and Sugawara, 1991). The intestinal con- centration of Cd-MT is not affected significantly by nutritional deficiencies in our present study. Both hepatic and renal concentrations of cadmium, after 60 mitt incubation of CdClz, are markedly increas- ed in iron deficiency. Studies on low oral dose of cadmium salts, showed that the concentration of cadmium accumulated by the kidney is also substan- tially higher than that in the liver (Lehman and Klaassen, 1986). Thus, the distinct difference in dis- tribution of cadmium between kidney and liver is influenced not only by the form of cadmium ad- ministered but also by the dose (Miller et al., 1968; Lehman and Klaassen, 1986; Scheuhammer, 1988). There is no experimental evidence which suggests any change in circulating MT levels in iron delicien- cy. Therefore, it is difficult to conclude that the in- creased renal deposition of cadmium from CdCl, in iron deficiency is due to changes in circulating MT.

The role of intestinal MT on the uptake of zinc, copper and cadmium has been discussed by several investigators (Squibb et al., 1976; Engstrom and Nordberg, 1979; Kello et al., 1979; McGivern and Mason, 1979; Sugawara and Sugawara, 1987; Bevan and Foulkes, 1989; Ohta and Cherian, 1991). The absorption mechanisms of these metals are not as well characterized as those for iron, which involves internalization of the iron-bound transferrin- receptor complex (Finch and Huebers, 1982), or as those for calcium, which uses specific calcium chan- nels (Wasserman and Fullmer, 1983; Bean, 1989). However, intense immunohistochemical staining for MT has been demonstrated in the mucosal cells of intestine incubated with Cd-MT (Ohta and Cherian, 1991). These results suggest that small amounts of exogenous Cd-MT can be taken up from the intes- tinal lumen, released to circulation and then deposited in the kidney, although the exact mecha- nism of Cd-MT uptake from the lumen is unclear. An increase in time-dependent uptake of intact MT is also observed in the present study after incuba- tion with Cd-MT.

It has also been shown that induction of intesti- nal MT synthesis with oral feeding of zinc salts can decrease the deposition of cadmium in liver, in in situ experiments, when rat intestine was incubated with CdClz (Ohm and Cherian, 1991). This may be due to the strong binding of Cd to intestinal MT, thereby, decreasing the release of cadmium from the intestine and its transport to organs. In both zinc and iron deficiencies, the hepatic cadmium levels are increased after incubation with CdCl,. The results suggest that under these mineral nutritional deticien- ties, the uptake and transport of cadmium to the liver may not be related directly to the endogenous intestinal MT but may be related to metabolic changes in the intestine. The fractionation of intes- tinal cytosol from control and zinc or iron deflci- ent rats, after incubation with CdCl,, suggests that most of the cadmium is associated with high mo- lecular weight (> 60 000 Da) proteins, which is also reported by others in zinc deficiency (Kowarski, et al., 1974; Hahn and Evans, 1975). On the other hand, a major portion of cadmium, after incuba- tion with Cd-MT in the intestine, was still bound to MT.

In summary, the present study demonstrates that

H. Ohta. M.G. Cherian / Toxicology 97 (1995) 71-80 79

in both zinc and iron deficiency, the uptake and tis- sue concentration of cadmium are increased when incubated with CdCl,. The hepatic deposition of cadmium is increased in both zinc and iron delicien- ties, while renal deposition is more markedly in- creased in iron deficiency. This study also demonstrates that Cd-MT is absorbed intact from the intestine, but in smaller amounts than cadmi- um salts. In iron deficiency, the intestinal accumula- tion of cadmium from Cd-MT is increased, and most of this cadmium is recovered in the cytosolic fraction. In addition, the renal accumulation of cad- mium is high, from Cd-MT incubation, in iron de- ficiency. Thus, some of the interactions between essential metals and cadmium can be explained at the intestinal absorption level.

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