Chieko Sugawara

Effects of subcutaneous and oral cadmium on iron metabolism: Role of ceruloplasmin and metallothionein

Male ICR mice were either given water containing Cd at a level of 192–200 ppm for 45 days (ingestion group), or were injected subcutaneously once a week with Cd (1 mg/kg) as CdCl2 for 7 weeks (injection group). The control group was given Cd-free water. In both Cd groups, the hematocrit and hemoglobin values did not change markedly. In the ingestion group, the Fe concentration decreased greatly in the liver, kidney, spleen, and duodenum. These decreases may be due to depression of Fe absorption from the intestine. In the injection group, Fe increased in the liver, spleen, and duodenum, although it decreased in the kidney. By Sephadex G-200 gel filtration, Fe-proteins in the hepatic supernatants were located in the void volume region of this gel column in both Cd groups. Apparently, Fe was not a component of metallothionein (MT) protein. The hepatic MT induction by Cd resulted in an increase in hepatic supernatant Cu. Serum Cu and ceruloplasmin (Cp) activity were stimulated only in the injection group. The enhancement of Cp activity may possibly be due to the increase in hepatic Cu which was accompanied by an increase in hepatic Fe, rather than a decrease. Our observations suggest that Fe metabolism is influenced differentially by the administration route of Cd.

Gastrointestinal absorption of Cd-metallothionein and cadmium chloride in mice

CdCl2 or Cd-metallothionein (MT) (6 μg Cd with 2.25 μCi (83.25 KBq)109Cd) was given orally to mice, which were sacrificed at 30 min and 2 h after intubation. Although109Cd in Cd-MT was excreted rapidly into the urine, its absorption was found to be significantly less than that of CdCl2. The poor absorption was due to a decrease of Cd-MT uptake into the intestine. Cadmium chloride taken up into the mucosa could stimulate MT synthesis even 30 min after its intubation. However, the percentage of MT-bound Cd in the Cd of intestinal supernatants was lower with CdCl2 (62% at 30 min and 2 h) than with Cd-MT (78% and 84% at 30 min and 2 h, respectively). These results suggest that the transport mode of lumenal Cd-MT to mucosal cells is different from that of lumenal CdCl2. Lumenal Cd-MT is probably internalized into intestinal cells in an intact form. Furthermore, the Cd-MT may pass through the basolateral membrane in this form. This hypothesis was supported by the different distributions of Cd in the liver and kidney after Cd-MT and CdCl2 intubations.

Iron depletion prevents adenine nucleotide decomposition and an increase of xanthine oxidase activity in the liver of the Long Evans Cinnamon (LEC) rat, an animal model of Wilson's disease.

The Long Evans Cinnamon (LEC) rat, which accumulates excess Cu in the liver as in patients with Wilsons disease, is a mutant strain displaying spontaneous hepatitis. It was reported that Fe, like Cu, increases in the liver and that the severity of hepatitis is modified by Fe in the diet. In this experiment, oxidative stress increased by Fe was investigated before the onset of hepatitis. To examine the effect of Fe on the progress into hepatitis, LEC female rats were fed an Fe-regular (Fe 214 microg/g; Fe(+) group) or an Fe-restricted (Fe 14 microg/g; Fe(-) group) diet from 53 days of age for 35 days. Fischer rats were also fed as control animals. Adenine nucleotide decomposition was determined as an index of oxidative stress based on xanthine oxidase activity. The size of the hepatic pool of adenine nucleotides (ATP+ADP+AMP) was significantly smaller in LEC rats than Fischer rats. The energy charge (ATP+0.5ADP)/(ATP+ADP+AMP) was smaller in Fe(+) groups than in Fe(-) groups. In the LEC rat liver, the Fe concentration in the Fe(+) group was 160% of that in Fe(-) group and the correlation coefficient between the hepatic Fe concentration and the energy charge was significant. In this strain, an increase of xanthine oxidase activity resulted in an increase of xanthine, an oxidized metabolite of hypoxanthine in the liver. The results suggest the involvement of the Fe in the progression into hepatitis in the LEC rat, even if the dietary Fe concentration is similar to that of commercial diet.

Comparative study of effect of acute administration of cadmium and silver on ceruloplasmin and metallothionein: involvement of disposition of copper, iron, and zinc

Male ICR mice were subcutaneously injected with either aqueous Ag (1.5 or 5.0 mg/kg) or Cd (1.5 or 2.5 mg/kg) for 2 consecutive days. Body fluids and livers were collected 24 hr after the second dose. In the hepatic supernatant, Ag and Cd were recovered at 2 and 36-46% of the total dose, respectively. Ag-metallothionein (MT), which is associated with Ag, Cu, and Zn, and Cd-MT, which is associated with Cd, Cu, and Zn, were induced in the liver by the injection of Ag and Cd, respectively. The supernatant Ag and Cd existed in the MT fraction at 34-61 and 97% levels, respectively. Cu concentration in the hepatic supernatant was increased by the Ag and Cd injections. The increased Cu was due to the appearance of Ag-MT and Cd-MT, respectively. Microsomal concentrations of Cu increased in the Cd groups, but decreased in the Ag groups. Serum ceruloplasmin (Cp) activity was remarkably increased by the injection of Cd, but severely decreased by the injection of Ag. These opposing changes in Cp activity induced by Cd and Ag may be due not to the sequestering of Cu in MT, but to the alteration of microsomal Cu concentration and/or the difference in affinity of the induction metals to MT. Hepatic Fe concentration was increased by the Ag injection, but was decreased by the Cd injection. These changes may not be related to induction of MT, but to Cp synthesis in the liver.

The effect of cadmium on vitamin A metabolism

It has been reported that a number of toxic agents impair normal vitamin A (VA) metabolism (A. D. Bruin, 1976, In Biochemical Toxicology of Environmental Agents, pp. 937–980. Elsevier, Amsterdam). A study was conducted to explore the effect of cadmium chloride (CdCl2) on VA metabolism. Female rats of the Wistar strain were given drinking water containing 50 ppm of cadmium (Cd) for 237 days. Cadmium ingestion did not affect the food intake and body weight, but a significant decrease in serum VA was observed, concurrently with an increase in liver VA. In studying ingestion of tritiated VA alcohol (all trans-[15-3H]retinol), it was found that the absorption of VA alcohol from the intestine, the release of newly absorbed VA from the liver to serum, and the conversion of VA to water-soluble metabolites in the liver were not influenced by Cd. These findings suggest that Cd interfered with the release of VA, especially stored VA, to serum.

Role of mucosal metallothionein preinduced by oral Cd or Zn on the intestinal absorption of a subsequent Cd dose

Intestinal absorption of Cd is one of the main channels of Cd entry into the body leading to systemic toxicity. When Cd is absorbed, it first comes into contact with the gastrointestinal mucosa, particularly the duodenum mucosa. Therefore, the mucosal behavior of Cd is one of the most important factors, not only for determining the absorption rate, but also as concerns the interactions between Cd and other metals.

Response of hepatic function to hepatic copper deposition in rats fed a diet containing copper.

Fischer rats were a fed diet supplied with copper chloride (150–600 ppm) for 60 d from weaning. Serum (glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities were increased with the increase of Cu concentration in the diet. Biliary excretion of Cu was related to the dietary Cu level. Depositions of hepatic and renal Cu were also related to the dietary Cu level in a dose-dependent manner. In particular, hepatic (155.2±13.3 μg/g) and renal (44.9±4.4 μg/g) Cu concentrations increased abruptly in the Cu-600 ppm group. In the liver, about 60% of Cu was distributed in the soluble fraction (100,000 g supernatant). In the Cu-600 ppm group, 25% of cystosolic Cu was bound to metallothionein (MT). Our results suggest that chronic exposure to Cu appears to have a deleterious effect on the hepatic function, and further, that even in rats with normal biliary Cu excretion, clearance of Cu from the liver may be marginal when dietary Cu is near the 600-ppm level. Although Cu is an essential nutrient, an overload of Cu should be avoided.

Effect of silver on ceruloplasmin synthesis in relation to low-molecular-weight protein

Male mice received a single subcutaneous injection of 10 mg/kg body weight of Ag as AgNO3 and were killed at 2, 6, 10 and 24 h after the injection. Serum ceruloplasmin (Cp) significantly decreased at all times. These low Cp values were accompanied by low serum Cu levels. There was a significant (P less than 0.01) positive correlation between the two substances. A significant enhancement of hepatic Cu concentration was due to the appearance of low Mr protein induced by the Ag administration. The LMW protein induced by Ag was associated with three metals (Ag, Zn and Cu), the Mr values of which ranged from 8300-10 300. In the hepatic supernatant fraction, Ag was recovered at 0.9, 2.7, 9.4 and 8.7% of the doses at 2, 6, 10 and 24 h, respectively. Unexpectedly, the proportion of Ag existing in the low-Mr protein was only 65% of total supernatant Ag, even at 24 h. The decrease in Cp activity with a concomitant increase in hepatic Cu suggests that the increased hepatic Cu was not utilized for the addition to apo-Cp.

A copper deficient diet prevents hepatic copper accumulation and dysfunction in Long-Evans Cinnamon (LEC) rats with an abnormal copper metabolism and hereditary hepatitis

Long-Evans Cinnamon (LEC) rats that develop spontaneous hepatitis due to an inherently abnormal Cu metabolism have recently been established. This investigation concerns the effects of a Cu-deficient diet on the Cu metabolism linked to hepatic injury in LEC rats. The hepatic Cu concentration at 30 days after birth was 94±4 Cu μg/g liver in LEC rats, whereas that of Fischer rats at the same age was 7±1 Cu μg/g. From 30 days after birth, all rats were fed a semisynthetic diet with two different levels of Cu, 0.5 or 30 μg/g food, for 35 days. In LEC rats fed a Cu-deficient diet (0.5 μg/g), the hepatic Cu concentration was 39±7 μg/g. The Cu-normal diet (30 μg/g) LEC group had a concentration of 357±15 μg/g in the hepatic Cu. The group had significantly higher aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and γ-glutamyl transferase (GGT) levels than did the LEC rats given the Cu-deficient diet. These results suggest that the occurrence of acute hepatitis in LEC rats can be prevented by feeding the animals a Cu-deficient diet.

Binding of cadmium chloride and Cd-metallothionein to mucosal brush border membrane of the rat small intestinal tract

Even though Cd is categorized as a metal with low absorption from the gastrointestinal tract, its total body burden predominantly results from its gastric absorption. Oral Cd also eventually triggers an endemic disease, Itai-Itai disease, in certain areas of Japan. Cd absorption is influenced by several endogenous or exogenous factors such as aging, sex, and food components. To resolve the mechanism of Cd absorption from the gastrointesinal tract, metallothionein (MT) is one of the most interesting endogenous substances. Although the protein induced by Cd exists mainly in the mucosal cytosol fraction as well as in those of liver or kidneys, compared to the function of renal or hepatic MT, its function seems to be slightly different. So far, it is reasonable to assume that the protein is involved in the prevention of the transfer of Cd from the mucosa to the body. However, the behavior of MT in/on the mucosal brush border membrane (BBM) is unclear. In order to gain a better understanding of this point, the authors estimated the Cd binding of CdCl/sup 2/ and Cd-Mt to BBM isolated from control and Cd-exposed rats.