Naoki Sugawara

Decreased hepatobiliary secretion of inorganic mercury, its deposition and toxicity in the Eisai hyperbilirubinemic rat with no hepatic canalicular organic anion transporter

Eisai hyperbilirubinemic (EHB) rats, a new mutant strain inbred from Sprague-Dawley (SD) rats, show no inherent expression of the canalicular multidrug resistance protein (cMrp) and lack canalicular multispecific organic anion transporter (cMOAT) activity. A sample of 203Hg (40 microCi with 40 microg Hg/kg) was injected intravenously (i.v.) into four male SD and EHB rats. Biliary excretion of reduced-glutathione (GSH) was 426 and 2 microg/bile for 15 min in the SD and EHB rats, respectively. Biliary excretion of 203Hg for 45 min in EHB rats significantly decreased to 1/4 of that of the SD rats. However, there was no difference in the hepatic uptake of 203Hg between the two strains. Other rats were injected subcutaneously (s.c.) with HgCl2 solution (at 0.2 and 1.6 mg/kg) containing 203Hg. Some 4 days after the injection of 0.2 mg/kg, about 3 and 13% of the total dose was found in the liver in SD and EHB rats, respectively. The hepatic supernatant Hg was recovered mainly in the void volume of a Sephadex column. Some 2 days after the injection of 1.6 mg/kg, these values were 3 and 23% in SD and EHB rats, respectively. The increased retention stimulated hepatic metallothionein (MT) induction and increased the proportion of Hg in the MT region on the Sephadex column. On the other hand, biliary excretion of 203Hg for 15 min in EHB rats was about 1/6-1/4 of that in SD rats. With the injection of 1.6 mg/kg, hepatic and renal functions worsened in EHB rats. In particular, severe necrosis was found in the renal tubules. Our results suggest that biliary secretion of inorganic Hg may be partly regulated by the ATP-dependent transport system, the glutathione S-conjugate export pump (GS-X pump) composed of Mrp and MOAT. Significantly decreased excretion stimulates hepatic retention of inorganic Hg. However, the hepatic lesions are less predictive. The MT induction may reduce the toxicity of metal to the liver cells.

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.

Biliary excretion of exogenous cadmium, and endogenous copper and zinc in the Eisai hyperbilirubinuric (EHB) rat with a near absence of biliary glutathione

Mutant Eisai hyperbilirubinuric (EHB) rats derived from an inbred strain of Sprague-Dawley (SD) rats are characterized by a near absence of biliary excretion of glutathione (GSH) due to inherently impaired ATP-driven organic anion transport. Cd (0.1 mg/kg bw from CdCl2) was injected intravenously into EHB rats and control SD rats. Output of biliary excretion of Cd was followed over 15-min intervals up to 60 min. Cd was excreted rapidly and reached the maximum level (73.2 ng/15 min) in the period from 15 to 30 min in SD rats. Its excretion in EHB rats, however was one-fortieth (only 1.8 ng/15 min) of that in SD rats. Biliary concentrations of two endogenous metals, Cu and Zn were also measured. The output of Cu in EHB rat bile (50 ng/15 min before Cd injection) was about one-fifth of that in SD rat bile (270 ng/15 min). The output was not influenced by the Cd injection in the two groups. There was a slight difference of Zn output between the two groups. The biliary excretion of GSH was 500 to 700 micrograms/15 min and only 1 to 2 micrograms/15 min in SD and EHB rats, respectively. Sixty min after Cd injection, the Cd concentrations in the serum, liver and kidney were slightly higher in EHB rats than in SD rats. There was no difference in the hepatic metallothionein (MT-I and-II) concentration between SD (34 micrograms/g liver) and EHB (33 micrograms/g liver) rats. The renal Cu concentration was about four times in the higher in the EHB rat than in the SD rat. These results suggest that reduced biliary excretion of Cd is mainly, but that of Cu is only partly, based in reduced canalicular transport of GSH due to lack of an ATP-driven organic anion transport system, not MT induction in EHB rats. It seems likely that biliary excretion of Cd is regulated mainly by the canalicular anion transport in rats.

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.