Cinnamon-like Coat Color Research Articles

Effect of glutathione depletion on removal of copper from LEC rat livers by tetrathiomolybdate

Tetrathiomolybdate (TTM) is a powerful and selective copper (Cu) chelator that is used as a therapeutic agent for Wilson disease. TTM is the sole agent that can remove Cu bound to metallothionein (MT) in the livers of Long–Evans rats with a cinnamon-like coat color (LEC rats). However, the administration of excess TTM causes the deposition of Cu and molybdenum (Mo) in the liver. In the present study, the effect of hepatic glutathione (GSH) depletion on the removal of Cu from the livers of LEC rats was evaluated to establish an effective therapy by TTM. Pretreatment with l-buthionine sulfoximine (BSO), a depletor of GSH in vivo, reduced the amounts of Cu and Mo excreted into both the bile and the bloodstream, and increased the amounts of Cu and Mo deposited in the livers of LEC rats in the form of an insoluble complex 4 h after the TTM injection. The results suggest that GSH depletion creates an oxidative environment in the livers of LEC rats, and the oxidative environment facilitates the insolubilization of Cu and Mo in the livers of LEC rats after the TTM injection. Therefore, the effect of TTM on the removal of Cu from the liver was reduced in the oxidized condition. Wilson disease patients and LEC rats develop liver injury caused by oxidative damage. From a clinical viewpoint, increasing in the GSH concentration is expected to enhance the effect of TTM.

Effects of soy protein isolate on LEC rats, a model of Wilson disease: mechanisms underlying enhancement of liver cell damage

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.

Roles of metallothionein in copper homeostasis: responses to Cu-deficient diets in mice

Metallothionein (MT) protects the body from both harmful non-essential and excessive essential metals. Copper (Cu) is an essential metal, and its concentration in the body is regulated at a constant level between excess and deficient ones. Cu accumulating in the livers of Wilson disease patients and its animal model, Long-Evans rats with a cinnamon-like coat color (LEC) rats, is in the form of Cu,Zn–MT, MT being an antioxidant. Contrary to the efficient production of MT in response to excessive accumulation of Cu in LEC rats, Cu-binding to MT only occurs marginally under normal conditions. However, the present study revealed that Cu binds to MT more with a severe Cu-deficiency. Namely, male C57BL/6J mice were fed a Cu-deficient diet (0.037 mg Cu/g) and deionized water containing trientine, and then the concentration and distribution of Cu were determined. It was suggested that the cessation of biliary excretion and limitation of the Cu supply to ceruloplasmin are the first responses on feeding of a Cu-deficient diet, followed by an increase in Cu–MT with maintenance of the Cu concentration in the liver. These results suggest that MT causes the recruitment of Cu in a Cu-deficient environment by sequestering Cu from degraded Cu-enzymes and delivering it to Cu chaperones.

Overexpression of cyclin E and cyclin-dependent kinase 2 is correlated with development of hepatocellular carcinomas

Mammalian cell cycle progression is regulated by the combined action of cyclins/cyclin-dependent kinases (CDKs) and CDK inhibitors. Abnormal expression as well as interaction of these proteins may result in malignant transformation of cells. To further address the role of these cell cycle proteins in hepatocellular carcinomas, we analyzed the expression of cyclin E and CDK2. A panel of livers with human hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis were used as a human experimental system. The inbred LEC (Long–Evans with a cinnamon-like coat color) rats were used as an animal experimental HCC model. Immunohistochemical staining of serial paraffin sections was performed using antibodies to cyclin E and CDK2. The results showed that cyclin E and CDK2 were concurrently overexpressed in hepatocellular carcinomas both in human and rat livers. Western blot analysis and CDK2 kinase assay demonstrated expression levels of cyclin E and CDK2 and CDK2 kinase activity, respectively, and both were shown to increase along with the development of hepatocellular carcinomas. Analysis of the correlation between expression of cyclin E and CDK2 and clinicopathological parameters revealed a significant correlation between expression of cyclin E and tumor grade ( P=0.013), and PCNA index ( P=0.006) as well as CDK2 expression ( P=0.015). Overexpression of CDK2 tended to be associated with poorly differentiated HCCs. The results suggest that overexpression of cyclin E and CDK2 plays an important role in the development of hepatocellular carcinoma.

Relationship between copper, zinc and metallothionein in hepatocellular carcinoma and its surrounding liver parenchyma

Background/Aim: Accumulation of copper (Cu) in hepatocellular carcinoma (HCC), especially in small tumors, is greater than that in the surrounding liver parenchyma. Metallothionein (MT) is considered to be present as Cu-MT, Zn, Cu-MT or Zn-MT. The aim of this study was to determine the presence and localization of Cu-MT and Zn-MT in HCC and surrounding liver parenchyma. Methods: In 16 HCC patients, surgically resected specimens including HCC and surrounding liver parenchyma were evaluated. Results: The level of Cu present in small HCC (<4 cm in diameter) was significantly greater than that in the surrounding liver parenchyma ( p<0.05). However, the level of Cu in large HCC (⪢4 cm in diameter) was similar to that in the surrounding liver parenchyma. Analysis by Sephadex G-75 gel filtration revealed that the peak fraction due to Cu was identical to that due to MT in 14 (87.5%) of 16 HCC, the peak fraction due to Cu and Zn was identical to that due to MT in 2 (12.5%) HCC, and the peak fraction due to Zn was identical to that of MT in none of 16 HCC. Conclusions: Accumulation of Cu in small HCC, in which Cu was present as Cu-MT or Zn, Cu-MT, was greater than that in the surrounding liver parenchyma. Cu accumulation and the presence of MT in the liver may be related to carcinogenesis of HCC, because of the similarity of these findings in the experimental data of Long-Evans rats with a cinnamon-like coat color who develop HCC spontaneously.

Excretion of copper complexed with thiomolybdate into the bile and blood in LEC rats

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long–Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was removed with ammonium tetrathiomolybdate (TTM), and the fate of the Cu complexed with TTM and mobilized from the liver was determined. TTM was injected intravenously as a single dose of 2, 10 or 50 mg TTM/kg body weight into LEC and Wistar (normal Cu metabolism) rats, and then the concentrations of Cu and molybdenum (Mo) in the bile and plasma were monitored with time after the injection. In Wistar rats, most of the Mo was excreted into the urine, only a small quantity being excreted into the bile, while Cu excreted into the urine decreased. However, in LEC rats, Cu and Mo were excreted into the bile and blood, and the bile is recognized for the first time as the major route of excretion. The Cu excreted into both the bile and plasma was accompanied by an equimolar amount of Mo. The relative ratio of the amounts of Cu excreted into the bile and plasma was 40/60 for the low and high dose groups, and 70/30 for the medium dose group. The systemic dispositions of the Cu mobilized from the liver and the Mo complexed with the Cu were also determined for the kidneys, spleen and brain together with their urinal excretion. Although Mo in the three organs and Cu in the kidneys and spleen were increased or showed a tendency to increase, Cu in the brain was not increased at all doses of TTM.

Metabolic fate of the insoluble copper/tetrathiomolybdate complex formed in the liver of LEC rats with excess tetrathiomolybdate

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, can be removed from the MT with tetrathiomolybdate (TTM). However, the insoluble Cu/TTM complex formed with excess TTM is known to be deposited in the liver. The metabolic fate of the insoluble Cu/TTM complex was investigated in the present study. LEC rats were injected with TTM at the dose of 10 mg/kg body weight for 8 consecutive days and were fed with a standard or low Cu diet for 14 days after the last injection. About 95% of the Cu in the liver became insoluble together with Mo. The concentration of Cu in the liver supernatants of rats fed with the standard diet increased significantly compared with that in rats dissected 24 h after the last injection (control rats), while the concentration in rats fed with the low Cu diet remained at a comparable level to that in the controls. The rate of Cu accumulation in the livers of rats fed with the standard diet did not differ before and after the treatment, suggesting that there was no rebound effect by treatment with TTM. These results suggest that the insoluble Cu/TTM complex is resolubilized in the liver, and that the solubilized complex is excreted into the bile and blood, i.e., the insoluble Cu/TTM complex is not the source of Cu re-accumulation in the form bound to MT in the liver after TTM treatment. It was concluded that, once Cu is complexed with TTM, the metal is excreted either immediately in the soluble form or slowly in the insoluble form into the bile and blood.

Biological regulation of copper and selective removal of copper: therapy for Wilson disease and its molecular mechanism

Copper (Cu) is an essential trace element and constitutes the active center of the redox Cu enzymes such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD), ceruloplasmin and cytochrome c oxidase. Among hereditary diseases due to a defect in the metabolism of Cu, Menkes disease (caused by a Cu deficiency) and Wilson disease (caused by the excessive accumulation of Cu) have been shown to be caused by the mutation of genes encoding Cu-binding ATPase for the efflux of Cu, ATP7A and ATP7B, respectively. Following the identification of these causative genes, intracellular Cu transporters (Cu chaperones) specific for the Golgi apparatus, mitochondria and Cu, Zn-SOD were discovered, and these findings have facilitated the study of the underlying mechanisms of the biological regulation of Cu. Apart from these physiological and biochemical studies, toxicological studies have elucidated the underlying mechanisms of the occurrence of acute hepatitis caused by the accumulation of Cu accumulating in the liver of an animal model for Wilson disease, LEC rats. In these toxicological studies, two biological aspects of metallothionein (MT), i.e., antioxidant and prooxidant depending on the Cu/Zn ratio in Cu-containing MT have been proposed. The present article overviews the recent findings on the biological regulation of Cu and on the toxicological aspect of Cu. It is known that Cu forms a stable ternary complex with molybdenum and sulfur under reductive conditions in the body. On the basis of this observation, tetrathiomolybdate (TTM) has been applied to remove Cu from the liver of Long-Evans rats with a cinnamon-like coat color (LEC) rats. Precise mechanisms underlying the complex formation between Cu bound to MT and TTM were presented, and an appropriate protocol for the chelation therapy was also proposed together with the mechanisms underlying the occurrence of side-effects.

Identification of the zinc-binding protein specifically present in male rat liver as carbonic anhydrase III

A zinc (Zn)-binding protein that is present specifically in the livers of male adult rats was detected by HPLC with in-line detection by mass spectrometry (ICP MS). The Zn-binding protein was purified on Sephadex G-75 and G3000SW HPLC columns, and was identified as carbonic anhydrase III (CAIII) based on the amino acid sequence of a peptide obtained on lysyl endopeptidase digestion. CAIII is expressed as one of the major Zn-binding proteins in the livers of male rats in an age-dependent manner, a comparable amount of Zn to that of copper, Zn-superoxide dismutase (Cu,Zn-SOD) being bound to CAIII at 8 weeks of age. Castration at 4 or 8 weeks of age was shown to reduce Zn bound to CAIII to 47.5% of the sham-operated control level, suggesting that the sex-dependent expression of CAIII is partly regulated by a sex hormone, androgen. The concentration of CAIII in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was also estimated as Zn bound to CAIII and shown to be lower than that in Wistar rats before the onset of hepatitis. The concentration of CAIII was decreased specifically by repeated injections of cupric ions without the Cu,Zn-SOD concentration being affected.

Comparative mechanism and toxicity of tetra- and dithiomolybdates in the removal of copper

Tetrathiomolybdate (TTM) can be used as a specific chelator to remove copper (Cu) accumulating in the form bound to metallothionein (MT) in the livers of Wilson disease patients and Long–Evans rats with a cinnamon-like coat color (LEC rats). However, an adverse effect, hepatotoxicity, was observed occasionally on its clinical application. The mechanism underlying the adverse effect of TTM has been studied in comparison with dithiomolybdate (DTM), and a safer and more effective therapy by TTM was proposed based on the mechanism. The activity of glutamic-pyruvic transaminase (GPT) in serum was shown to increase significantly on the treatment of Wistar rats with sulfide produced through hydrolytic degradation of TTM and DTM, the latter being more easily degraded. The hydrolytic degradation of TTM was enhanced under acidic conditions. Cu in Cu-containing enzymes such as Cu,Zn-superoxide dismutase (SOD) in liver and ceruloplasmin (Cp) in plasma was decreased by excessive thiomolybdates, the Cu being found in the plasma in the form of a Cu/thiomolybdate/albumin complex. The decreased amounts of Cu in SOD and Cp were explained by the sequestration of Cu from their chaperones by thiomolybdates rather than the direct removal of Cu from the enzymes. Although both TTM and DTM remove Cu from MT, DTM is not appropriate as a therapeutic agent for Wilson disease due to its easy hydrolysis and production of sulfide.

A Marked Increase in Free Copper Levels in the Plasma and Liver of LEC Rats: An Animal Model for Wilson Disease and Liver Cancer

Most of copper present in rat plasma and liver binds to caeruloplasmin and metallothionein, respectively, and is not redox active. However, free forms of copper including loosely bound forms to other molecules are redox active. We assessed the free copper in Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease and liver cancer. Compared to those of control rats, the liver and plasma of LEC rats showed a marked elevation of free copper, especially at the stage of acute hepatitis, in parallel with an increase of total copper levels in the livers and a decrease of plasma caeruloplasmin (ferroxidase I) activity. At the onset of jaundice, the total copper levels, however, decreased in liver, but increased in plasma, while free copper levels in both liver and plasma remained higher. Free iron levels in both liver and plasma were also determined and did not change significantly, except for the case of plasma in jaundiced rats. The data are consistent with a proposal in which increased levels of redox active free copper in the liver of LEC rats catalyze Fenton-type reactions, producing a large flux of hydroxyl radicals that would play an important role in the observed liver dysfunction, leading to acute hepatitis, and, finally, hepatocarcinoma. This is the first demonstration that the free copper may participate in the pathophysiology of the LEC rats and Wilson disease.

Metal-Induced Hydroxyl Radical Generation by Cu+-Metallothioneins from LEC Rat Liver

Reactions of LEC (Long-Evans rats with a cinnamon-like coat color) rat liver Cu(I)-metallothioneins (MTs) with HgCl2or K3Fe(CN)6were investigated by ESR spectroscopy and generation of hydroxyl radicals was demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When Cu(I)-MTs were incubated with more than one equivalent mole HgCl2or K3Fe(CN)6to Cu+bound to MTs, strong signals due to Cu2+appeared. ESR spectra, which were a combination of the DMPO-OH adduct signal and a six-line signal, were observed in the reaction of Cu(I)-MTs with HgCl2, whereas no oxygen radical signal was seen with K3Fe(CN)6. The DMPO-OH signal intensity was greater in the presence of SOD while the signal disappeared in the presence of catalase. The results suggest that addition of HgCl2causes the liberation of cuprous ions from MTs followed by a reaction with oxygen, leading to hydroxyl radical formation through a Fenton-type Haber-Weiss reaction.

LEC rats: An overview of recent findings

The Long Evans rat with a cinnamon-like coat color, (LEC) rat established from a closed colony of the Long-Evans strain, spontaneously develops hepatitis as a result of abnormal copper accumulation in the liver and, eventually, hepatoma after 1 year of age. Numerous studies using the LEC rat as an animal model of Wilson's disease and hepatocarcinogenesis have been reported. Recently, the causative gene for abnormal copper accumulation, which is a rat homologue of the human Wilson's disease gene, was cloned and the defect of the gene was demonstrated. Therefore, the LEC rat represents a bona fide model of Wilson's disease from a genetic point of view. Moreover, other abnormalities such as immunodeficiency also have been found in the LEC rat. Incomplete maturation of T cells is observed in LEC rats, but the underlying mechanism for this remains unknown. Thus, LEC rats can be used as an ideal animal model for at least two different purposes. One is based on the defect in the gene responsible for the copper transporting ATPase (Atp7b), and, therefore, the investigation of LEC rats can provide useful information on the pathophysiology of Wilson's disease and lead to possible gene therapy. The other is triggered by abnormally accumulated copper and iron and/or a defect in the immune system. Free copper and iron accumulation, especially, may facilitate Fenton chemistry to produce reactive oxygen species, which represent likely candidates for carcinogenesis. Here, we review recent studies concerning the LEC rat from both genetic and biochemical points of views and propose perspectives for future investigations. J. Trace Elem. Exp. Med. 10:135–145, 1997. © 1997 Wiley-Liss, Inc.

LEC rats: An overview of recent findings

The Long Evans rat with a cinnamon-like coat color, (LEC) rat established from a closed colony of the Long-Evans strain, spontaneously develops hepatitis as a result of abnormal copper accumulation in the liver and, eventually, hepatoma after 1 year of age. Numerous studies using the LEC rat as an animal model of Wilson's disease and hepatocarcinogenesis have been reported. Recently, the causative gene for abnormal copper accumulation, which is a rat homologue of the human Wilson's disease gene, was cloned and the defect of the gene was demonstrated. Therefore, the LEC rat represents a bona fide model of Wilson's disease from a genetic point of view. Moreover, other abnormalities such as immunodeficiency also have been found in the LEC rat. Incomplete maturation of T cells is observed in LEC rats, but the underlying mechanism for this remains unknown. Thus, LEC rats can be used as an ideal animal model for at least two different purposes. One is based on the defect in the gene responsible for the copper transporting ATPase (Atp7b), and, therefore, the investigation of LEC rats can provide useful information on the pathophysiology of Wilson's disease and lead to possible gene therapy. The other is triggered by abnormally accumulated copper and iron and/or a defect in the immune system. Free copper and iron accumulation, especially, may facilitate Fenton chemistry to produce reactive oxygen species, which represent likely candidates for carcinogenesis. Here, we review recent studies concerning the LEC rat from both genetic and biochemical points of views and propose perspectives for future investigations. J. Trace Elem. Exp. Med. 10:135–145, 1997. © 1997 Wiley-Liss, Inc.

Dietary copper (Cu)‐ and age‐dependent changes of C‐metallothionein in relation to the level of nitric oxide (NO) in the liver of LEC (Long‐Evans Cinnamon) rats

To understand the unique pathophysiological process of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop hereditary and spontaneous hepatic injury -4 months after birth, the levels of four biometals, such as Cu, Fe, Zn, and Mn together with metallothionein (MT) and nitric oxide (NO as NO2− plus NO3−), were determined in the liver of male and female animals given normal (Cu level: 0.9–1.0 mg/100 g solid) or Cu-deficient (Cu level: <0.1 mg/100 g solid) diet by atomic absorption spectrometry, radioimmunoassay, and the Griess method, respectively. Levels of Cu and MT, which in turn form Cu-MT, increased, whereas the NO level was decreased in the aging process of both male and female rats given a normal diet. Therefore, the accumulated Cu-MT was proposed to scavenge NO, since NO levels in the liver of LEC rats were lowered compared with those of normal Wistar rats given the same diet. In female LEC rats, enhancements of both Cu and MT accumulations were proposed to relate closely to the development of hepatoma as estimated by histochemical staining. In addition, alterations of Fe, Zn, and Mn levels in the liver were obtained, which may contribute the development of jaundice, hepatitis, and hepatoma of LEC rats. J. Trace Elem. Exp. Med. 10:89–100, 1997. © 1997 Wiley-Liss, Inc.

Introduction for the special issue for the LEC rats (the Long-Evans rat with a cinnamon-like coat color)

Introduction for the special issue for the LEC rats (the Long-Evans rat with a cinnamon-like coat color)

High expression of heparin-binding EGF-like growth factor in rat hepatocarcinogenesis.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family and is highly expressed in hepatoma tissues but not in normal liver. However, it is unknown when HB-EGF is induced during hepatocarcinogenesis and what are the mechanisms underlying its high expression in hepatoma. To address this issue, the expression of HB-EGF was investigated during hepatocarcinogenesis in LEC (Long-Evans with a cinnamon-like coat color) rats, which spontaneously develop hepatitis and hepatoma. LEA (Long-Evans with an agouti coat color) rats were used as controls. Furthermore, the induction of HB-EGF mRNA by various agents was investigated in a rat hepatoma cell line and hepatocytes in primary culture. Expression of HB-EGF mRNA in the liver was very low at the stage of acute and chronic hepatitis and markedly increased at the stage of hepatoma in LEC rats. Non-involved tissues adjacent to hepatoma showed low expression of HB-EGF mRNA. Immunochemical studies revealed positive staining in hepatoma tissues. Induction of HB-EGF mRNA by several growth factors was observed in a hepatoma cell line but not in normal hepatocytes. Our results suggest that HB-EGF is associated with the early progression steps of hepatoma.

Mechanisms of selective copper removal by tetrathiomolybdate from metallothionein in LEC rats

Copper (Cu) was selectively removed from metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color) in vivo and in vitro by tetrathiomolybdate (TTM). Female LEC rats were injected intraperitoneally with TTM at a dose of 10 mg/kg body weight for 8 consecutive days. More than 2 3 of the Cu accumulating in the liver was removed by TTM treatment 24 h after the last injection. Although most Cu was bound to MT in the soluble fraction before TTM treatment, the Cu remaining in the liver was present almost exclusively in the nonsoluble fraction together with molybdenum (Mo). Cu,Zn,Cd-MT was separated from the liver of LEC rats that had been injected with cadmium (Cd) and reacted with TTM at mol ratios of 0, 0.25, 0.50, 1.0, 2.0 and 4.0 to Cu bound to MT for 10 min at 37 °C. When TTM was added at a mol ratio of less than 1.0, a Cu,Zn,Cd-MT/TTM complex was detected, while addition of TTM at a mol ratio of greater than 1.0 selectively removed Cu from MT and produced a Cu/TTM complex via liberation of Zn,Cd-MT from the Cu,Zn,Cd-MT/TTM complex. Excessive TTM appeared to facilitate polymerization of the Cu/TTM complex to insoluble polymers. The dose-related formation of differing MTATTM complexes explains the findings observed in vivo.

Changes in free radical-metabolizing enzymes and lipid peroxides in the liver of Long-Evans with cinnamon-like coat color rats

We report changes in free radical-metabolizing enzymes and the increased generation of lipid peroxides associated with extreme metal accumulation in the liver of the Long-Evans with cinnamon-like coat color (LEC) rat, a new mutant strain displaying hereditary hepatitis and subsequent hepatocellular carcinoma. The activity of free radical-metabolizing enzymes and lipid peroxides, and the concentration of metal in the liver were determined sequentially after birth. Mn-superoxide dismutase activity significantly increased immediately after the onset of hepatitis in LEC rats, whereas no remarkable change was observed in control rats. Cu, Zn-superoxide dismutase activity in LEC rats was similar to that in control rats. Glutathione reductase activity increased, while glutathione peroxidase activity was lower in LEC rats than in control rats throughout the observation periods. Lipid peroxides, estimated by thiobarbituric acid reaction, also increased 4- to 5-fold immediately after the onset of hepatitis in LEC rats. Copper concentration was 30- to 50-fold higher in the liver of LEC rats than in control rats, and the iron content also increased significantly before and after the onset of hepatitis. These findings suggested that an oxidant injury generated by toxic metals could be one of the factors responsible for hepatocellular damage in this unique hereditary hepatitis.

Enhanced synthesis of metallothionein as a possible cause of abnormal copper accumulation in LEC rats

Long-Evans rats with a cinnamon-like coat color (LEC) is an inbred strain accumulating copper (Cu) in the liver abnormally and showing spontaneous hepatitis and hepatoma. The present study was intended to clarify how Cu accumulates in the LEC rat liver. For this purpose, the distribution profiles of Cu and zinc (Zn) and the inducibility of metallothionein (MT) synthesis were examined in the liver between Cu-loaded Long Evans agouti (LEA, the original strain of LEC) rats and were compared with those in control LEC rats. LEA rats (female, five weeks old) were injected subcutaneously with CuCl 2 daily at a dose of 3 mg Cu/kg body weight for 2, 4, 6, and 9 days. The concentration of Cu (124 μg/g) accumulated in the LEA rat liver after four injections was comparable to that in control LEC rats. Only 20% of Cu in the liver of LEA rats was recovered in the supernatant fraction in the form of MT, while Cu in the LEC rat liver (113 μg/g) was recovered mostly in the supernatant fraction, and was bound to MT. Although the increased concentration of Cu in the LEA rat liver was further elevated after additional injections of Cu, the amount of MT did not increase further. The MT mRNA content in the LEA rat liver remained lower than that of LEC rats even after further injections of Cu. Therefore, the present results suggest that LEC rats can accumulate Cu at a high concentration in the liver because of their extremely high inducibility of MT.