Evans Cinnamon Research Articles

Hepatocyte Transplantation in the Long Evans Cinnamon Rat Model of Wilson's Disease

Wilson's disease (WD), caused by a mutation in the P-type copper transporting ATPase (Atp7b) gene, results in excessive accumulation of copper in the liver. Long Evans Cinnamon rats (LEC) bear a mutation in the atp7b gene and share clinical characteristics of human WD. To explore hepatocyte transplantation (HT) as therapy for metabolic liver diseases, 8-week-old LEC rats (n = 12) were transplanted by intrasplenic injection of hepatocytes from donor Long Evans (LE) rats. Immunosuppression was maintained with intraperitoneal tacrolimus. The success of HT was monitored at 24 weeks of life. Serum aminotransferases and bilirubin peaked at 14-21 weeks in both HT rats and nontransplanted controls, but at 24 weeks, survival was 97% in LEC-HT versus 63% in controls. All transplanted rats showed restored biliary copper excretion and reduced liver iron concentration associated with increased ceruloplasmin oxidase activity. Liver tissue expressed atp7b mRNA (11.9 +/- 13.6%) indicative of engraftment of normal cells in 7 of 12 HT rats, associated with a reduced liver copper concentration compared to untreated LEC rats. Periportal islets of normal appearing hepatocytes, recognized by atp7b antibody, were observed in transplanted livers while lobular host cells showed persistent pleomorphic changes and inflammatory infiltrates. In conclusion, transplantation of normal hepatocytes prevented fulminant hepatitis, reduces chronic inflammation, and improved 6-month survival in LEC rats. Engraftment of transplanted cells, which express atp7b mRNA, repopulated the recipient liver with normal functional capacity.

Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4

The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.

Efficacy of zinc supplementation in preventing acute hepatitis in Long–Evans Cinnamon rats

Long-Evans Cinnamon (LEC) rats are characterized by an abnormal hepatic deposition of copper (Cu) due to a lack of the Cu-transporter P-type adenosine triphosphatase: accordingly, the strain is a good animal model of Wilson's disease. The effect of oral zinc (Zn) acetate treatment on the development of acute hepatitis and the biochemical parameters of Cu-induced liver damage was studied in 5-week-old LEC rats (n=52). Rats receiving 50 or 80 mg/ml/day Zn acetate by gavage and control rats receiving a daily dose of glucose solution 0.02 g/ml by gastric intubation were killed at 1, 2 or 8 weeks after the start of treatment. Treatment with Zn acetate resulted in the prevention of acute hepatitis: 10 of the 13 untreated rats developed signs and symptoms compatible with acute hepatitis between the 6th and 7th week of treatment. Tissue metallothionein (MT) significantly increased in the treated rats and positively correlated with Zn concentrations within the liver. Control rats had a significantly higher iron concentration in the liver and kidneys compared with supplemented rats, after both short- and long-term experiments. 8-hydroxy-2'-deoxyguanosine amounts were significantly lower in untreated rats. Zn acetate prevents acute hepatitis, by increasing tissue MT concentrations, reducing Cu absorption and interfering with Fe metabolism.

A new strain of rat for functional analysis of PINA

Long Evans cinnamon (LEC) rat is an animal model for human Wilson disease (WD) due to a deletion in Atp7b, the copper transporter defective in WD patients. Previously, we have demonstrated presence of an alternative product termed PIneal Night-specific ATPase (PINA) generated by an intronic promoter in Atp7b gene. Analysis of LEC rat in this study demonstrates that PINA is absent in the LEC pineal establishing its usefulness for investigating PINA function. Studies of the LEC pineal, however, revealed an additional defect in serotonin N-acetyltransferase (NAT), the key enzyme in melatonin production. Linkage studies confirm that the NAT phenotype is entirely independent of PINA mutation in the pineal gland of LEC rats, and sequence analysis demonstrates that NAT defect is due to a point mutation in NAT coding region. In addition, we demonstrate that the cinnamon coat color of the LEC rat is unlinked to PINA and NAT deficiencies in these animals. To facilitate further functional analysis of PINA in pineal physiology, we crossed LEC rats with PVG rats that are wildtype for PINA, NAT and coat color, and obtained rats that are defective only in PINA/Atp7b locus (termed LPP rats) and normal for NAT activity and coat color. Furthermore, we have identified the deletion breakpoint of Atp7b gene in LPP rats, which allows simplified genotyping of mutant animals. The separation of PINA mutation from both NAT and coat color mutations in the new LPP rats will permit better functional studies of PINA in pineal circadian physiology.

Ferrous and ferric iron accumulates in the brain of aged Long–Evans Cinnamon rats, an animal model of Wilson's disease

The Long–Evans Cinnamon (LEC) rat, which accumulates excess copper (Cu) in its liver, is an animal model of Wilson's disease. We evaluated and compared the distributions of Cu, ferrous (Fe 2+), and ferric (Fe 3+) iron in four-brain regions, namely, in the cerebral cortex, cerebellum, substantia nigra (SN), and striatum of LEC and Long–Evans Agouti rats at 30 and 55 weeks. Cu levels were elevated in the striatum of LEC rats, and Fe 2+ and Fe 3+ were higher in the striatum and SN of LEC rats. Ratios of Fe 2+ to Fe 3+ were >1 in four regions, and were highest in the striatum and SN of LEC rats. Cu and iron levels were found to be augmented during aging, and we suggest that these accumulations may exert deleterious effects in aged LEC rats. This study is the first report that demonstrates regional differences of Fe 2+ and Fe 3+ accumulation in the brain of aged LEC rats. Further studies are required to elucidate the mechanisms of Cu and iron accumulations and of their effects.

A novel H28Y mutation in LEC rats leads to decreased NAT protein stability in vivo and in vitro

Nocturnal melatonin production is reportedly controlled by the rhythms of serotonin N-acetyltransferase (NAT, or arylalkylamine N-acetyltransferase). While analyzing the melatonin synthetic pathways of Long Evans cinnamon (LEC) rats mutant for PINA, a pineal night-specific ATPase defective in Wilson disease, we discovered that NAT activity and protein levels are greatly reduced in LEC rats, and that the highly conserved histidine 28 is mutated to tyrosine. To study the effect of H28Y, we isolated a new strain of rat termed LPN that is mutant for NAT but wildtype for both PINA and coat color. Compared with control rats, the LPN rats displayed low NAT protein levels and enzyme activities. These results suggest that the H28Y mutation in NAT is the cause of reduced NAT levels in vivo. The identical H28Y mutation was also found in Sprague-Dawley rats from Zivic-Miller, suggesting it may be a common mutation in rodents. When analyzed in bacterial cells and HEK293 cells, the mutation resulted in reduction of both NAT protein stability and catalytic activity, confirming that the in vivo NAT phenotype in LPN rats was due to the H28Y mutation. Further analysis of the NAT-H28Y will focus on the mechanisms of the increased degradation both in vitro and in vivo, which will facilitate our understanding of how melatonin synthesis is controlled at the molecular level.

Whole animal copper flux assessed by positron emission tomography in the Long – Evans cinnamon rat – a feasibility study

Copper is an essential trace element. However, excess copper can lead to oxidation of biomolecules and cell damage and copper levels must be carefully controlled. While copper homeostasis has been studied extensively at the cellular level, short-term body copper fluxes are poorly understood. Here, we assessed for the first time the feasibility of measuring whole body copper flux by positron emission tomography, using 64Cu. A comparative approach comparing the Long-Evans cinnamon (LEC) rat to the wild type was chosen. LEC rats are an accepted model for Wilson disease, an inherited disorder of copper excretion in humans. In LEC rats as well as in Wilson patients, the copper transporting ATPase, ATP7B, is defective. This ATPase is primarily expressed in the liver and serves in copper secretion via the bile. Dysfunction of ATP7B leads to accumulation of copper in the liver. A control and an LEC rat were transgastrically injected with 10 microg of 64Cu and the copper flux followed for three hours by whole animal PET and concomitant collection of bile, as well as the analysis of tissue following tomography. As seen by PET, the administered copper was largely trapped in the stomach and the proximal intestine, and without a significant difference between control and LEC rat. Due to an insufficient dynamic range of the PET technology, copper which was systemically absorbed and primarily transported to the liver could only be followed by sampling and by beta-counting. Biliary copper excretion ensued after 15 min in the control rat, but was absent in the LEC rat. Biliary excretion reached saturation one hour after copper administration. The trapping of orally administered copper in the gastrointestinal tract may be an important mechanism to prevent copper toxicity under conditions of a sudden, excessive copper load, which cannot be alleviated by increased biliary secretion. This trapping does however limit the utility of PET to measure whole animal copper flux.

Reply

We appreciate the comments of Luedde et al.1 We read the interesting article reported by Luedde et al.2 In the partial hepatectomy (PH) experiments using knockout mice for various cyclin-dependent kinase (Cdk) inhibitors, the authors have shown that a loss of p18INK4C alone did not influence Cdk4 activity, but the lack of p21CIP1/WAF1 led to Cdk 4 activation. On the other hand, we have reported that the kinase activity of Cdk4 was higher in p18INK4C-negative hepatocellular carcinomas (HCCs) than in p18INK4C-positive ones.3 Based on the results, we concluded that p18INK4C expression might play an important role in the development of HCC through the upregulation of Cdk4 activity. These two reports appear to be contradictory. However, our experiments are different from the study of Leudde and colleagues. The reasons are as follows. Liver regeneration after PH reflects the proliferation of normal hepatocytes, and the process is completed after one week. Thus, changes induced by liver regeneration after PH are quite short, and are reversible. On the other hand, proliferation of HCC is a chronic and continuous process in transformed cells (cancer cells), and it is irreversible. Therefore, although the kinetic changes of cell cycle-related molecules in liver regeneration after PH provide important hints for the mechanism of hepatocarcinogenesis, they may not be directly applied to the proliferation of cancer cells. To solve this problem, it may be necessary to study Cdk 4 activity in carcinogen-induced HCC of p18INK4C knockout animals. It has been shown that the expression of p21CIP1/WAF1, p27 KIP1 and p57KIP2 were frequently downregulated in HCC.4, 5 Therefore, we agree with the suggestion that the effect on Cdk4 activity in HCC may be caused by a simultaneous loss of p18INK4C, p21CIP1/WAF1 and/or p27KIP2. In the future, it will be needed to determine what Cdk inhibitors influence Cdk4 activation in HCC. In addition, we would like to address the following point. Luedde et al. have stated that a loss of p18INK4C expression alone did not influence Cdk4 activation after PH. However, a clear band corresponding to the retinoblastoma protein phosphorylated at Ser-780 was visible 36 and 48 hours after PH in p18INK4C knockout animals (Fig. 5C in the text of Luedde et al.2). This result suggests that a loss of p18INK4C alone may also influence Cdk4 activation in liver regeneration after PH. We previously reported that the overexpression of Cdk4 was detected in HCC of Long Evans cinnamon rats and human.6, 7 In addition, Pascale et al.8 have also demonstrated that the overexpression of cyclin D1/Cdk4 complex occurred in chemically induced HCC of Fischer 344 rats. Conversely, it has been shown that Cdk6 protein was not increased in HCC.4, 5 Therefore, we assume that the amount of Cdk4 (cyclin D/Cdk4) may be higher than that of Cdk 6 (cyclin D1/Cdk6), and p18INK4C may dominantly interact with Cdk4 rather than with Cdk6 in p18INK4C-positive HCCs, suggesting that p18INK4C in HCC may contribute only to the up-regulation of Cdk4. Finally, expression levels of p18INK4C, p21CIP1/WAF1, p27KIP1, p57KIP2 and other Cdk inhibitors should be evaluated to examine their functional collaboration in the development of HCC. Tsutomu Masaki M.D.*, Asahiro Morishita M.D.*, Shigeki Kuriyama M.D.*, * Third Department of Internal Medicine, Kagawa University School of Medicine, Kagawa, Japan.

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long–Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.

Protection from spontaneous hepatocellular damage by N-benzyl- d-glucamine dithiocarbamate in Long–Evans Cinnamon rats, an animal model of Wilson's disease

The Long–Evans Cinnamon (LEC) rat is a mutant strain that accumulates excessive tissue copper (Cu) and models the clinical symptoms and biological features of Wilson's disease in humans. We compared the effects of three metal chelating agents, N-benzyl- d-glucamine dithiocarbamate (BGD), d-penicillamine (D-PEN), and triethylenetetramine (TETA), on the biliary and urinary excretions of Cu using LEC rats. The animals were treated ip with each chelating agent (1 mmol/kg body weight) and then the bile and urine samples were collected for 3 h. Because single treatment with BGD markedly stimulated biliary excretion of Cu, the protective effect of repeated BGD injection on spontaneous hepatocellular damage was further examined. Separate groups received two weekly injections of BGD starting at 11 weeks of age and were compared to saline-injected controls. Serum alanine aminotransferase (ALT) activity and bilirubin level were significantly increased in control LEC rats by 19 weeks of age and histopathological analysis demonstrated extensive hepatic damage in these rats. However, repeated BGD injections prevented the increases in serum ALT and bilirubin and blocked the histopathological changes in the liver. Furthermore, although Cu rapidly accumulated in the liver, kidney, spleen, and serum of control LEC rats during the test period, repeated BGD injection largely prevented these increases. These results indicate that BGD treatment is effective in blocking excessive Cu accumulation in LEC rats that, in turn, provides protection from spontaneous liver damage.

Protection from spontaneous hepatocellular damage by N-benzyl--glucamine dithiocarbamate in Long?Evans Cinnamon rats, an animal model of Wilson's disease

The Long–Evans Cinnamon (LEC) rat is a mutant strain that accumulates excessive tissue copper (Cu) and models the clinical symptoms and biological features of Wilson's disease in humans. We compared the effects of three metal chelating agents, N -benzyl- d -glucamine dithiocarbamate (BGD), d -penicillamine (D-PEN), and triethylenetetramine (TETA), on the biliary and urinary excretions of Cu using LEC rats. The animals were treated ip with each chelating agent (1 mmol/kg body weight) and then the bile and urine samples were collected for 3 h. Because single treatment with BGD markedly stimulated biliary excretion of Cu, the protective effect of repeated BGD injection on spontaneous hepatocellular damage was further examined. Separate groups received two weekly injections of BGD starting at 11 weeks of age and were compared to saline-injected controls. Serum alanine aminotransferase (ALT) activity and bilirubin level were significantly increased in control LEC rats by 19 weeks of age and histopathological analysis demonstrated extensive hepatic damage in these rats. However, repeated BGD injections prevented the increases in serum ALT and bilirubin and blocked the histopathological changes in the liver. Furthermore, although Cu rapidly accumulated in the liver, kidney, spleen, and serum of control LEC rats during the test period, repeated BGD injection largely prevented these increases. These results indicate that BGD treatment is effective in blocking excessive Cu accumulation in LEC rats that, in turn, provides protection from spontaneous liver damage.

No prevention of liver and kidney tumors in Long–Evans Cinnamon rats by dietary curcumin, but inhibition at other sites and of metastases

Long–Evans Cinnamon (LEC) rats, an inbred mutant strain which accumulates copper due to an aberrant copper-transporting ATPase gene, develop acute hepatitis, chronic liver injury and liver tumors as a result of copper-induced oxidative stress, lipid peroxidation and DNA damage. Curcumin, an antioxidant and anti-inflammatory agent, has shown anticancer properties in many rodent models. We investigated the modulating role of curcumin in liver and kidney carcinogenesis in LEC rats. Two groups of 4-week-old LEC rats ( n=60 each) were fed either a standard diet (control) or received 0.5% curcumin in the diet for life. In untreated LEC rats, the rate of acute liver failure, the incidence of liver tumors and of kidney tumors were 32, 100 and 10% respectively, which was not altered by curcumin treatment. However, curcumin reduced tumor incidence at other organ sites (15% versus 0%; P=0.025) and suppressed formation of metastases (18% versus 0%; P=0.01). Median survival time was decreased from 88.7 to 78.1 weeks in curcumin-treated rats ( P=0.002). The lack of chemoprevention of liver and kidney tumors in LEC rats by curcumin may be caused by enhanced toxicity and oxidative stress due to excess copper. We conclude that curcumin should be contra-indicated for patients suffering from inherited and acquired metal storage diseases that include patients with hepatitis C virus infection.

Animal models of copper-associated liver disease

Recent advances in molecular biology have made possible the identification of genetic defects responsible for Wilson's disease, Indian childhood cirrhosis and copper toxicosis in Long Evans Cinnamon rats, toxic milk mice, and Bedlington terriers. The Wilson's disease gene is localized on human chromosome 13 and codes for ATP7B, a copper transporting P-type ATPase. A genetic defect similar to that of Wilson's disease occurs in Long Evans Cinnamon rats and toxic milk mice. Familial copper storage disorders in Bedlington and West Highland white terriers are associated with early subclinical disease, and copper accumulation with subsequent liver injury culminating in cirrhosis. The canine copper toxicosis locus in Bedlington terriers has been mapped to canine chromosome region CFA 10q26. Recently, a mutated MURR1 gene was discovered in Bedlington terriers affected with the disease. Idiopathic childhood cirrhosis is biochemically similar to copper toxicosis in Bedlington terriers, but clinically much more severe. Both conditions are characterized by the absence of neurologic damage and Kayser-Fleisher rings, and normal ceruloplasmin levels. A recent study added North Ronaldsay sheep to the list of promising animal models to study Indian childhood cirrhosis. Morphologic similarities between the two conditions include periportal to panlobular copper retention and liver changes varying from active hepatitis to panlobular pericellular fibrosis, and cirrhosis. Certain copper-associated disorders, such as chronic active hepatitis in Doberman pinschers and Skye terrier hepatitis are characterized by copper retention secondary to the underlying disease, thus resembling primary biliary cirrhosis in humans. Copper-associated liver disease has increasingly being recognized in Dalmatians. Copper-associated liver diseases in Dalmatians and Long Evans Cinnamom rats share many morphologic features. Fulminant hepatic failure in Dalmatians is characterized by high serum activities of alanine aminotransferase and aspartate aminotransferase, and severe necrosis of centrilobular areas (periacinar, zone 3) hepatocytes. Macrophages and surviving hepatocytes contain copper-positive material. Liver disease associated with periacinar copper accumulation has also been described in Siamese cats. Many questions regarding copper metabolism in mammals, genetic background, pathogenesis and treatment of copper-associated liver diseases remain to be answered. This review describes the similarities between the clinico-pathological features of spontaneous copper-associated diseases in humans and domestic animals.

Effect and possible role of Zn treatment in LEC rats, an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long–Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.

Cofactors of mitochondrial enzymes attenuate copper-induced death in vitro and in vivo.

Copper toxicity contributes to neuronal death in Wilson's disease and has been speculatively linked to the pathogenesis of Alzheimer's and prion diseases. We examined copper-induced neuronal death with the goal of developing neuroprotective strategies. Copper catalyzed an increase in hydroxyl radical generation in solution, and the addition of 20 microM copper for 22 hours to murine neocortical cell cultures induced a decrease in ATP levels and neuronal death without glial death. This selective neuronal death was associated with activation of caspase-3 and was reduced by free radical scavengers and Z-Val-Ala-Asp fluoromethylketone, consistent with free radical-mediated injury leading to apoptosis. Pyruvate dehydrogenase is especially vulnerable to inhibition by oxygen free radicals, and the upstream metabolites, pyruvate, phosphoenolpyruvate, and 2-phosphoglycerate were elevated in cortical cells after toxic exposure to copper. One approach to protecting pyruvate dehydrogenase from oxidative attack might be to enhance binding to cofactors. Addition of thiamine, dihydrolipoic acid, or pyruvate reduced copper-induced neuronal death. To test efficacy in vivo, we added 1% thiamine to the drinking water of Long Evans Cinnamon rats, an animal model of Wilson's disease. This thiamine therapy markedly extended life span from 6.0 +/- 1.6 months to greater than 16 months.

Chemical forms of metallothionein and cellular oxidative stress in human hepatocellular carcinoma: A comparative study with the hepatitis of LEC rat

AbstractMetallothionein (MT) has been described as a cell‐protective protein that binds several heavy metals. In mammals, MT plays an important physiological role that includes detoxifying heavy metals, accumulating essential trace elements in cells to maintain homeostasis, and scavenging reactive oxygen species or free radicals. In contrast, the generation of hydroxyl radical (·OH) has been reported in the reaction of hydrogen peroxide and copper (Cu)‐MT separated from the livers of Long–Evans Cinnamon (LEC) rats, an animal model of human hepatic injury with unusual accumulation of Cu‐MT in the liver. On the other hand, Cu has been reported to accumulate in human hepatocellular carcinoma (HCC). Thus, we investigated whether the induction of MT, accumulation of Cu, and enhancement of oxidative stress are relevant to the development of human HCC, similarly to the observations in the liver of LEC rats. Although the kinetic behaviors of metal and MT levels were different in the livers of humans and LEC rats, Cu‐MT induction in the tumor portion of human HCC tissues was suggested to occur similarly to that in the liver of LEC rats. In addition, it was found that oxidative stress in terms of the level of thiobarbituric acid‐reactive substances was enhanced in the tumor portion of human HCC, corresponding well with increasing in the age of LEC rats, which developed jaundice and hepatitis. On the basis of these results, we conclude that the induction of Cu‐MT in human HCC might be related to the hepatic oxidative stress, which in turn develops hepatic injury involving carcinoma. J. Trace Elem. Exp. Med. 15:31–45, 2002. ©2002 Wiley‐Liss, Inc.

Histological changes in monoaminergic neurons of Long–Evans Cinnamon rats

The Long–Evans Cinnamon rat, an animal model of Wilson’s disease, is an inbred mutant strain with spontaneous hepatitis isolated from Long–Evans rats. The copper concentration in the brains of Long–Evans Cinnamon rats at 4 weeks of age was lower than that of controls, but higher than that of controls at 20 weeks of age. We investigated the tyrosine hydroxylase and 5-hydroxytryptamine immunoreactive fiber densities in the brains of Long–Evans Cinnamon rats aged 4, 10, and 20 weeks by immunohistochemistry, comparing them with Long–Evans Agouti rats used as controls. Tyrosine hydroxylase immunoreactive fiber densities in the cingulate cortex, hippocampus and cerebellum in Long–Evans Cinnamon rats were significantly lower than those of Long–Evans Agouti rats at 4 and 10 weeks of age. On the other hand, 5-hydroxytryptamine immunoreactive fiber densities in the cingulate cortex, caudate-putamen, hypothalamus, and hippocampus in Long–Evans Cinnamon rats were significantly higher than those of controls at 4, 10 and 20 weeks of age. In the cingulate cortex and caudate-putamen, 5-hydroxytryptamine immunoreactive fiber densities became gradually higher with age. The number of aberrant 5-hydroxytryptamine immunoreactive fibers in the cingulate cortex, caudate-putamen, hypothalamus and hippocampus in LEC rats was significantly higher than that of controls. The number of another type of aberrant 5-hydroxytryptamine immunoreactive fibers, which were detected only at 20 weeks of age in the caudate-putamen in LEC rats was significantly higher than that of controls. These results suggest that age-dependent changes in copper concentrations of Long–Evans Cinnamon rats were related to changes in monoaminergic neuron systems.

Cu-Metallothioneins (Cu(I)8-MTs) in LEC Rat Livers 13 Weeks after Birth Still Act as Antioxidants

Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long–Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)8-MTs). Titration of Cu(I)8-MTs with CuCl2 indicates that Cu(I)8-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg2+-induced hydroxyl radical generation from Cu(I)8-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl2. No ESR signal due to Cu2+ was also detected with LEC rat liver cytosol, whereas strong Cu2+ signal appeared by the addition of HgCl2. The rate constants for the reaction of Cu(I)8-MTs with superoxide and hydroxyl radicals were estimated to be 2 × 106 and ≥1012 M−1s−1, respectively, from competition kinetics. Cu2+-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)8-MTs from LEC rat livers just before hepatitis still act as antioxidants.

Correction of Liver Disease Following Transplantation of Normal Rat Hepatocytes into Long–Evans Cinnamon Rats Modeling Wilson's Disease

To establish the efficacy of cell therapy in Wilson's disease, we used the Long–Evans Cinnamon (LEC) rat model with atp7b gene mutation and copper toxicosis. Several groups of LEC rats were established, including animals pretreated with retrorsine to exacerbate copper toxicosis and inhibit proliferation in native hepatocytes followed by partial hepatectomy to promote liver repopulation. Hepatocytes from normal, syngeneic LEA rats were transplanted intrasplenically. Animal survival, biliary copper excretion, and hepatic copper were determined. The magnitude of liver repopulation was demonstrated by measuring serum ceruloplasmin and hepatic atp7b mRNA. Long-term survival in LEC rats treated with retrorsine, partial hepatectomy, and cell transplantation was up to 90%, whereas fewer than 10% of animals pretreated with retrorsine, without cell therapy, survived, P < 0.001. Liver repopulation occurred gradually after cell transplantation, ranging from <25% at 6 weeks, 26 to 40% at 4 months, and 74 to 100% at 6 months or beyond. Liver repopulation restored biliary copper excretion capacity and lowered liver copper levels. Remarkably, liver histology was completely normal in LEC rats with extensive liver repopulation, compared with widespread megalocytosis, apoptosis, oval cell proliferation, and cholangiofibrosis in untreated animals. These data indicate that liver repopulation with functionally intact cells can reverse pathophysiological perturbations and cure Wilson's disease.

Development of a Stable Isotope Approach for the Inductively Coupled Plasma–Mass Spectrometry Determination of Oxidized Metallothionein in Biological Materials

The use of isotope dilution analysis (IDA) with inductively coupled plasma–mass spectrometry (ICP–MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope (109Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements (114Cd/111Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP–MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP–MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive 109Cd. The final procedure was applied to the quantification of MT in Long–Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference 109Cd methodology. A good agreement between the analytical values obtained by both methods was observed.