Cinnamon Rats Research Articles

Low susceptibility to N-ethyl-N-nitrosourea-induced transplacental carcinogenesis in Long-Evans Cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, is resistant to a variety of chemical carcinogenesis except liver and colon. In the present study, N-ethyl-N-nitrosourea (ENU)-induced transplacental carcinogenesis was examined in male and female LEC, Long-Evans Agouti (LEA), a sibling line of the LEC rat, and F344 rats (n=21). ENU was administered to pregnant rats as a single s.c. injection at a dose of 60 mg/kg body weight on the 17th day after conception. Cerebral/spinal gliomas and trigeminal/spinal nerve schwannomas developed in both LEA and F344 rats at 30 weeks of age, but no nervous system tumors developed in LEC rats, the difference being statistically significant. Lung adenomas also developed in LEA and F344 rats, but not in LEC rats. Semiquantitative RT-PCR demonstrated that metallothionein (MT)1a, MT2 and O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA levels in the liver of LEC rats were higher than those in F344 and LEA rats. In addition, Western blot analysis showed that MT (MT1 plus MT2) in the liver of LEC rats was also higher than that in other strains. Present results suggest that high levels of MT and/or MGMT contribute to the resistance to nitrosamine-induced carcinogenesis in LEC rats.

Bile salt–induced pro-oxidant liver damage promotes transplanted cell proliferation for correcting Wilson disease in the Long-Evans Cinnamon rat model #

Insights into disease-specific mechanisms for liver repopulation are needed for cell therapy. To understand the efficacy of pro-oxidant hepatic perturbations in Wilson disease, we studied Long-Evans Cinnamon (LEC) rats with copper toxicosis under several conditions. Hepatocytes from healthy Long-Evans Agouti (LEA) rats were transplanted intrasplenically into the liver. A cure was defined as lowering of copper to below 250 microg/g liver, presence of ATPase, Cu++ transporting, beta polypeptide (atp7b) messenger RNA (mRNA) in the liver and improvement in liver histology. Treatment of animals with the hydrophobic bile salt, cholic acid, or liver radiation before cell transplantation produced cure rates of 14% and 33%, respectively; whereas liver radiation plus partial hepatectomy followed by cell transplantation proved more effective, with cure in 55%, P < 0.01; and liver radiation plus cholic acid followed by cell transplantation was most effective, with cure in 75%, P < 0.001. As a group, cell therapy cures in rats preconditioned with liver radiation plus cholic acid resulted in less hepatic copper, indicating greater extent of liver repopulation. We observed increased hepatic catalase and superoxide dismutase activities in LEC rats, suggesting chronic oxidative stress. After liver radiation or cholic acid, hepatic lipid peroxidation levels increased, indicating further oxidative injury, although we did not observe overt additional cytotoxicity. This contrasted with healthy animals in which liver radiation and cholic acid produced hepatic steatosis and loss of injured hepatocytes. We concluded that pro-oxidant perturbations were uniquely effective for cell therapy in Wilson disease because of the nature of preexisting hepatic damage.

Early effects of copper accumulation on methionine metabolism.

Wilson's disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.

Development of Inflammatory Bowel Disease in Long-Evans Cinnamon Rats Based on CD4+CD25+Foxp3+ Regulatory T Cell Dysfunction

A mutant strain with defective thymic selection of the Long-Evans Cinnamon (LEC) rat was found to spontaneously develop inflammatory bowel disease (IBD)-like colitis. The secretion of Th1-type cytokines including IFN-gamma and IL-2 from T cells of mesenteric lymph node cells (MLNs) and lamina propria mononuclear cells, but not spleen cells, in LEC rats was significantly increased more than that of the control Long-Evans Agouti rats through up-regulated expression of T-bet and phosphorylation of STAT-1 leading to NF-kappaB activation. In addition, the number of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells of the thymus, MLNs, and lamina propria mononuclear cells from LEC rats was significantly reduced, comparing with that of the control rats. Moreover, bone marrow cell transfer from LEC rats into irradiated control rats revealed significantly reduced CD25(+)Foxp3(+) Treg cells in thymus, spleen, and MLNs compared with those from control rats. Indeed, adoptive transfer with T cells of MLNs, not spleen cells, from LEC rats into SCID mice resulted in the development of inflammatory lesions resembling the IBD-like lesions observed in LEC rats. These results indicate that the dysfunction of the regulatory system controlled by Treg cells may play a crucial role in the development of IBD-like lesions through up-regulated T-bet, STAT-1, and NF-kappaB activation of peripheral T cells in LEC rats.

Development of Cell Therapy Strategies to Overcome Copper Toxicity in the LEC Rat Model of Wilson Disease

Therapeutic replacement of organs with healthy cells requires disease-specific strategies. As copper toxicosis due to ATP7B deficiency in Wilson disease produces significant liver injury, disease-specific study of transplanted cell proliferation will offer insights into cell and gene therapy mechanisms. We used Long-Evans Cinnamon (LEC) rats to demonstrate the effects of liver preconditioning with radiation and ischemia reperfusion, followed by transplantation of healthy Long-Evans Agouti rat hepatocytes and analysis of hepatic atp7b mRNA, bile copper, liver copper and liver histology. LEC rats without cell therapy or after transplantation of healthy cells without liver conditioning accumulated copper and showed liver disease during the study period. Liver conditioning incorporating hepatic radiation promoted transplanted cell proliferation and reversed Wilson disease parameters, although with interindividual variations and time lags for improvement, which were different from previous results of liver repopulation in healthy animals. Cell therapy will correct genetic disorders characterized by organ damage. However, suitable mechanisms for inducing transplanted cell proliferation will be critical for therapeutic success.

Bovine lactoferrin potently inhibits liver mitochondrial 8-OHdG levels and retrieves hepatic OGG1 activities in Long-Evans Cinnamon rats

To assess the effect of lactoferrin on oxidative liver damage and its mechanism, we used Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant-like hepatitis and lethal hepatic failure. Four-week-old female LEC rats were divided into the untreated and treated groups. The latter was fed bovine lactoferrin at 2% mixed with conventional diet. The cumulative survival rates were 75.0% vs. 100% at 14 weeks, 37.5% vs. 91.7% at 15 weeks, and 12.5% vs. 91.7% at 16 weeks, respectively, for untreated and treated rats (P=0.0008). The 8-OHdG levels in liver mitochondrial DNA and malondialdehyde in plasma and liver tissues were significantly lower in treated than untreated rats (P<0.001, =0.017 and 0.034, respectively). Mitochondrial DNA mutations were more common in untreated rats. OGG1 mRNA and protein expression levels were significantly lower in untreated than treated rats (P=0.003 and 0.007, respectively). Hypermethylation of the second CpG island located upstream of OGG1 gene was observed in untreated rats. Our findings indicated that lactoferrin inhibits oxidative liver damage in LEC rats. Lactoferrin could be potentially useful for the treatment of oxidative stress-induced liver diseases.

β-Galactoside α2,6-Sialyltransferase I Cleavage by BACE1 Enhances the Sialylation of Soluble Glycoproteins

BACE1 (beta-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid beta-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of alpha2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of alpha2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.

Functional analysis of phenolsulfonphthalein transport system in Long–Evans Cinnamon rats

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long–Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long–Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.

The role of TNF-α in the development of hepatic toxicity in Long-Evans Cinnamon rats

The role of TNF-α in the development of hepatic toxicity in Long-Evans Cinnamon rats

A New Amphiphilic Derivative, N‐{[4‐(Lactobionamido)methyl]benzylidene}‐ 1,1‐dimethyl‐2‐(octylsulfanyl)ethylamine N‐Oxide, Has a Protective Effect Against Copper‐Induced Fulminant Hepatitis in Long–Evans Cinnamon Rats at an Extremely Low Concentration Compared with Its Original Form α‐Phenyl‐N‐(tert‐butyl) Nitrone

An amphiphilic alpha-phenyl-N-(tert-butyl) nitrone (PBN) derivative, N-{[4-(lactobionamido)methyl]benzylidene}-1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide (LPBNSH), newly synthesized from its original form PBN in hopes of clinical use, was intraperitoneally administered to Long-Evans Cinnamon (LEC) rats every 2 days at the concentrations of 0.1, 0.5, 1.0, and 2.0 mg/kg. We found that LPBNSH protected against copper-induced hepatitis with jaundice in LEC rats at concentrations of 0.1 and 0.5 mg/kg, which were extremely low compared with that of PBN. It also effectively prevented the loss of body weight, reduced the death rate, and suppressed the increase in serum aspartate aminotransferase and alanine aminotransferase values arising from fulminant hepatitis with jaundice at the same concentrations. Similar results were observed when PBN was administered at the concentration of 150 mg/kg. Immunohistochemical analysis of 8-hydroxy-2'-deoxyguanosine and measurement of thiobarbituric acid-reactive substances in the liver showed that LPBNSH largely suppressed the formation of these oxidative products at same concentrations. No difference in the abnormal accumulation of copper in the liver between the LPBNSH administered and control groups was observed. From these results, it was concluded that LPBNSH exhibited liver-protective effects against fulminant hepatitis with jaundice at ca. 1/1000, 500 the molar concentration of PBN and, therefore, was clinically promising.

Perspectives for Gene Therapy of Wilson Disease

Wilson disease is a rare autosomal-recessive copper overload disorder due to mutations of the Wilson disease gene ATP7B. The disease typically manifests at late childhood or in young adults with hepatic and/or neurological symptoms. Being fatal without medical treatment or liver transplantation the long-term outcome of Wilson disease depends on the adherence to an effective treatment. Because current medical treatment options are not effective in all Wilson disease patients and adherence to therapy is a problem, gene therapy might represent an alternative curative future therapy. In the rat model of Wilson disease adenoviral and lentiviral gene transfer studies could prove that viral gene transfer is therapeutically effective and can reverse clinical symptoms. However, both approaches were limited by a more or less transient transgene expression. As several tactics can be used to overcome these current limitations, gene therapy approaches may become more efficient than standard medical treatment for Wilson disease in the future. This review discusses both, existing vectors and strategies and prospective developments towards liver-directed gene therapy, although there is still a long way to go until gene therapy can be used for safe treatment of Wilson disease in humans.

Maturational arrest of thymocyte development is caused by a deletion in the receptor-like protein tyrosine phosphatase κ gene in LEC rats

The Long-Evans Cinnamon (LEC) rat has a spontaneous mutation, T helper immunodeficiency ( thid), which causes a markedly reduced CD4 + thymocyte population. Here we positionally clone the locus and identify a deletion in the gene encoding a receptor-like protein tyrosine phosphatase κ ( Ptprk) that led to complete loss of the transcript. The rat Ptprk gene exhibits 98% identity with the human and mouse counterparts and is expressed most abundantly in the CD4 −CD8 − double-negative stage. The downregulation of Ptprk in mouse immature thymocytes by RNA interference mimicked the thid phenotype. These results indicate that thid maps to the Ptprk locus and that functional Ptprk is crucial for lineage commitment or progression of CD4 + T cells. We also found that Ptprk appears to function in parallel with or downstream of Th-POK/cKrox (also known as ZBTB7B), a master regulator of T cell lineage decision.

Possible role of zinc and iron in the development of hepatic abnormality in Long‐Evans Cinnamon rats

Possible role of zinc and iron in the development of hepatic abnormality in Long‐Evans Cinnamon rats

Mild zinc deficiency and dietary phytic acid accelerates the development of fulminant hepatitis in LEC rats

Restriction of copper intake delays hepatic copper accumulation in Long-Evans Cinnamon (LEC) rats, which are animal models of Wilson's disease. Involvement of zinc is suggested to develop hepatitis in the disease; however, this has not been clarified. The aims of this study were to investigate the effects of mild zinc deficiency on the development of hepatitis and to determine the relationship between the absorption and hepatic levels of copper, zinc and iron. Male LEC and F344 (wild type atp7b) rats were fed a low zinc, phytate-containing or control diet. The onset of hepatitis (Experiment 1), and absorptive rates of copper, zinc and iron and hepatitis indices in 4 weeks (Experiment 2) were observed. The onset of fulminant hepatitis in LEC rats was much earlier in the low zinc and phytate groups (mean 94.6 +/- 2.74 days and 82.8 +/- 3.56 days old, respectively) than in the control group (136 +/- 2.11 days old) with worse hepatitis indices. Hepatic copper levels were much higher in LEC rats than F344 rats, but were not largely different among the diet groups without prominent changes in copper absorption. Hepatic levels and intestinal absorption of zinc and iron were lower in the phytate group than in the control group. Mild zinc deficiencies caused by a low zinc or phytate-containing diet accelerate the onset of hepatitis in LEC rats without increasing copper absorption, and zinc and iron metabolism may be involved in the earlier onset of jaundice of LEC rats.

Abnormal hepatobiliary and circulating lipid metabolism in the Long-Evans Cinnamon rat model of Wilson's disease

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7α-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.

Down-Regulation of Intestinal Multidrug Resistance-Associated Protein 2 in Long-Evans Cinnamon Rats

Wilson's disease is an inherited, autosomal recessive disorder of copper accumulation and toxicity. Lifelong chelation therapy is essential in all Wilson's disease patients. Intestinal absorption of some compounds is limited partly because they are preferentially transported in the secretory direction. Several ATP-binding cassette (ABC) transporters are expressed in the apical membrane of the small intestine and secrete various drugs into the lumen. In this study, we investigated the characteristics of the intestinal efflux ABC transporters in LEC rats. We found that the expression of multidrug resistance-associated protein 2 (Mrp2) in the jejunum of Long-Evans Cinnamon (LEC) rats, an animal model for Wilson's disease, is decreased.

Characterization of Hepatobiliary Organic Anion Transporters in Long-Evans Cinnamon Rats

The liver plays important roles in the detoxification of xenobiotics. Hepatobiliary transporters contribute to hepatic uptake and efflux processes of xenobiotecs. Expressions of these transporters may be modulated under the condition of hepatic failure. Long-Evans Cinnamon (LEC) rats provide a pertinent model for basic and clinical studies on hepatitis. However, only a few reports describing the properties of hepatobiliary transporters in LEC rats have appeared in the literature. We investigated the expression levels of hepatobiliary transporters in LEC rats by real-time RT-PCR. We found that hepatic expressions of three sinusoidal organic anion transporters, Ntcp, Oatp1a1 and Oatp1a4, were decreased in LEC rats. However, no significant difference of the expressions of Mrp2 and Bsep, organic anion transporters located on canalicular membrane, were found between Wistar rats and LEC rats.

Aldo-keto reductase 1 family B7 is the gene induced in response to oxidative stress in the livers of Long-Evans Cinnamon rats

The Long-Evans Cinnamon (LEC) rat strain (Atp7b m/m), which accumulates copper in the liver due to mutations in the Atp7b gene, is a useful model for investigating the relationship between oxidative stress and hepatocarcinogenesis. To determine the effect of this mutation on oxidative stress marker genes, we performed oligonucleotide array analysis (Affymetrix), and compared the results in Atp7b m/m rats with those of a sibling line with the Atp7b w/w genotype. We focused our studies on the expression of the aldo-keto reductase 1 family B7 (AKR1B7)-like protein gene, since this gene codes for reductase enzymes involved in the detoxification of oxidizing compounds (e.g., aldehydes) and was differentially expressed in Atp7b m/m and Atp7b w/w rat liver. Akr1B7 mRNA expression was significantly increased in comparison with the expression of 4 other known oxidative stress responsive genes, haem-oxygenase-1 (HO-1), thioredoxin (Trx), aldehyde reductase (AKR1A1), and glucose-6-phosphate dehydrogenase (G6PDH). By searching binding motifs, five nuclear factor kappa B (NF-kappaB) binding sites were located in the 5'-upstream region of the akr1b7 gene. Transient co-transfection with both I-kappaBalpha and the Akr1b7 6 kb promoter (p6.0-AKR-Luc) inhibited luciferase activity of p6.0-AKR-Luc in HepG2 cells. Cuprous ion however did not affect the transcription activity induced by p6.0-AKR-luc. Gel-shift assay showed that the DNA binding activity of NF-kappaB increased in the livers of LEC rats, suggesting that the oxidative stress is mediated through NF-kappaB. The results indicate conclusively that in LEC rat liver, akr1b7 might be up-regulated by oxidative stress mediated through NF-kappaB, but not that mediated directly by copper.

Effects of copper metabolism on neurological functions in Wistar and Wilson’s disease model rats

Behavioral functions of Wistar and Long-Evans Cinnamon (LEC) rats, Wilson’s disease animal model, were compared by measuring the open-field, acoustic startle reflex and prepulse inhibition (PPI), and shuttle-box avoidance learning tests with or without oral supplementation with copper or d-penicillamine, copper chelator. All of the LEC rats, irrespective of the treatment, exhibited higher locomotor activity, a decreased habituation to startle response or a lower PPI, compared with Wistar rats. The copper content of all brain regions examined, except for the medulla oblongata of LEC rats, was significantly lower than those in Wistar rats. Besides, in the region of the striatum and the nucleus accumbens of the LEC rats, lower content of norepinephrine, and higher content of dopamine and serotonin were observed compared with Wistar rats. Although copper supplementation did not affect the brain copper content, it reduced the PPI in both Wistar and LEC rats. In contrast, d-penicillamine supplementation decreased both the brain copper content and locomotor activity, and enhanced the startle amplitude only in Wistar rats. These findings suggest that an imbalance in copper homeostasis affects monoamine metabolism and behavioral functions.

Iron removal by phlebotomy for the prophylaxis of fulminant hepatitis in a Wilson disease model of Long-Evans Cinnamon Rats

Long-Evans Cinnamon rats are an animal model of Wilson disease with a compound load of copper and iron. However, the mechanisms of the high mortality rate from fulminant hepatitis in the rats remain undetermined. In this study, phlebotomy as an alternative for an iron-deficient diet was investigated to modify hepatitis in the rats. One group of female rats was treated with phlebotomy twice a week, and the other group received blood tests only every 2 weeks. Liver specimens were examined for biochemical non-heme iron and copper, and histochemical trivalent iron. Although all rats examined had icteric hepatitis around the age of 20 weeks, the mortality of fulminant hepatitis was lower in phlebotomized rats than in control rats (1/18 versus 8/20, p<0.05). Phlebotomy not only ameliorated the severity of hepatitis, but also reduced serum malondialdehyde. Non-heme iron in the liver was the greatest in victims of fulminant hepatitis in the control group. There was no difference in the hepatic copper content between the two groups. The prognosis of hepatitis in the rats with Wilson disease was hepatic non-heme iron-dependent, and iron removal by phlebotomy was an effective prophylaxis against fulminant hepatitis.