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Lipid peroxidation is highly associated with chronic degenerative diseases such as cancer. 4-hydroxy-2-nonenal is one of the major products of lipid peroxidation. 4-hydroxy-2-nonenal can interact with biomolecules, changing their conformation and activity. This study presents 4-hydroxy-2-nonenal-protein adducts formation in the first stages of Long-Evans Cinnamon rat hepatitis, a well recognized model for oxidative stress-associated hepatocarcinogenesis. 4-hydroxy-2-nonenal-protein adducts appeared in hepatocyte cytoplasm before the beginning of hepatitis and their presence was very strong during hepatitis, while a transient perinuclear expression of 4-hydroxy-2-nonenal-protein adducts was shown mainly at early hepatitis stages. 4-hydroxy-2-nonenal-protein adducts formation correlated to the expression of the tumour marker glutathione S-transferase P-form. These results show that lipid peroxidation modification of proteins might be implicated in the first stages of hepatocyte cancer initiation in Long-Evans Cinnamon rats. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Background and Aim: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Medici V, Sturniolo GC, Santon A, D'Incà R, Bortolami M, Cardin R, Basso D, Albergoni V, Irato P. Efficacy of zinc supplementation in preventing acute hepatitis in LEC rats. Liver International: DOI: 10.1111/j.1478-3231.2005.01108.x. © Blackwell Munksgaard 2005 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. [ABSTRACT FROM AUTHOR]

Abstract: We assessed the prevention of hepatic fibrogenesis by water-extract of Panax notoginseng Buck F.H. Chen. (Arialiaceae) root (PNS) in Long-Evans rats with cinnamon coat color (LEC rats). LEC rats were divided into three groups A, fed on a basal diet (BD); B, fed on BD plus 1% PNS; and C), fed on BD plus 0.005% lycopene as a control. All rats were sacrificed at 26 weeks of age. The percentage of the total area involved by fibrosis was 1.46 ± 0.47 in group A, 0.83 ± 0.10 in B (P = 0.0030, B vs A) and 0.91 ± 0.45 in C (P = 0.0035, C vs. A). The percentage of the total area that was stained for α-SMA was 0.56 ± 0.34 in group A, 0.15 ± 0.02 in B (P = 0.0016, B vs. A and 0.11 ± 0.01 in C (P = 0.0025, C vs. A. In group B, malondialdehyde (MDA) in the liver was lower than in group C (P = 0.007). In group C, the concentration of iron in the liver was lower than in group A (P = 0.0053). Thus, PNS suppressed fibrogenesis through reduced generation of lipid peroxides. The mechanisms of this preventive effect of fibrogenesis with PNS were suggested to inhibit the stellate cell activity. Second objective of this study was to determine whether PNS affects hepatic microvascular dysfunction elicited by gut ischemia and reperfusion (I/R), since gut I/R causes hepatic microvascular dysfunction, and to investigate the role of nitric oxide (NO). Male Wistar rats were exposed to 30 min of gut ischemia followed by 60 min of reperfusion. Intravital microscopy was used to monitor the number of non-perfused sinusoids (NPS). In another set of experiments, PNS (1 g/kg per day intragastrically) was administered to rats for 7 days. In some experiments, dexamethasone (ST) (2 mg/kg per day intravenously) was administered. In control rats, gut I/R elicited increases in the number of NPS, and plasma TNF-α and ALT activities, and these changes were mitigated by the pretreatment with PNS. Pretreatment with an NO synthase inhibitor diminished the protective effects of PNS on the increase in NPS and plasma TNF-α levels, but not its effect on the increase in plasma ALT activities. Pretreatment with PNS increased plasma nitrite/nitrate levels. The responses caused by gut I/R were attenuated by the pretreatment with ST. Pretreatment with an NO synthase inhibitor did not affect the effect of ST. These results suggest that PNS attenuates the gut I/R-induced hepatic microvascular dysfunction and inflammatory responses such as TNF-α production in the early phase via enhancement of NO production, and sequential hepatocellular damage via its anti-inflammatory effect. [Copyright &y& Elsevier]

We investigated the effects of dietary D-penicillamine on copper and zinc contents in the liver, serum, and urine of Long-Evans Cinnamon (LEC) rats, as well as the gene expression of hepatic metallothionein-1 and hepatic metallothionein protein level in these animals. Young male LEC and Fischer rats were fed a basal or D-penicillamine (5 g D-penicillamine/kg diet) diet for 16 days. The urinary output of copper and zinc was markedly increased by the D-penicillamine diet, and the copper and zinc contents in the liver were significantly decreased in LEC rats. Excess copper accumulation in the liver induced hepatic metallothionein in LEC rats, but the D-penicillamine diet depressed the level of hepatic metallothionein-1 mRNA as well as the hepatic metallothionein protein level. When fed the basal diet, serum ornithine carbamoyltransferase activity was higher in LEC rats than in Fischer rats, but a significant decrease in serum ornithine carbamoyltransferase activity rats was observed in LEC rats on feeding the D-penicillamine diet. These results indicated that dietary D-penicillamine significantly reduced excess copper and zinc in the livers of LEC rats through urinary excretion, and the decrease in hepatic copper and zinc contents caused a decrease in the levels of metallothionein-1 mRNA and metallothionein protein in LEC rats. [Copyright &y& Elsevier]

Copper concentrations in the livers of rats in the SPI and SPIM diet KEY WORDS: LEC rats soy protein isolate copper Wilson's disease liver cell damage Rat with a cinnamon-like coat color; SPI, soy protein isolate; SPIM, SPI with Copper, liver cell damage, soy protein isolate, Wilson's disease, LEC rats. [Extracted from the article]

The aims of the present work were to determine the effect of long-term treatment with zinc (Zn) on metallothionein (MT) concentrations and to study the levels of both MT-1 and MT-2 mRNAs in Long-Evans Cinnamon (LEC) rat liver. We also identified apoptotic cells comparing two cytochemical techniques. Thirteen rats received 50 mg zinc acetate daily by gavage, 13 rats received no treatment, and both groups were killed after 60 days. Finally four rats were killed 35 days after birth (T0). The results demonstrate that the Zn-treated group had higher levels of MT than both the untreated and basal ones. Quantification of mRNA indicates that the level of the Zn-treated group was significantly higher than the untreated group. Confocal fluorescent staining with monoclonal antibody (Mab) against single-strand DNA localised the hepatic cells that had chromatin condensation and nuclear fragmentation typical of apoptosis, especially in the untreated group sections. The intensity and quantity of fluorescence decreased in both the treated and basal groups. The higher sensitivity of Mab staining compared to TUNEL, which revealed both apoptotic and necrotic cells, reflects the different action mechanism of the two techniques. These findings confirm, in LEC rats, the important role of Zn in cellular protection in relation to MT expression and apoptotic processes as cellular responses to DNA damage by free radicals. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

We assessed the prevention of hepatic fibrogenesis by a herbal medicine Sho-saiko-to or a carotenoid lycopene in Long-Evans rats with cinnamon coat color (LEC rats). LEC rats were divided into three groups: A (n=40), fed on a basal diet (BD); B (n=25), fed on BD plus 1% Sho-saiko-to; and C (n=40), fed on BD plus 0.005% lycopene. All rats were sacrificed at 76 weeks of age. The liver tissues were stained with Azan–Mallory and α-smooth muscle actin (α-SMA). The malondialdehyde (MDA) in the liver was measured for the assay of lipoperoxides. The percentage of the total area stained was determined morphometrically. The percentage of the total area involved by fibrosis was 1.35±0.56 in group A, 0.72±0.34 in B (P=0.0020, B vs. A) and 0.78±0.75 in C (P=0.0031, C vs. A). The percentage of the total area that was stained for α-SMA was 0.61±0.57 in group A, 0.11±0.05 in B (P=0.0017, B vs. A) and 0.12±0.06 in C (P=0.0021, C vs. A). In group B, MDA in the liver was lower than in group C (P=0.009). In group C, the concentration of iron in the liver was lower than in group A (P=0.0059). In conclusion, Sho-saiko-to suppressed fibrogenesis through reduced generation of lipid peroxides. Hepatic fibrogenesis was also suppressed by lycopene. The mechanisms of this preventive effect of fibrogenesis with Sho-saiko-to and lycopene were suggested to inhibit the stellate cell activity. [Copyright &y& Elsevier]

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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