A Transplantable Cell Line Derived from Spontaneous Hepatocellular Carcinoma of the Hereditary Hepatitis LEC Rat

A serially transplantable rat hepatocellular carcinoma (HCC) line was established. The primary spontaneous HCC which developed in a 506-day-old male hereditary hepatitis LEC rat was inoculated into young LEC rats. Only this HCC of 18 primary HCCs was successful in serial transplantation. The established cell line was histologically identical to the primary HCC showing a well-differentiated type with a trabecular structure of tumorous hepatocytes. The characteristic of albumin production was maintained. Chromosome analysis revealed rather widely dispersed polyploid chromosome numbers with a modal value at 96. Every metaphase contained two to five unusually large marker chromosomes.     

Binding of Cu to metallothionein in tissues of the LEC rat with inherited abnormal copper accumulation

Long-Evans Cinnamon (LEC) rats aged 16 ± 4 weeks with histopathological alterations of liver and kidney, exhibited elevated Cu levels in liver, kidney and spleen which were 52, 27 and 5 times higher than those of the respective tissues of age-matched Wistar rats. About 61% of hepatic and about 38% of renal Cu was recovered in the cytosolic fraction. Metallothionein (MT) levels were found to correlate with the cytosolic Cu concentrations in liver and kidney. According to differential MT analysis, about 68 and 82% of hepatic and renal MT was loaded with Cu. The portion of MT which binds Cu was negatively correlated with the ratio of cytosolic Zn/Cu in all organs investigated. Despite high MT levels and the high percentage of Cu binding to MT, particularly in liver and kidney, considerable amounts of Cu remained unbound to MT. This non-MT bound Cu showed good correlation with the total cytosolic Cu content, and might play a crucial role in the pathogenesis of Cu toxicosis. Received: 21 August 1996 / Accepted: 18 December 1996     

Abnormal CD45RC expression and elevated CD45 protein tyrosine phosphatase activity in LEC rat peripheral CD4+ T cells

LEG rats are known to show a maturational arrest in the development of CD4⁺8⁺ to CD4⁺8^? cells in the thymus. Despite the blockade of maturation of CD4⁺8^? thymocytes, CD4⁺ T cells were observed in peripheral lymphoid organs, and these cells exhibit a defect in interleukin-2 (IL-2) production upon concanavalin A (Con A) stimulation. Although peripheral CD4⁺ cells in normal rat highly expressed CD45RC (CD45RC^high), the level of CD45RC expression was low (CD45RC^low) in LEC rat peripheral CD4⁺ cells. However, CD4⁺ cells from both strains highly expressed CD45 when those cells were stained by pan-CD45 mAb, suggesting that LEC rat CD4⁺ cells are deficient in expression of the CD45RC isoform, but not of CD45 molecules. When backcross rats from (F344 × LEC)F₁ × LEC were examined, the phenotype for CD45 expression pattern in CD4⁺ cells was clearly correlated with IL-2 production level in response to Con A stimulation. Thus, CD45RC^low cells exhibit a defect in IL-2 production, while CD45RC^high cells show normal IL-2 production. Protein tyrosine phosphatase (PTPase) activity in the membrane fraction of LEC rat CD4⁺ cells was threefold higher than that of normal rat CD4⁺ cells. Con A stimulation led to an increase in tyrosine phosphorylation levels, especially 100- and 40-kDa proteins, in normal rat CD4⁺ cells. In LEC rat CD4⁺ cells, however, the level of tyrosine phosphorylation in those proteins were very low. These results suggest that an elevated CD45 PTPase activity is responsive for a defect in IL-2 production in LEC rat peripheral CD4⁺ T cells.     

Gene Expression of Placental Glutathione S-Transferase in Hereditary Hepatitis and Spontaneous Hepatocarcinogenesis of LEC Strain Rats

Liver tissues of LEG rats, which develop fulminant hepatitis and hepatocellular carcinoma (hepatoma), were examined by Northern blot analysis using a cDNA probe of rat placental glutathione S -transferase (GST-P). GST-P gene expression was observed not only during hepatocarcinogenesis but also in fulminant hepatitis before development of chronic hepatitis and hepatoma in LEC rats. Cholangiofibrosis in LEC rats also showed high GST-P expression. A transplantable cell line derived from spontaneous LEC hepatoma exhibited a remarkably high expression. By contrast, very weak expression was observed in the livers of young LEC rats before development of hepatitis and control strain rats. Thus, spontaneous hepatic lesions in LEC rats may provide a new clue to elucidate the mechanism of GST-P gene expression.     

Neurofunctional endpoints assessed in human neuroblastoma SH-SY5Y cells for estimation of acute systemic toxicity

The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicity data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.     

Abnormal Hepatic Iron Accumulation in LEC Rats

The LEC (Long-Evans cinnamon) rat is a mutant strain displaying hereditary hepatitis and spontaneous hepatocellular carcinoma, and shows abnormal hepatic copper accumulation similar to that occurring in Wilson's disease. We evaluated the iron metabolism of LEC rats compared to LEA (Long-Evans agouti) rats. Hepatic iron and ferritin concentrations were remarkably increased depending on age in LEC rats but not in LEA rats. Increased hepatic iron is normally associated with decreased serum transferrin and total iron binding capacity in hepatic iron overload. In LEC rats, however, both serum transferrin and total iron binding capacity increased with increasing hepatic iron. This increase of serum transferrin and hepatic iron may be an additional important factor contributing to liver injury in LEC rats.     

Role of Copper Accumulation in Spontaneous Renal Carcinogenesis in Long-Evans Cinnamon Rats

Spontaneous renal cell tumors in totals of 223 male and female Long-Evans Cinnamon (LEC) rats of 51–120 weeks old, 157 male F344 rats of 51–120 weeks old, and 14 male Long-Evans Agouti (LEA) rats of 51–70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long-term treatment of LEC rats with d -penicillamine, a copper-chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.     

Lycopene from tomatoes partially alleviates the bleomycin-induced experimental pulmonary fibrosis in rats

In this study, lycopene extracted from tomatoes was evaluated to treat PF induced by BLM in rats. Sixty Sprague-Dawley rats were randomly divided into 3 groups of 20 rats each for a normal control group (group C), BLM-treated group (group M), and lycopene + BLM-treated group (group L). The rats in groups M and L were subjected to intratracheal instillation of BLM to induce PF; group C served as a sham control (intratracheal instillation of normal saline). Lycopene diluted with olive oil was administered at a dose of 5 mg/kg body weight once a day in group L after BLM instillation, and groups C and M were treated with the same amounts of olive oil. The pathologic alterations of lung tissues, the concentrations of TNF-α, NO, malonyldialdehyde, the activities of superoxide dismutase in plasma, and the expression of TNF-α in lungs were assessed on day 3, 7, 14, and 28 after BLM instillation. The results showed that the lung coefficients in group L were reduced (day 14, P < .01) as well as the extents of alveolitis (day 7 and 14, P < .05) and PF (day 14 and 28, P < .05) compared with group M. The concentrations of TNF-α (day 7, 14, and 28, P < .001), NO (day 14, P < .05), and malonyldialdehyde (day 3, P < .01) in plasma as well as the expression of TNF-α in lungs decreased, whereas the plasma superoxide dismutase activities increased (day 28, P < .05) in group L compared with group M. Our study demonstrates that lycopene can partially reduce the extent of PF induced by BLM in rats. These findings suggest that the suppression of oxidative stress, the reduction of plasma TNF-α and NO levels, and the down-regulation of TNF-α in lungs contribute to the alleviation of PF in rats administered lycopene.     

Hereditary Low Level of Plasma Ceruloplasmin in LEC Rats Associated with Spontaneous Development of Hepatitis and Liver Cancer

Both young (5 weeks old) and old (61 100 weeks old) hereditary hepatitis LEC rats showed a markedly low level of plasma ceruloplasmin (Cp) ferroxidase activity as compared with that of age-matched LEA and BN strain rats. This trait was genetically examined hy the use of (BN × LEC) F₁ hybrid and (F₁× LEC) backcross rats. The F₁ hybrids never developed hepatitis and showed a similar level of Cp to that found in the parental BN rats. Among the backcross rats with about 1:1 segregation rate for hepatitis, affected rats had a remarkably decreased level of Cp, as found in LEC rats, whereas unaffected rats exhibited a similar level of Cp to that of BN, F₁ and LEA rats. These results indicate that the low level of Cp is heritable in a single autosomal recessive mode in LEC rats. The observed tight link between the low Cp level and the hepatitis in LEC rats suggests that defective copper metabolism may he associated with the occurrence of hepatitis in LEC rats, since Cp is a copper-binding protein primarily involved in copper transport from the liver.