Copper and iron-induced oxidative damage in non-tumor bearing LEC rats

The copper and Iron status in the liver of non-tumor bearing Long-Evans Cinnamon (LEC) rats (average age 17 months) was investigated. A direct quantitation of loosely-bound copper and iron was also investigated by using a chelating agent, nitrilotriacetic acid (NTA-chelatable free copper and iron). Besides the total copper and iron contents, the level of NTA-chelatable free copper was also higher in LEC rats than In LEA rats (P<0.05). But for the free iron level there was no signiflcant difference between the two rat groups (P>0.05). The formation of thiobarbituric acid-reactive substances was higher In LEC rats than In LEA rats (P<0.01). The 4–hydroxy-2–nonenal (HNE)-modified proteins were also clearly demonstrated in LEC rat liver. The copper and iron which produced the most important effect In the process of oxidative damage in LEC rats could not be distinguished. Even though free copper, which could induce free radical injuries, was increased in LEC rats, neither tumor-induction nor preneo-plastic lesions in the experimental LEC rats were observed. Therefore it is speculated that the elevation of a free iron is another important factor. Copper and iron, both important translation metals In the body, may participate In the Induction of DNA damage and oncogenesls     

The WD Gene for Wilson's Disease Links to the Hepatitis of LEG Rats

LEC rats develop an autosomal recessive hepatitis and subsequently liver cancer associated with copper accumulation in the liver similar to that of Wilson's disease. Using 71 backcross [(WKAH x LEC) x LEC] rats, linkage analysis of the hepatitis with the WD gene for Wilson's disease revealed identical segregation and no recombination event between these two genes. This result indicates that the WD gene is a prime candidate for the hts gene responsible for the hepatitis of LEG rats, and suggests that the hepatitis of LEC rats may be caused by a defect in a copper-transporting ATPase expressed in the liver.     

水蛭提取液对体外培养牛晶状体上皮细胞生长抑制的实验研究

目的观察水蛭提取液对体外培养的牛晶状体上皮细胞（LEC）生长的影响，探讨水蛭用于防治后发性白内障的可能性。方法采用植块培养法，对牛晶状体前囊膜进行培养。利用水蛭水提醇沉提取液，观察传代培养48小时的LEC加入不同浓度提取液培养24和72小时后的生长情况，以MTT法测定OD值，并求出半效抑制量（ID50）。传代的LEC加入提取液高剂量（ID50）及低剂量（1／10ID50）培养24小时，观察贴壁抑制情况。结果水蛭水提醇沉提取液抑制LEC生长的24和72小时ID50分别为31．85mg／ml、30．69mg／ml；与空白组比较，水蛭水提醇沉液对LEC贴壁有明显抑制作用（P〈0．01）。结论水蛭水提醇沉液能同时抑制体外培养的LEC生长及贴壁，为水蛭提取液用于防治后发性白内障提供了实验依据。     

Effects of organic solvents on motor activity in mice

Groups of male mice were exposed via inhalation to methylene chloride, perchloroethylene, toluene, trichloroethylene or 1,1,1-trichloroethane. The exposures were started at 2300 h. Generation of vapor was stopped after 1 h. Motor activity of the animals during the exposures was measured with a Doppler radar. Several concentrations of each solvent were tested. Concentrations could be found for all solvents at which they initially increased the motor activity. When the generation of vapor was terminated and the concentration started to decline, a new phase of changes in motor activity was induced. At this phase, motor activity was in most cases influence in the opposite direction to that at the beginning of the exposure. Trichloroethylene concentrations could be found which gave no increase in activity at the start of exposure but a prominent decrease at termination. The lowest concentration at which effects could be seen was different for the different solvents. Perchloroethylene was more and 1,1,1-trichloroethane less potent than the other solvents in inducing motor activity. The time pattern of the motor activity alterations was specific for each solvent. Both the concentration and the rate of the concentration increase were responsible for the effects on motor activity. The differences between the solvents probably reflect differences in their site of action, their distribution and their biotransformation.     

Mechanism of enhanced lipid peroxidation in the liver of Long-Evans cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rat is a mutant strain of rats that accumulate copper (Cu) in the liver in much the same way as individuals who suffer from Wilson's disease (WD) and has been suggested as a model for this disease. Lipid peroxidation (LPO) is considered to be involved in the toxic action of Cu in the livers of LEC rats. We investigated the mechanism of LPO in the livers of LEC rats showing apparent signs of hepatitis. Several-fold higher LPO levels were observed in post-mitochondrial supernatant (S-9) fraction of livers from hepatitic LEC rats than in those from Wistar rats. To mimic living cells, we introduced NADPH-generating system (NADPH-gs) into the S-9 incubation system. Thus was ensured a constant supply of NADPH to vital enzymes that may be directly or indirectly involved in the generation and/or elimination of reactive oxygen species (ROSs), such as glutathione reductase (GSSG-R), which require NADPH for their reactions. The levels of LPO in liver S-9 from hepatitic LEC rats were further increased by incubating liver S-9 at 37 °C in the presence of NADPH-gs. This increase was inhibited by EDTA, butylated hydroxytoluene (BHT), and catalase (CAT), suggesting that some metal, most likely the accumulated Cu, and ROSs derived from hydrogen peroxide (H₂O₂) are involved in the increased levels of LPO in the livers of hepatitic LEC rats. The requirement of NADPH-gs for enhanced LPO in the livers of hepatitic LEC rats indicates the consumption of NADPH during reactions leading to LPO. It is known that H₂O₂, and consequently hydroxyl radical are generated during Cu–catalyzed glutathione (GSH) oxidation. The cyclic regeneration of GSH from GSSG by NADPH-dependent GSSG-R in the presence of NADPH-gs may cause sustained generation of hydroxyl radical in the presence of excess free Cu. The generation of H₂O₂ in S-9 fraction of livers from hepatitic LEC rats was observed to be significantly higher than that in S-9 fraction of livers from non-hepatitic LEC rats and Wistar rats. Moreover, in addition to the reported decrease in glutathione peroxidase (GPX) activity, we found that CAT activity was markedly decreased in LEC rats with hepatitis. The increased generation of H₂O₂ with reduced activities of GPX and CAT may result in cellular accumulation of H₂O₂ in the liver of hepatitic LEC rats. Taken altogether, it is suggested that the accumulated H₂O₂ undergoes the Fenton-type reaction with also accumulated free Cu, thus generating hydroxyl radical in the livers of hepatitic LEC rats and increasing LPO levels in these animals. Received: 20 April 1999 / Accepted: 2 September 1999     

Effects of d-Galactosamine Hydrochloride and Partial Hepatectomy on Spontaneous Hepatic Injury and Hepatocarcinogenesis in Long-Evans Cinnamon Rats

To examine the effect of nongenotoxic chemicals on hepatocarcinogenesis in Long-Evans Cinnamon (LEC) rats, we gave 6-week-old male and female LEC rats ( n =18) weekly subcutaneous injections of d -galactosamine hydrochloride (GalN, 300 mg/kg) in 0.9% NaCl or only 0.9% NaCl for 50 weeks, and killed them in week 62. GalN-treated male rats unexpectedly showed no lethal necrotizing hepatitis. GalN treatment increased the incidence of cholangiofibrosis in males and its severity in females, but did not cause significant increases of hepatocellular tumors in either sex. GalN treatment increased the 5-bromo-2'-deoxyuridine (BrdU)-labeling index of hepatocytes and plasma hepatocyte growth factor, and accelerated megalocytic alterations without reduction of the hepatic copper concentration. Next, male and female LEC rats were subjected to two-thirds partial hepatectomy (PH) or sham hepatectomy in week 8 ( n =12) or in week 14 ( n =9), and killed in week 62. PH in week 14 inhibited lethal hepatitis, but PH in week 8 was less effective. PH reduced the hepatic copper concentration to half that of controls. The present data suggest that induction of hepatocyte regeneration by repeated injections of GalN, or by PH just before the onset of jaundice has a significant effect in prevention of hepatic injury of LEC rats, but not enhancement of spontaneous hepatocarcinogenesis.     

Enhancing Effect of a Choline–deficient Diet on Alterations of Hepatic Drug–metabolizing Enzymes in Hepatitis– and Hepatoma–predisposed Rats (LEC Rats)

Marked alterations of hepatic drug–metabolizing enzymes were observed in hepatitis– and hepatoma–predisposed rats (LEC rats) fed a choline–deficient diet. The diet enhanced the development of hepatitis with severe jaundice. The levels of two major classes of cytochrome P–450, P–450_PB and P–450_MC, were markedly decreased. GST–Yp was dramatically increased, whereas GST–Ya, Ybl and Yb2 were decreased. LEA rats (the control rats to LEC) fed a choline–deficient diet mimicked LEC rats fed a normal diet in terms of the above enzyme alterations, indicating that hypomethylation is involved in the pathogenesis of hepatitis and hepatoma in LEC rats. Such hypomethylation may initiate the hepatocytes that spontaneously develop hepatitis and hepatoma.     

Accumulation of Abnormally High Ploid Nuclei in the Liver of LEC Rats Developing Spontaneous Hepatitis

Enlarged hepatocytes with huge nuclei were found in LEC rats with hereditary hepatitis. Flow cytometric analysis of the DNA content of nuclei from jaundiced LEC rats revealed the presence of very high polyploids, such as 32n and 64n. At the age of 12 weeks, before the onset of hepatitis, 8n polyploid nuclei were more frequent in LEC rats than in LEA rats, a sibling line of LEC rats. Binucleated hepatocytes were also more frequent in LEC rats than in LEA rats at week 4. Bi-, tri- and tetra-nucleated cells whose nuclei were sometimes different in size were observed when jaundice became manifest. The number of proliferating liver cells, determined by pulse labeling with 5-bromo- 2'-deoxyuridine (BrdU), was higher in LEC rats than in LEA rats at 2, 4, 8, 12 and 14 weeks, with a maximum at week 4. A remarkable increase of BrdU uptake was observed at week 16, when jaundice developed. The possible involvement of abnormal cytokinesis and kariokinesis in the manifestation of hepatitis was suggested.