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     

联合抗代谢药物预防兔后发性白内障的研究

目的 探讨联合抗代谢药物在预防兔后发性白内障（PCO）中的作用及其机制.方法 40只新西兰白兔随机分为两组。在兔晶状体超声乳化吸出术后，实验组右眼晶状体囊袋内注入含有02mg／ml丝裂霉素及12．5mg/ml 5-氟尿嘧啶的甲基纤维素，左眼晶状体囊袋内仪注入甲基纤维素设置空白对照组；对照组右眼晶状体囊袋内注入含有0．2mg/ml丝裂霉素的甲基纤维素，左眼晶状体囊袋内注入含有12．5mg/ml 5-氟尿嘧啶的甲基纤维素。部分动物囊袋内植入人工晶体。手术后对动物进行1～12个月临床观察，对不同阶段进行晶状体囊膜组织和人工晶体病理学检查、晶状体囊膜平片检查及人工晶体电镜扫描。结果 空白对照组在手术后2周出现后囊膜混浊，随着术后时间的延长，PCO明显加重；对照组在术后1个月发生不同程度PCO，而实验组观察12个月，未见PCO发生。结论 晶状体囊袋内联合抗肿瘤药物能有效杀伤晶状体上皮细胞（LEC）、晶状体赤道部生发区细胞，较单一抗肿瘤药物更可靠预防PCO的发生。晶状体囊袋内联合抗肿瘤药物可能为解决预防人类PCO提供一条新途径。     

LEC鼠自发性肝癌谷胱甘肽S-转移酶表达的意义

目的：观察LEC大鼠自发性肝癌谷胱甘肽S-转移酶(GST-P)的蛋白表达,探讨GST-P对原发性肝癌早期诊断的价值。方法：采用免疫组织化学方法检测LEC大鼠肝脏GST-P的表达。 结果：67周龄LEC鼠即肝癌组GST-P的表达率（96.97%）明显高于4~6个月龄LEC鼠即肝炎组（37.5%）及SD大鼠组（9.38%）的表达率(P<0.01),4~6个月龄LEC鼠GST-P的表达率高于SD大鼠组(P<0.05)。 结论：GST-P可作为大鼠原发性肝癌早期诊断的肿瘤标志物。     

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.     

Hepatocellular Carcinoma Induction in LEC Rats by a Low Dose of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

A food-borne heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5- f ]qninoxaline (MeIQx), induces hepatocellular carcinomas (HCCs) in F344 male rats at an incidence of 95%, when fed in the diet at 400 ppm for 61 weeks. In this study, the effect of a low dose of MeIQx was examined in Long-Evans with cinnamon-like coat color (LEC) rats, which have a mutation in Atp7b and suffer from hereditary hepatitis and HCCs, with high levels of copper accumulation in the liver. Rats of the LEC and Long-Evans with agouti coat color (LEA) sibling lines were given a diet containing 40 ppm MeIQx from the age of 23 weeks to 63 weeks, for a total administration period of 40 weeks. In LEC rats, HCCs were observed in 8/8 animals administered MeIQx, and 2/8 rats receiving a normal diet. The number of HCCs per rat (mean±SD) was 2.8±2.0 and 0.3±0.5, respectively. In the LEA rats, however, no tumors were induced by administration of MeIQx. These results indicate that damaged liver associated with compensatory cell proliferation is much more susceptible to chemical hepatocarcinogens, including MeIQx, than the normal 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.