Toxic effects of 2-methyl-4-dimethylaminoazobenzene in normal and partially hepatectomized rats

In order to obtain a more precise definition of the conditions under which 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) and liver cell proliferation play a role in the initiation of hepatocarcinogenesis, the toxicity of 2-Me-DAB for normal and partially hepatectomized rats was investigated. Continuous feeding of a basal low protein, low riboflavin diet supplemented with 2-Me-DAB was found to be highly toxic for male albino rats. All animals fed on such a diet died before 200 days. Sham operation and partial hepatectomy (PH) at 30 days of 2-Me-DAB feeding reduced the median survival time from 122 days to 107 and 94 days, respectively. Transfer to the basal diet after 30 days of 2-Me-DAB feeding and PH prolonged the median survival time to 216 days while 97% of the rats returned to the normal complete diet after the same treatments survived for more than 300 days. 2-Me-DAB was not necrogenic and there was no evidence of reparative proliferation or hepatic tumor formation in any group. Feeding rats with the 2-Me-DAB containing diet for 1 month delayed and strongly inhibited the mitotic response of the liver to the stimulus of partial hepatectomy. This is the result of a blockage of the cells in G1 as revealed by the fact that only 1% of the hepatocytes became labeled when 2-Me-DAB fed animals were injected with tritiated thymidine prior to sacrifice at 24 h post-hepatectomy, as compared to 40% in rats fed the normal or the control basal diet. This inhibitory effect of 2-Me-DAB is reversible however since rats returned to the normal diet for 1 or 2 months after 2-Me-DAB feeding showed percentages of mitoses and labeling indices comparable to those of control animals following PH. The number of abnormal mitoses was high (13%) in regenerating livers of rats fed 2-Me-DAB and the lesions responsible for this effect are apparently not repaired since 2-Me-DAB fed rats partially hepatectomized after being transferred to the normal diet for 1 or 2 months showed the same number of mitotic irregularities. The present results suggest that assays with 2-Me-DAB as 'pure initiator' or agent of selective toxicity should be pursued in attempts to improve existing experimental models of hepatocarcinogenesis.     

Fas-mediated apoptosis is attenuated in preneoplastic GST-P-positive hepatocytes isolated from diethylnitrosamine-treated rats

Previous reports have indicated that apoptosis is selectively decreased in enzyme-altered foci (EAF) in the livers of rats treated with a carcinogen. Here we have investigated the effects of an anti-Fas antibody (anti-Fas Ab) on EAF cells in vitro. Hepatocytes were isolated from rats treated repeatedly with diethylnitrosamine (DEN), whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Subsequently, primary cultures of GST-P-positive and GST-P-negative hepatocytes were established and exposed to anti-Fas Ab. Anti-Fas Ab (4 g/ml) preferentially induced apoptosis in GST-P-negative cells. Furthermore, GST-P-positive cells were shown to be resistant to p53-mediated apoptosis. We conclude that EAF hepatocytes are resistant to Fas-mediated apoptosis in vitro. This lack of response may explain the selective decrease in apoptosis in EAF.     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.     

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.     

The effect of N-nitroso carcinogens and triazenes on protein synthesis in cell-free systems

The effect of a series of carcinogenic nitrosamines, alkylnitrosoureas and alkaryltriazenes on enzymatic reactions involved in protein synthesis was studied in cell-free systems from rat liver. The addition of most compounds stimulated the formation of aminoacyl-tRNA complex in test systems from rat liver whereas analogous preparations from Escherichia coli did not show this effect. The polymerization of phenylalanine and the binding of aminoacyl-tRNA to ribosome were only slightly and apparently non-specifically inhibited in the presence of the test compounds. On the other hand, the binding of nRNA to ribosome was markedly stimulated after the addition of most carcinogens tested. It appears that the carcinogens intervene specifically with one of the early steps in peptide initiation. Since the binding of mRNA to ribosome is known to be an important rate-limiting step in protein synthesis, the N-nitroso carcinogens and triazenes may thus control the expression of genetic message at the translation level.     

A characteristic electrophoretic pattern of cytosolic polypeptides from hepatocyte nodules generated during liver carcinogenesis in several models

The cytosolic polypeptides of hepatocyte nodules in six models of liver carcinogenesis were analysed by SDS-polyacrylamide gel electrophoresis and their patterns compared with these of control and variously treated livers. The amount of a polypeptide of Mr 21,000 was about tenfold elevated in the cytosol of five of the six types of nodules and moderately elevated in the sixth. Certain other polypeptides, particularly one of Mr 26,000, also varied in amount, so that all of the nodules analysed could be distinguished from liver by their electrophoretic patterns. Some possible identities of the two polypeptides are discussed. Their study may have mechanistic as well as diagnostic importance.     

多烯磷脂酰胆碱对肝癌大鼠肝细胞保护作用的研究

探讨多烯磷脂酰胆碱(PPC)对肝癌大鼠肝脏细胞的保护作用.将72只Wistar大鼠随机分为正常对照组、肝癌模型组、联苯双酯阳性对照组、PPC低、中、高剂量组6组,模型组、阳性对照组及PPC低、中、高剂量组均采用二乙基亚硝胺(DEN)诱导构建肝癌大鼠模型,低、中、高剂量PPC组在模型组的基础上分别给予100、200、300 mg·(kg·d)-1ppC灌胃,治疗6周后,腹主动脉取血测定血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)以及总胆红素(TBIL)含量,然后处死大鼠,测定肝脏湿重,计算肝脏指数,同时测定肝匀浆中丙二醛(MDA),超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平.结果显示,与正常对照组相比,DEN诱导的肝癌模型组中肝脏指数、AST、ALT、TBIL、MDA水平显著升高(p＜0.01);SOD、GSH-Px活力则显著下降(p＜0.01).PPC和联苯双酯能依耐性的逆转DEN所致的上述改变,表现为肝脏指数、AST、ALT、TBIL、MDA显著降低,SOD、GSH-Px活力则显著上升,PPC高剂量组作用最为明显,随着PPC摄入量的增加,PPC对肝癌大鼠肝细胞的保护作用也相应增强.