Biological significance of AFP-positive cells in rat livers with hyperplastic nodules induced by 3'-Me-DAB; combined immunocytochemical and radioautographical studies

We investigated alpha-fetoprotein (AFP)-positive cells in 3'-Me-DAB hepatocarcinogenesis. In the early stage of 3'-Me-DAB hepatocarcinogenesis immunoreaction of AFP was detected in proliferating cholangiolar "oval" cells and in the late stage, AFP-positive cells, differing from oval cells, were detected in clusters in rat liver with hyperplastic nodules. The clusters of AFP-positive cells were morphologically classified into two different types of foci and one type of nodule. The combined techniques of immunocytochemistry and ratioautography revealed that AFP-positive foci and nodule had more 3H-thymidine labeling index than did areas without AFP-positive cells in liver with hyperplastic nodules; also one type of AFP-positive foci had a higher labeling index than the other type and in AFP-positive nodules there was active cell proliferation; but AFP-positive foci or nodules had less 3H-thymidine labeling than AFP-positive hepatoma cells. These findings suggested that AFP-positive foci and nodule had different biological significance in development of hepatocellular carcinoma and that AFP-positive foci with higher labeling index and AFP-positive nodule should be surveyed as preneoplastic population.     

Polychlorinated biphenyl(s) as a promotor in experimental hepatocarcinogenesis in rats

Summary The effects of dietary polychlorinated biphenyls (PCBs) on hepatocarcinogenesis in female rats of Donryu strain receiving 3-methyl-4-dimethyl-aminoazobenzene (3-Me-DAB) were investigated. Oral administration of PCBs after administration of 3-Me-DAB resulted in a high incidence (64%) of hepatocarcinoma. In contrast, administration of a similar amount of PCBs before, or together with 3-Me-DAB did not induce hepatic tumors. Administration of a slightly larger amount of PCBs alone did not induce liver tumors, while administration of a slightly larger amount of 3-Me-DAB alone caused only a low incidence (13%) of hepatocarcinoma. These results strongly suggest that PCBs exert a potent promoting action in experimental azo dye hepatocarcinogenesis.This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education and from the Ministry of Health and Welfare, JapanThe authors thank Prof. K. Tanaka for gift of Kanechlor-400, and for treating the excreta to prevent environmental pollution by PCBs     

大鼠肝癌发生过程中癌基因和抑癌基因的表达

目的 探讨癌基因ｒａｓ和抑制基因Ｐ５３蛋白在实验性肝癌中的表达与内在联系。方法 应用原位杂交和免疫组织化学（ＳＡＢＣ０法,在３８例化学致癌剂二乙基亚硝胺（ＤＥＮＡ）诱发大鼠原发性肝细胞癌及癌前增殖结节中。Ｐ５３蛋白和ｒａｓ家族基因蛋白表达。结果 化学诱癌生成为６８．４２％,癌前增生性病例为３１．５８％,在癌与增生性病变中ｒａｓ基因收搞表达分别为８８．４６％和７５％,两组无显著差异,但与癌的恶笥分化     

Hyperplastic foci reflect the risk of multicentric development of human hepatocellular carcinoma

Background/Aims: Identification of the risk factors of multicentric hepatocarcinogenesis is important for the clinical management of hepatocellular carcinoma. We investigated hyperplastic foci in non-cancerous liver parenchyma, and clarified their pathological features and clinical significance.Methods: Hyperplastic foci were defined as hypercellular areas, which architecturally and cytologically resembled early hepatocellular carcinoma or adenomatous hyperplasia but did not form macroscopically detectable nodules. Surgically resected livers from 155 patients with hepatocellular carcinoma were examined histopathologically and immunohistochemically.Results: Hyperplastic foci were found in 26 of 155 patients (16.8%). All the patients with hyperplastic foci had chronic liver diseases, and the incidence did not differ between those with chronic hepatitis and those with liver cirrhosis. Six of 92 (6.5%) patients with single primary hepatocellular carcinoma nodules, 8 of 42 (19.0%) with two nodules, and 12 of 21 (57.0%) with more than three nodules had hyperplastic foci. The incidence of hyperplastic foci showed a significant positive correlation with the multiplicity of hepatocellular carcinoma nodules. Immunohistochemically, hyperplastic foci were masses of proliferative hepatocytes similar to adenomatous hyperplasia and early hepatocellular carcinoma.Conclusions: Hyperplastic foci reflect the risk of multicentric hepatocarcinogenesis. Our results suggest strongly that hyperplastic foci are precursors of adenomatous hyperplasia or hepatocellular carcinoma.     

Carcinogen-induced mdr overexpression is associated with xenobiotic resistance in rat preneoplastic liver nodules and hepatocellular carcinomas.

We have previously reported the isolation of a human breast cancer cell line resistant to doxorubicin (adriamycin; AdrR MCF-7 cells) that has also developed the phenotype of multidrug resistance (MDR). MDR in this cell line is associated with increased expression of mdr (P glycoprotein) gene sequences. The development of MDR in AdrR MCF-7 cells is also associated with changes in the expression of several phase I and phase II drug-detoxifying enzymes. These changes are remarkably similar to those associated with development of xenobiotic resistance in rat hyperplastic liver nodules, a well-studied model system of chemical carcinogenesis. Using an mdr-encoded cDNA sequence isolated from AdrR MCF-7 cells, we have examined the expression of mdr sequences in rat livers under a variety of experimental conditions. The expression of mdr increased 3-fold in regenerating liver. It was also elevated (3- to 12-fold) in several different samples of rat hyperplastic nodules and in four of five hepatomas that developed in this system. This suggests that overexpression of mdr, a gene previously associated with resistance to antineoplastic agents, may also be involved in the development of resistance to xenobiotics in rat hyperplastic nodules. In addition, although the acute administration of 2-acetylaminofluorene induced an 8-fold increase in hepatic mdr-encoded RNA, performance of a partial hepatectomy either before or after administration of 2-acetylaminofluorene resulted in a greater than 80-fold increase in mdr gene expression over that in normal untreated livers. This represents an important in vivo model system in which to study the acute regulation of this drug resistance gene.     

In vivo and in vitro test for growth potential of liver cells from rats during early stage of hepatocarcinogenesis by 3′-methyl-4-dimethylaminoazobenzene

Summary A rapid increase in the fraction of small liver cells was observed in the liver of rats during the early stage of hepatocarcinogenesis by 3-methyl-4-dimethylaminoazobenzene (3-Me-DAB). The change in cell population was represented by the decrease in glucose-6-phosphatase activity and by the increase in number of -glutamyltranspeptidase-positive cells. When DNA synthesis of liver cells from rats fed 3-Me-DAB was measured by autoradiography in primary culture, it began to increase 2 weeks after the start of the carcinogen feeding, reaching a plateau level after 3 weeks. Liver cells from rats fed 3-Me-DAB for 2 weeks or over demonstrated a remarkable resistance to the cytotoxic effect of the carcinogen (0.24 mM) in primary culture. Furthermore, liver cells from rats fed 3-Me-DAB for 3 weeks or over proliferated in the presence of the carcinogen in primary culture. When liver cells from 3-Me-DAB-fed and control rats were transplanted into syngeneic rat spleens, the former cells proliferated more vigorously than did the latter. The growth potential of liver cells from 3-Me-DAB-fed rats tended to be enhanced with time in the carcinogen feeding. Hepatocellular carcinomas developed in the host spleens implanted with liver cells from a rat fed 3-Me-DAB for 8 weeks. As described above, liver cells from rats fed 3-Me-DAB demonstrated much greater proliferative ability than normal control cells in vivo and in vitro.Abbreviations used HCC hepatocellular carcinoma - 3-Me-DAB 3-methyl-4-dimethylaminoazobenzene - GGT -glutamyltranspeptidase     

Survey of iron-accumulative macroregenerative nodules in cirrhotic livers

Recently, macroregenerative nodules in cirrhotic livers have been suspected to be among the putative precancerous lesions in human hepatocarcinogenesis. We examined the morphologies of 99 macroregenerative nodules in 44 cirrhotic livers with special emphasis on stainable iron. In 26 macroregenerative nodules (26%), stainable iron selectively accumulated within the macroregenerative nodules themselves, and little or no iron was found in the surrounding regenerative nodules. In nine macroregenerative nodules (9%), an appreciable amount of stainable iron was present in both the macroregenerative nodules and the surrounding regenerative nodules. In the remaining 64 macroregenerative nodules (65%), stainable iron was absent in both the macroregenerative nodules and the surrounding regenerative nodules. Hyperplastic hepatocellular foci were present within 19 (73%) of the 26 iron-accumulative macroregenerative nodules, one (11%) of the nine iron-positive macroregenerative nodules and 20 (31%) of the 64 iron-negative macroregenerative nodules. These findings suggest that iron-accumulative macroregenerative nodules are frequently associated with hyperplastic hepatocellular foci and should be included among the precancerous lesions in human hepatocellular carcinoma.     

Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.

A knowledge of the biological characteristics of carcinogen-induced hyperplastic nodules of rat liver may be important in the understanding of cancer development. Although its biological role remains to be elucidated, the level of microsomal epoxide hydrase (epoxide hydrolase, EC 3.3.2.3) is 5- to 7-fold greater in hyperplastic nodules nodules induced by feeding the hepatocarcinogen 2-acetylaminofluorene than in liver of control rats. After removal of the carcinogen from the diet, the high level of the enzyme is maintained in those nodules that persist and in the hepatocellular carcinomas that subsequently develop. The availability of antibody to the epoxide hydrase made it possible to use electron microscopic immunocytochemistry to localize this enzyme in the cells of hyperplastic nodules. The immunocytochemical procedure provides direct visual evidence for the presence of this enzyme in smooth endoplasmic reticulum and also in rough endoplasmic reticulum (including the nuclear envelope) of the nodule's parenchymal cells.     

Preneoplastic lesions of human liver

In a consecutive autopsy series of 95 males, parenchymal hyperplastic nodules of clear cells occurred in 11.6% and liver cell dysplasia (LCD) in 7.4%. These two possible preneoplastic lesions never occurred together. Of the men, 50.5% had been alcoholics or had drunk more than an average of 80 g of alcohol daily, and 65.3% had been smokers, as established by interviewing a family member or a close friend of the deceased. Hyperplastic nodules of clear cells were found in association with liver cirrhosis (p less than 0.005), liver enlargement (p less than 0.025), and heavy alcohol consumption (p less than 0.05), and tended (n.s.) to be associated with smoking. The largest nodules of clear cells were found in nodular regenerative hyperplasia of the liver in a male who had used testosterone. In another case focal nodular hyperplasia coexisted. Hyperplastic nodules showed no association with the occurrence of cavernous hemangioma or benign bile duct tumors. Males with hyperplastic nodules of clear cells were significantly (p less than 0.01) younger (average 49.3 years) than those with LCD (average 61.1 years). Males with LCD were slightly (p less than 0.05) older, tended to have a small-sized liver, but were otherwise comparable with males without precursor lesions. The present results indicate that both hyperplastic nodules and liver cell dysplasia are common findings in male autopsy series comprising alcoholics and smokers. Hyperplastic nodules were associated with some of the predisposing factors of primary hepatocellular carcinoma.     

Specific lectin bindings to oval cells and proliferated bile ductules

Histochemical evaluation of lectins was performed to examine the carbohydrate residues of oval cells induced by administration of α-naphthylisothiocyanate (ANIT), 2-acetylaminofluorene (2-AAF) and 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) in comparison with those of normal bile ducts and proliferated bile ductules induced by bile duct ligation. The normal bile ducts showed intense binding of Ricinus communis agglutinin, concanavalin A and wheat germ agglutinin, and weak binding of Ulex europaeus agglutinin I (UEA I). A few cells in the portal bile ducts showed binding of peanut agglutinin (PNA). Two different binding patterns were observed in oval cells and proliferated bile ductules. One group showed increased binding of PNA, while the other showed intense binding of UEA I. In both groups, binding of other lectins was similar to those of the normal bile duct. The first group included oval cells induced by 2-AAF and 3′-Me-DAB, and the second included both oval cells induced by ANIT and proliferated bile ductules induced by ligation. These results suggest that oval cells and proliferated bile ductules have their own specific carbohydrate residues and that oval cells induced by the carcinogens might be a cell population different from those induced by non-carcinogens and proliferated bile ductules by ligation. The authors wish to thank Dr. T. Itoshima and Dr. Y. Okada for their invaluable suggestions, and Mrs. T. Emi for her assistance in preparation of light microscopic examination.     

Genetic susceptibility to murine hepatocarcinogenesis is associated with high growth rate of NDEA-initiated hepatocytes

Summary The murine hybrids (C57BL/6JxC3Hf)F1 (B6C3) and (C57BL/6JxBALB/c)F1 (B6C), which have a high and low spontaneous and induced incidence of hepatocellular tumors, respectively, were treated with a single dose of NDEA at 1 week of age followed by TCPOBOP, a phenobarbital-like promoter of liver carcinogenesis, or by vehicle, and sacrificed at 30 weeks of age. The frequency per liver of hepatocellular nodules was similar in the two hybrids. However, in male mice the mean volume of nodules was about 10-fold greater in B6C3 than in B6C mice receiving NDEA followed by vehicle, and the treatment with TCPOBOP after NDEA stimulated nodule growth, with a much greater response in B6C3 mice. In female mice no differences in the mean volume of nodules were seen between hybrids after NDEA and vehicle, whereas upon NDEA and TCPOBOP treatment the mean volume of nodules was 25-fold greater in B6C3 than in B6C females. In addition, a few hepatocellular adenomas and carcinomas were observed, mostly in animals treated with NDEA and TCPOBOP, and they were 3-fold more numerous among B6C3 than B6C mice. TCPOBOP alone induced the same biochemical and hyperplastic effects in the liver of both hybrids. Using DNA probes homologous to Moloney murine leukemia virus, intracisternal A particle and virus-like 30S sequences, no correlation was apparent between the expression of any of these endogenous retroviral families and the strain susceptibility to hepatocarcinogenesis. We hypothesize that the different susceptibility to hepatocarcinogenesis between B6C3 and B6C mice is related to a higher growth rate of B6C3 than B6C initiated liver cells.Abbreviations used IAP intracisternal A particle - Mo-MuLV Moloney murine leukemia virus - NDEA diethylnitrosamine - TCPOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene - VL30 virus-like 30S     

Inhibitory effects of synthetic acidic retinoid and polyprenoic acid on the development of hepatoma in rats induced by 3'-methyl-N, N-dimethyl-4-aminoazobenzene

A study was conducted to investigate the inhibitory effects of acidic retinoid (trimethylmethoxyphenyl analog of retinoic acid ethylester or TMMP) and polyprenoic acid (3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid or E-5166) on the development of hepatoma induced by 3'-methyl-N, N-dimethyl-4-aminoazobenzene (3'-MeDAB) in rats. Morphometric analysis of liver specimens was employed to evaluate the antitumor effects of the compounds in detail, and revealed significant decreases in the number and area of tumors in the TMMP- and E-5166-treated groups. As for adverse effects, retarded growth and marked hypertriglyceridemia were observed only in TMMP-treated rats. During the hepatocarcinogenesis, cellular retinoid-binding protein, F-type or CRBP(F) and cellular retinoic acid-binding protein or CRABP newly appeared in the tumor tissue, particularly in hyperplastic nodules which are the precancerous state of hepatoma. These results suggest that the polyprenoic acid is a good candidate for clinical chemoprevention of hepatoma, targetting its precancerous stage when intracellular receptors for acidic retinoid have emerged.     

Combined supplementation of vanadium and beta-carotene suppresses placental glutathione S-transferase-positive foci and enhances antioxidant functions during the inhibition of diethylnitrosamine-induced rat liver carcinogenesis

BACKGROUND AND AIM: The present study was designed to investigate the chemopreventive effects of combined vanadium (V; 0.5 p.p.m.) and beta-carotene (BC; 120 mg/kg of basal diet) on diethylnitrosoamine (DEN)-induced and phenobarbital (PB)-promoted rat hepatocarcinogenesis. METHODS: All rats were subjected to two-thirds partial hepatectomy (PH) at the fourth week. After PH they were administered either trioctanoin alone (groups A', B', C' and D') or a single injection of DEN in trioctanoin at a dose of 10 mg/kg of body weight (groups A, B, C and D). Two weeks after the DEN treatment PB was administered (0.05% in basal diet) to all the DEN-treated rats and continued until the end of the experiment. Supplementation of V (groups B and B'), BC (groups C and C') or both V and BC (groups D and D') at the doses stated previously were started 4 weeks before DEN administration (at week 0) and continued until the 16th week. RESULTS: It was observed that in the DEN-treated and PB-promoted group (group A) the expression of the numbers and areas of the placental form of glutathione S-transferase (GST-P)-positive altered hepatic foci (AHF) was maximum. Treatment with V (group B) and BC (group C) significantly reduced the expression of GST-P-positive AHF by 29.5% and 42.8%, respectively. An additive protection action (65.7%) was noticed in group D, which received both V and BC for the entire period of the experiment. It was also observed that supplementation of V and BC for the entire period of the experiment significantly reduced the number and size of the hyperplastic nodules, while the combination treatment worked as an additive effect, reducing the number and size of the hyperplastic nodules to 22% from 89%. Moreover, a significantly reduced level of cytosolic glutathione (P < 0.001) and glutathione-S-transferase (P < 0.001) activity and stabilization of aerobic metabolism and hepatic architecture of the cells as compared with carcinogen control were observed in the V + BC-treated group. CONCLUSION: The present study suggests that V, an essential trace element, may be useful in combination with BC, an antioxidant, in the inhibition of experimentally induced rat hepatocarcinogenesis.     

Effects of malotilate on ethanol-inhibited hepatocyte regeneration in rats

The effects of malotilate, a hepatotrophic drug, on the inhibition of hepatocyte regeneration induced by ethanol were studied in rats. Following acute ethanol administration, both DNA synthesis and commencement of the S phase of the cell cycle in hepatocytes after partial hepatectomy were delayed. Malotilate prevents this ethanol-induced delay. Upon chronic ethanol administration, DNA synthesis after partial hepatectomy was reduced and commencement of the S phase was retarded. Treatment with malotilate clearly lessened the reduction in DNA synthesis in the resected liver, and retardation of the cell cycle was partially inhibited. These results indicate that malotilate prevents the ethanol-induced inhibition of hepatocyte regeneration at the dose tested and that this drug may be effective for the treatment of alcoholic liver disease.     

Effects of malotilate treatment on the serum markers of hepatic fibrogenesis in liver cirrhosis

Malotilate, a hepatotropic agent, was given to 39 cirrhotic patients for more than 32 weeks. The serial changes in the serum levels of hepatic fibrogenesis markers, such as procollagen type III N-terminal peptides (P-III-N-P) and immunoreactive prolyl hydroxylase beta-subunit (IR-BPH) were analyzed. Serum albumin levels, transaminase and choline esterase activities and the Normotest values were found to be significantly improved by malotilate treatment. The levels of both serum markers of hepatic fibrogenesis were also significantly reduced by malotilate. The prognoses of the decompensated liver cirrhosis patients treated with malotilate were significantly better than those who did not receive malotilate. These results indicate that the effects of malotilate on chronic liver diseases are not simply biocosmetic, but rather are related to an improvement in the basal changes of the liver, including a decrease in the fibrogenetic stimulus. These effects of malotilate improved the prognosis of liver cirrhosis.     

In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis

ＮＴＲＯＤＵＣＴＩＯＮＩｔｉｓｗｅｌｋｎｏｗｎｔｈａｔｓｅｘｈｏｒｍｏｎｅｐｌａｙｓａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｒｅｇｕｌａｔｉｏｎｏｆｃｅｌｇｒｏｗｔｈ,ｏｒｇａｎｄｅｖｅｌｏｐｍｅｎｔａｎｄｃａｒｃｉｎｏｇｅｎｅｓｉｓ．Ｂｕｔｔｈｅｍｏｓｔ...     

Changes of integrin expression in rat hepatocarcinogenesis induced by 3'-Me-DAB

AIM:To investigate the expression of integrins in rats liver during 3'-Me -DAB induced hepatocarcinogenesis and to find out the relationship between integrins and liver cancer metastasis.METHODS:The expressions of integrins alpha(1), alpha(2), alpha(3) and alpha(5) and epidermal keratin (EK) were observed by immunohisto chemical PAP method.RESULTS:In the normal liver tissues, hepatocytes express integrins alpha(1) and alpha(5) and in the bile duct epithlium, EK. In liver cirrhosis, hepatocytes highly express integrins alpha(1), alpha(2), alpha(3) and alpha5and in hyperplastic bile duct epithelium, integrins alpha(1), alpha(5) and EK. Expression of integrins alpha(1), alpha(2), alpha(3) and alpha(5) were obviously decreased in the preneoplastic nodules and primary carcinoma but expressions of integrins alpha(1) and alpha(5) in metastasis in the lung and diaphragma were higher than those in primary carcinoma.CONCLUSION:Integrins alpha(1) and alpha(5) may play a major role in chemically induced hepatocarcinogenesis and metastasis in rats.     

Detection of malotilate toxicity in vitro with peripheral blood mononuclear cells as targets. A preliminary report

An in vitro assessment of susceptibility to malotilate was made with peripheral blood mononuclear cells (PBMCs) as targets. PBMCs were incubated with malotilate in the presence or absence of the NADPH generating system and hepatic microsomes. Malotilate cytotoxicity to PBMCs, assessed by trypan blue dye exclusion and lactate dehydrogenase (LDH) release into the culture media, was found to be markedly increased by the addition of the NADPH generating system, indicating that metabolites play a significant role in toxicity. Once the significant role of malotilate metabolites in cytotoxicity was established, we applied this system to patients with suspected malotilate-induced liver injury. It was demonstrated that cytotoxicity was greater in PBMCs which were obtained from patients with chronic active hepatitis with a parallel malotilate-induced liver injury than in those from two different control groups; normal volunteers and patients with chronic active hepatitis who had had long-term malotilate treatment without any adverse reactions. This in vitro system seems to be of value in predicting potential malotilate toxicity in subjects who might be susceptible to this drug.     

Relationship between alpha-fetoprotein positive cells and precancerous lesions in rat liver during 3'-methyl-4-dimethylaminoazobenzene carcinogenesis.

Localization of characteristic precancerous lesions was compared with that of alpha-fetoprotein (alpha-FP) positive cells during hepatocarcinogenesis in Wistar rats fed 0.06% 3'-methyl-4-dimethylaminoazobenzene in basal diet. Proliferative activity of liver cells was simultaneously followed by deoxycytidylate deaminase. The first alpha-FP positive hepatocytes were found after 3 weeks of carcinogenesis in certain parts of the capsule 0.1--0.2 mm thick. In the seventh week of carcinogenesis their number increased and they were present in groups also subcapsularly. At this stage, hyperplastic islets began to be formed in the liver parenchyma. After 14 weeks of carcinogenesis, alpha-FP positive cell clusters and islets were also found in the deeper layers of liver parenchyma in regions with proliferation of transitory cells. Hyperplastic basophilic foci and islets occurred regularly, however, these were not identical with alpha-FP positive sites and their incidence was more frequent.