Decreased Expression of Bcl-x Protein during Hepatocarcinogenesis Induced Exogenously and Endogenously in Rats

Dysregulations of apoptosis have been widely recognized as important events in multi‐stage carcinogenesis. Bcl‐x, a member of the Bcl‐2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl‐x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short‐term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg/kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S‐transferase (GST‐P)‐positive, putative preneoplastic lesions were induced, and Bcl‐x protein expression was decreased in 24.7% of such lesions. The incidence of GST‐P‐positive lesions with decreased Bcl‐x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm², respectively. In GST‐P‐positive lesions larger than 0.3 mm², both apoptosis induction and cell proliferation activity were enhanced when Bcl‐x protein expression was decreased. In the long‐term exogenous models, rats were given 10 mg/kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg/kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl‐x protein expression. In the endogenous model, rats were fed a choline‐deficient, l ‐amino acid‐defined diet for 16 or 80 weeks and sacrificed. Bcl‐x protein expression was decreased both in GST‐P‐positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl‐x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.     

Inhibitory effects of lemon grass (Cymbopogon citratus,Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats

Effects of lemon grass extract (LGE) on hepatocarcinogenesis were examined in male Fischer 344 rats, administered diethylnitrosamine (DEN) at three weekly intraperitoneal doses of 100 mg/kg body weight and partially hepatectomized at the end of week 5. LGE was given at dietary concentrations of 0, 0.2, 0.6 or 1.8% from the end of week 4 for 10 weeks. All rats were sacrificed at the end of week 14. LGE reduced the number of putatively preneoplastic, glutathione S-transferase placental form-positive lesions and the level of oxidative hepatocyte nuclear DNA injury, as assessed in terms of 8-hydroxydeoxyguanosine production. In contrast, LGE did not affect the size of the preneoplastic lesions, hepatocyte proliferative activity, activities of phase II enzymes or hepatocyte extra-nuclear oxidative injury. These results suggest inhibitory effects of LGE on the early phase hepatocarcinogenesis in rats after initiation with DEN.     

Inhibition by Green Tea Extract of Diethylnitrosamine–initiated but Not Cholinedeficient, L–Amino Acid–defined Diet–associated Development of Putative Preneo–plastic, Glutathione S–Transferase Placental Form–positive Lesions in Rat Liver

The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline–deficient, L–amino acid–defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/Kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rate were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN–initiation, the apparent numbers of glutathione S–transferase placental form–positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8–hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA–diet–associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine–initiated hepato–carcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis–associated model.     

Cleistocalyx nervosum Extract Ameliorates Chemical-Induced Oxidative Stress in Early Stages of Rat Hepatocarcinogenesis

Purpose: To study the effect of Cleistocalyx nervosum extract (CE) on diethylnitrosamine (DEN) andphenobarbital (PB) induced oxidative stress in early stages of rat hepatocarcinogenesis. Materials and Methods:Male Wistar rats were divided into 4 groups, with Group 1 as a negative control and Group 2 was a positivecontrol receiving DEN injections once a week and PB in drinking water for 6 weeks. Two weeks before DENinitiation and PB treatment, Groups 3 and 4, were fed with 500 and 1000 mg/kg of CEs, respectively, for 8weeks. Results: A number of GST-P-positive foci, preneoplastic lesions, in the liver were markedly increased incarcinogen administered rats, but was comparatively decreased in rats treated with 1000 mg/kg of CE. The CEreduced malondialdehyde in serum and in the livers of rats treated with DEN and PB. Moreover, CE significantlyincreased the activities of glutathione peroxidase and catalase in rat liver. Conclusions: CE appeared to exertits chemopreventive effects by modulating antioxidant status during DEN and PB induced early stages ofhepatocarcinogenesis in rats.     

Inhibition by N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid of exogenous and endogenous development of putative preneoplastic, glutathione S-transferase placental form-positive lesions in the livers of rats

The effects of N-(4-hydroxyphenyl)retinamide (4-HPR) and all-trans- retinoic acid (tRA) on the exogenous and endogenous models of rat liver carcinogenesis respectively using diethylnitrosamine (DEN) and a choline-deficient, L-amino acid-defined (CDAA) diet were studied. For the exogenous study, male Fischer 344 rats, 6 weeks old, were given a single i.p. dose of 200 mg/kg body wt of DEN, partially hepatectomized at week 3, administered 4-HPR at doses of 0, 0.04, 0.08 and 0.16% or tRA at 0, 0.004, 0.008 and 0.015% in diet from week 2 for 6 weeks, and killed at the end of week 8. For the endogenous study, rats were fed the CDAA diet containing 4-HPR or tRA for 12 weeks and killed at the end of week 12. 4-HPR decreased the numbers and sizes of the glutathione S-transferase placental form-positive foci, assayed as putative preneoplastic lesions, the levels of 8-hydroxyguanine (8-OHG), a parameter of oxidative DNA damage, and the bromodeoxyuridine labeling indices (BrdU L.I.) by all three doses in the DEN-initiated case and, more prominently, in the CDAA diet-associated case. In contrast, while tRA failed to exert inhibitory effects apparently on foci development, 8-OHG formation or BrdU labeling in the DEN-initiated case, it reduced the numbers and sizes of the foci, the 8-OHG levels and the BrdU L.I. by all three doses in the CDAA diet-associated case. Furthermore, both 4-HPR and tRA inhibited the CDAA diet-associated induction of hepatocyte necrosis and connective tissue increase but not intrahepatocellular fat accumulation. These results indicate that 4-HPR exerts chemopreventive effects against the exogenous and endogenous rat liver carcinogenesis, while tRA can inhibit only the latter.      

Modulation of diethylnitrosarnine-initiated placental glutathione S-transferase positive preneoplastic and neoplastic lesions by clofibrate, a hepatic peroxisome proliferator

The effect of clofibrate (CF) on proliferation of diethylnitrosamine(DEN)-initiated glutathione S-transferase placental form (GST-P)-positivepreneoplastic and neoplastic lesions was studied in male F344rats. Animals were given a single i.p. injection of 200 mg/kgbody weight of DEN, and then from 2 weeks later were given adiet containing 0.3% CF (group 1), or no supplement (group 2)until week 64. Group 3 received an injection of 0.9% NaCl insteadof DEN and then a diet containing 0.3% CF, like group 1. Animalsin all groups were subjected to partial hepatectomy at week3 and killed at weeks 8, 20, 32, 49 or 64. The results showedthat development of GST-P-positive lesions was significantlyless in group 1 than in group 2 from week 8 (P < 0.05). However,in group 1, morphologically distinguishable GSTP-negative preneoplasticlesions increased from week 20 (P < 0.05), and the totalnumber of GST-P-positive and -negative lesions was significantlygreater than that in group 2 from week 32 (P < 0.05). Theinduction of hepatocellular carcinoma (HCC) was greater in group1 than in group 2 from week 49. All the HCCs induced in group2 were GST-P-positive, whereas 38.9% (7/18) of those in group1 were GST-P-negative. In group 3, only a few GST-P-positiveand/or -negative preneoplastic lesions developed by week 64.These results suggest that CF has tumor-promoting activity,and that GST-P-positive cells induced by DEN changed to GST-P-negativecells on subsequent treatment with CF.     

Suppressive effect of Astragalus membranaceus Bunge on chemical hepatocarcinogenesis in rats

Astragalus membranaceus (AM) has been widely used for treating liver diseases in traditional Chinese medicine. Experimental evidence indicates that it has antitumor potential. In this study, the effect of AM on hepatocarcinogenesis induced by diethylnitrosamine (DEN), two-thirds partial hepatectomy, and 2-acetylaminofluorene (2-AAF) (DEN-PH-AAF) was evaluated using glutathione S-transferase placenta form (GST-P) as marker. First, rats were injected intraperitoneally (i.p.) with DEN (200 mg/kg in saline), a two-thirds partial hepatectomy was carried out 2 weeks later, and the rats were then placed on a basal diet containing 0.02% AAF from week 3 to week 8 to induce hepatocarcinogenesis. The rats were given AM (90 mg/kg or 180 mg/kg body weight) by gavage from week 3 to week 8 (treatment groups). The formation of GST-P-positive foci and the expression of GST-P protein and mRNA caused by DEN-PH-AAF were reduced in the treatment groups, which clearly suggests that AM is effective in delaying DEN-PH-AAF-induced hepatocarcinogenesis.     

Suppression of glutathione S-transferase placental form-positive foci development in rat hepatocarcinogenesis by Chlorella pyrenoidosa

The modifying effects of dietary administration of dried Chlorella pyrenoidosa powder (C. pyrenoidosa) on the development of glutathione S-transferase placental form-positive foci (GST-P-positive foci), which are putative preneoplastic lesions, in male F344 rats were investigated using a medium-term liver bioassay system. In rats given 10% C. pyrenoidosa in a basal diet, the number and area of GST-P-positive foci in the rat livers, which diethylnitrosamine (DEN) initiated and 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) promoted, were significantly decreased compared with those fed a basal diet not containing C. pyrenoidosa. The inhibition percentage of the number and area of GST-P-positive foci > or =0.2 mm in diameter was 67.6 and 74.2%, respectively (p<0.01). Furthermore, C. pyrenoidosa significantly decreased the number of GST-P-positive foci induced by MeIQx alone. The inhibition percentage of the number of GST-P-positive foci <0.2 mm in diameter was 52% (p<0.01). These results suggest that C. pyrenoidosa has chemopreventive effects against hepatocarcinogenesis in rats. C. pyrenoidosa appears to be a promising chemopreventive agent for human liver neoplasia and carcinogenesis induced by heterocyclic amines such as MeIQx.     

Presence of a no-observed effect level for enhancing effects of development of the alpha-isomer of benzene hexachloride (alpha-BHC) on diethylnitrosamine-initiated hepatic foci in rats

The dose dependence of the promoting effects of the alpha-isomer of benzene hexachloride (alpha-BHC) on hepatocarcinogenesis was investigated in a medium-term rat liver bioassay (Ito test). A total of 195 F344 male rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at the start of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the administration of DEN, alpha-BHC were fed to rats at doses of 0, 0.01, 0.1, 0.5, 1, 2, 4, 7.5, 15, 30, 60, 125 and 500 ppm in diet for 6 weeks. All surviving animals were killed at week 8, and their livers were examined immunohistochemically for detection of glutathione S-transferase placental form (GST-P)-positive foci, surrogate preneoplastic lesions. Quantitative values for numbers and areas were dose-dependently increased in rats given alpha-BHC at 0.5-500 ppm. However, those for groups treated with 0.01 and 0.1 ppm were decreased, albeit not significantly in comparison to the controls. Cytochrome P450 3A2 (CYP3A2) protein levels and activities showed a good correlation to the number and area of GST-P-positive foci. These results support evidence of hormesis and indicate a no-observed effect level for alpha-BHC promoting potentials may exist regarding rat liver carcinogenesis, which correlates with expression of CYP3A2 in the liver.     

Inhibition of Early-phase Exogenous and Endogenous Liver Carcinogenesis in Transgenic Rats Harboring a Rat Glutathione S-Transferase Placental Form Gene

Hepatocarcinogenesis initiated with N -nitrosodiethylamine (DEN) and that initiated by feeding of a choline-deficient, l -amino acid-defined (CDAA) diet were compared in transgenic male Wistar rats harboring a rat glutathione S -transferase placental form ( GST-P ) gene (GST-P-Tg rats) and non-transgenic (N-Tg) rats. Eight-week-old GST-P-Tg and N-Tg rats were administered DEN intraperitoneally at 100 mg/kg body weight, subjected to a selection procedure with 2-acetylaminofluorene and CCl₄, and killed at the end of weeks 5 and 12. Other groups were fed the CDAA diet for 12 weeks and killed. Five weeks after the DEN treatment, numbers and sizes of γ-glutamyltransferase (GGT)- or GST-P-positive lesions and 8-hydroxyguanine (8-OHG) levels in the livers were significantly less in GST-P-Tg rats than in N-Tg rats. The lesion numbers were unchanged between the ends of weeks 5 and 12 in GST-P-Tg rats, but decreased in N-Tg rats. The lesion sizes were increased in GST-P-Tg rats, but unchanged in N-Tg rats. While the proliferating cell nuclear antigen labeling indices (PCNA L.I.) in and surrounding the lesions were decreased, more prominently in GST-P-Tg rats than in N-Tg rats, the 8-OHG levels were also decreased but similarly in both cases. After 12 weeks on the CDAA diet, the lesion incidences, numbers and sizes, 8-OHG levels, PCNA L.I. in and surrounding the lesions, and liver injury were significantly less in GST-P-Tg rats than in N-Tg rats. These results indicate that insertion of a rat GST-P transgene alters the early phase of exogenous and endogenous rat hepatocarcinogenesis, presumably due to enhanced detoxification by GST-P expressed both transiently during the initiation and chronically in the altered hepatocyte populations.     

Reduction of hepatocarcinogenesis by ursodeoxycholic acid in rats

Ursodeoxycholic acid (UDCA) is used worldwide for treatment of primary biliary cirrhosis and chronic liver diseases. However, its action on hepatocarcinogenesis remains to be explored. To clarify its effect, in vivo and in vitro experiments were performed. Ninety Fisher 344 rats were fed a standard diet (Group 1, n = 30), a standard diet supplemented with 0.1% UDCA (Group 2, n = 30) and 0.3% UDCA (Group 3, n = 30). The rats were given an i.p. injection of diethylnitrosamine (DEN) weekly for 6 weeks. Fifteen additional rats were fed 0.3% UDCA supplemented diet without DEN treatment (Group 4). The rats were killed at 5, 10 and 18 weeks after the last injection of DEN. The number of liver tumor and percentage of the GST-P-positive hepatocytes were significantly reduced by UDCA treatment. The PCNA-positive cells were decreased by administration of UDCA at 18 weeks. The increased number of apoptotic cells was observed in the GST-P-negative area at 5, 10 and 18 weeks and in the GST-P-positive area at 18 weeks in the UDCA group. Expression of Bax in mitochondria and cytochrome c in cytosol was increased by UDCA treatment. Caspase 3 activity was also increased in the UDCA groups. The addition of UDCA into the culture of Huh7 and Fao hepatocellular carcinoma (HCC) cells induced apoptosis in a dose-dependent manner. The data of the present study suggest that UDCA treatment reduces hepatocarcinogenesis via inducing apoptosis of 'initiated hepatocytes' as well as inhibiting proliferation.     

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of gamma-glutamyltransferase in rat liver preneoplastic and neoplastic lesions

Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.     

Dose-dependent enhancing effects of quinacrine on induction of preneoplastic glutathione S-transferase placental form positive liver cell foci in male F344 rats

Dose-dependent modifying effects of quinacrine on induction of preneoplastic liver cell foci were investigated in male F344 rats. Six week old animals were injected i.p. with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg, and starting 2 weeks later, rats were given quinacrine at dietary levels of 20, 100 and 500 p.p.m. for 6 weeks. Groups without either DEN or quinacrine treatment were used as controls. At week 3 following DEN administration, all animals were subjected to two-thirds partial hepatectomy, and after killing the animals at week 8, development of preneoplastic liver cell foci was investigated using the glutathione S-transferase placental form (GST-P) as a marker. The numbers and unit areas of GST-P-positive foci per cm2 were significantly increased in the DEN/quinacrine (500 p.p.m.) group as compared to DEN-alone group values. An increase in number was also evident in the 100 p.p.m. but not the 20 p.p.m. treated group, no lesions being induced by quinacrine alone (500 p.p.m.). Electron microscopic study confirmed that quinacrine dose-dependently induces lipidosis in hepatocytes, i.e. markedly myeloid lamellar cytoplasmic inclusion bodies were observed. The results thus demonstrated that quinacrine treatment enhances GST-P-positive liver cell foci development in a dose-dependent way, this effect presumably being related to the induction of lipidosis.     

Chemoprevention by amiloride against experimental hepatocarcinogenesis induced by N-nitrosomorpholine in Sprague-Dawley rats

The effects of prolonged administration of the diuretic amiloride on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and the labeling index of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks and received s.c. injections of 2.5 mg/kg or 5.0 mg/kg body weight of amiloride every other day until the end of the experiment at week 16. Preneoplastic and neoplastic lesions staining positively for glutathione-S-transferase, placental type (GST-P) were examined immunohistochemically. In week 16, administration of amiloride at 2.5 mg/kg body weight significantly reduced the size (as mean area) of GST-P-positive lesions, and amiloride at 5.0 mg/kg body weight significantly reduced the number (as no./cm2) and sizes (as mean area and percent of parenchyma) of GST-P-positive lesions. Amiloride at both dosages also significantly decreased the labeling index of enzyme-altered lesions and amiloride at higher dosage significantly decreased the labeling index of adjacent hepatocytes. These findings indicate that amiloride inhibits hepatocarcinogenesis and suggest that this effect may be closely related to amiloride's inhibition of cell proliferation in enzyme-altered lesions and/or adjacent liver.     

Possible tumor development from double positive foci for TGF-alpha and GST-P observed in early stages on rat hepatocarcinogenesis

Expression of TGF-alpha during promotion of neoplastic development from GST-P-positive foci in rat chemical hepatocarcinogenesis was investigated. One-hundred male F344 rats were given a single intraperitoneal injection of DEN (200 mg/kg bodyweight) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0 or 500 p.p.m. was fed to the rats for 46 weeks. Groups of 10 rats were killed at weeks 4, 8, 16, 32, 48 and their livers were immunohistochemically examined for expression of GST-P and TGF-alpha. TGF-alpha-positive foci and single positive cells were observed from week 4, partially overlapping with GST-P-positive foci but being much fewer. Numbers of TGF-alpha-positive lesions did not increase from weeks 4-48, but their areas showed increment at weeks 32 and 48, especially with PB administration. Almost all of the tumors observed at weeks 16, 32 and 48 were positive for TGF-alpha (98%). In addition, epidermal growth factor receptor overexpression was observed in most TGF-alpha-positive lesions (foci and tumors). The proliferating cell nuclear antigen labeling index in double positive foci for GST-P and TGF-alpha was significantly higher than that in TGF-alpha-negative foci. In conclusion, TGF-alpha may be closely related with promotion from altered foci to neoplasms in rat hepatocarcinogenesis. Our data suggest that double positive foci for GST-P and TGF-alpha in the early stages of rat hepatocarcinogenesis may develop into tumors with promotion.     

Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis: direct measurement of communication in vivo

We have developed a simple method to measure gap-junctional intercellular communication (GJIC), by means of microinjection/dye transfer assay, in liver slices freshly removed from the rat. Using this method and immunostaining of connexin 32 (cx32), the major liver gap junction protein, we studied sequential changes of GJIC during chemical hepatocarcinogenesis in male Fischer-344 rats under a modified Solt-Farber protocol (3 weeks 4 day exposure regimen). Four weeks after commencement of the protocol, there was a substantial decrease in GJIC in the liver parenchyma, which was free from focal lesions. The decrease in GJIC persisted up to at least the 15th week of treatment, while a decrease in the number of immunoreactive cx32 spots was evident only at 4 weeks of post-protocol commencement. Most enzyme-altered (GST-P-positive) focal lesions showed markedly lower GJIC and a significantly lower number of cx32-positive spots than surrounding hepatocytes. Most GST-P-positive foci showed a selective lack of GJIC with surrounding heptocytes. Hepatocellular carcinomas arising 1 year after the carcinogenic regimen had significantly reduced communicational capacity accompanied by a large decrease in cx32 expression. These results suggest that a progressive decrease in homologous as well as heterologous GJIC in preneoplastic lesions occurs during rat hepatocarcinogenesis, and that preneoplastic lesions with the most prominent disorders in GJIC may be more likely to develop into carcinomas.     

突变型p53蛋白在二乙基亚硝胺诱发大鼠肝癌前病变中的表达

目的　探讨 p53基因与肝细胞癌变的关系。方法　用免疫组化方法 ,观察二乙基亚硝胺( DEN)诱发大鼠肝癌前病变组织及对照组肝组织中突变型 p53( mp53)蛋白、胎盘型谷胱甘肽 S转移酶 ( GST- P)的表达及其与细胞增殖状态的关系。结果　实验组第 5周时仅在 2 / 5例癌前病变肝组织中检测到 mp53蛋白的表达 ,此蛋白仅存在于部分较大的肝细胞增生结节 ( HN)或 GST- P阳性灶内 ;mp53蛋白阳性的 HN或 GST- P阳性灶 ,其细胞的溴化脱氧尿嘧啶标记指数 ( Brd U- LI)较阴性者高( P<0 .0 5)。结论　 p53基因的突变可发生于肝细胞癌变的早期阶段 ,与肝细胞的异常增殖和癌变有关 ,并有可能成为新的肝癌前肿瘤基因标记物。     

An acyclic retinoid, NIK-333, inhibits N-diethylnitrosamine-induced rat hepatocarcinogenesis through suppression of TGF-alpha expression and cell proliferation

The present study was designed to determine the effects of NIK-333, a synthetic acyclic retinoid, on N-diethylnitrosamine (DEN)-induced hepatocarcinogenesis in male F344 rats. Animals were given DEN dissolved in drinking water at a concentration of 40 p.p.m. for 5 weeks and then provided with drinking water free of DEN for 15 weeks to induce hepatocellular neoplasms. NIK-333 was administered orally (once a day) to rats at doses of 10, 40 and 80 mg/kg body wt for 14 weeks, starting 1 week after the completion of administration of DEN. At 20 weeks after the start of DEN administration, histopathological evaluation was carried out on all animals. The effects of NIK-333 on the cell proliferation activity of non-tumorous areas and liver tumor cells and the immunohistochemical expression of transforming growth factor-alpha (TGF-alpha) were also evaluated. NIK-333 at 40 and 80 mg/kg body wt significantly inhibited hepatocarcinogenesis (P < 0.05). In addition, NIK-333 at the same doses decreased DEN-induced overexpression of TGF-alpha in hepatocellular neoplasms (adenomas and carcinomas) and their surrounding tissue. Furthermore, NIK-333 significantly inhibited cell proliferation activity in the lesions and in non-tumorous areas (P < 0.01). Our results suggest that NIK-333 inhibits DEN-induced hepatocarcinogenesis through suppression of TGF-alpha expression and cell proliferation.     

Enhancement of GST-P-positive liver cell foci development by nivalenol, a trichothecene mycotoxin.

In order to elucidate whether T-2 toxin (T-2) and nivalenol (NIV), the naturally occurring trichothecene mycotoxins in food and feed, are carcinogenic or possess an ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity, a medium-term liver bioassay was carried out. F344 male rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg), and then fed the test trichothecenes in diet (2 and 5 p.p.m. T-2 or 6 p.p.m. NIV) for 6 weeks beginning 2 weeks after the injection. Some control groups received DEN alone. For synergism between AFB1 and the trichothecenes, DEN-initiated rats as above were given a single i.p. injection of AFB1 (0.5 mg/kg) 2 weeks later and were fed a NIV-containing diet (6 p.p.m.) for 6 weeks. The other control group received the vehicle alone. Control rats not initiated with DEN were also treated with AFB1, NIV or T-2 alone as above. All rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and killed at week 8, and liver sections were analyzed by glutathione S-transferase placental form (GST-P) expression. In rats that did not receive DEN, AFB1 alone enhanced both the numbers and areas of GST-P-positive foci as reported earlier, while NIV or T-2 alone induced no marked changes. In rats initiated with DEN, AFB1 caused a marked expression of GST-P, and thus the hepatocarcinogenicity of AFB1 was reconfirmed. The expression of GST-P foci in rats fed T-2 or NIV was found to be at background level, indicating that the hepatocarcinogenicity was not predicted for the trichothecene mycotoxins such as T-2 and NIV by this medium-term bioassay system. In the group initiated by DEN followed by AFB1, on the other hand, an elevation of both the numbers and areas of GST-P-positive foci was observed by the subsequent feeding of rats with NIV, and this elevation was statistically significant from the sum totals of individual data of AFB1 or NIV alone. From this evidence, it is predicted that NIV causes an enhancing effect on AFB1-induced hepatocarcinogenesis.