Pre- and post-initiation modulating effects of green tea ingestion on rat hepatocarcinogenesis

The purpose of this study was to investigate the effects of green tea ingestion on hepatocarcinogenesis before and after its initiation. Male Sprague-Dawley rats were fed an AIN76A diet with or without green tea. Initiation was induced by a single dose (200 mg/kg) of diethylnitrosamine at week 4 and 0.02% (w/w) 2-acetylaminofluorene was supplied in the diets. The control group had free access to water for 13 weeks (CTR13). Tea infusion was provided from the beginning of the experiment for 13 weeks (PRE13) or from the post-initiation stage until week 13 (POST13). Three other groups (CTR24, PRE24 and POST24) were added to examine the longer-term effects (24 weeks) with the same experimental design. The percentage area of liver sections that were positive for hepatic placental glutathione S-transferase (GST-P), which was used as a marker of preneoplastic lesions, was smaller in PRE13 (20.2 +/- 5.0%, mean +/- SD) and POST13 (26.0 +/- 4.8%) than in CTR13 (33.2 +/- 5.8%, p<0.05). Over the longer period, the GST-P lesions were significantly smaller for both PRE24 and POST24 (21.6 +/- 8.5% and 22.2 +/- 4.0%, respectively) than for CTR24 (28.6 +/- 5.1%, p<0.05), but there was no significant difference between PRE24 and POST24. The liver content of thiobarbituric acid reactive substances was significantly lower in the tea groups than in the controls (p<0.05). However, no significant differences were observed among groups of GST activity. The results show that tea consumption exhibits a stronger short-term initiation-inhibiting ability in liver carcinogenesis, but over a longer period, the preventive effects of green tea ingestion do not differ in post- and pre-initiation.

Effects of Vitamin E and Dehydroepiandrosterone on The Formation of Preneoplastic Lesions in Rat Hepatocellular Carcinogenesis

This study is designed to examine the effects of dietary supplementation with vitamin E and dehydroepiandrosterone (DHEA) on the formation of preneoplastic lesions in diethylnitrosamine (DEN) induced rat hepatocarcinogenesis. All Weaning male Sprague-Dawley rats were initiated by a single dose of DEN (200 mg/kg body weight), subjected to twothirds partial hepatectomy 3 weeks later and were sacrificed 8 weeks after DEN initiation. Two weeks after initiation, rats were fed Purina purified rodent diet 5053 (Ralston Purina Rat chow, USA) with 1.5% (15,000 IU/kg diet) vitamin E, 0.5% DHEA and both of those supplemented diet for 6 weeks. Placental glutathione S-transferase (GST-P) positive foci, the activities of catalase, total-glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and thiobarbituric acid reactive substances (TBARS) contents were decreased significantly by vitaimin E supplement. On the other hand GST-P positive foci number, Cu/Zn-superoxide dismutase (SOD) and glucose 6-phosphatase (G6Pase) activities weren't changed by vitamin E supplement. It might suggest that protective effect of vitamin E against hepatocarcinogens is not involved in the formation of the GST-P positive foci but related to the expansion of that. It seemed that vitamin E supplement helped endogenous defense system in carcinogenesis by decreasing TBARS contents, H2O2, organic peroxides. Therefore, vitamin E seemed to protect cell from free radical damage in carcinogenesis . By DHEA supplement liver weight and liver/body ratio were increased, the area and number of GST-P positive foci, the activities of catalase, GR, total GPx, GST and the TBA RS contents were decreased significantly. On the other hand Cu/Zn-SOD and G6Pase activities weren't changed by DHEA supplement. In hepatocarcinogenesis the activities of antioxidant enzymes weren't increased by DHEA supplement. DHEA did not increase the oxidative stress, while DHEA seems to have anticarcinogenic effect in rats hepatocarcinogenesis.

Effects of Dietary Levels of Corn and Tuna Oils on the Formation of Preneoplastic Lesions in Rat Hepatocellular Carcinogenesis

This study is conducted to determine the effects of dietary levels of corn and tuna oils on the formation of preneoplastic lesions in die-thylnitrosamine (DEN) induced rat hepatocarcinogenesis. Weanling male Sprague-Dawley rats were fed 2.5, 5, 15, 25% (w/w) corn or tuna oils. Hepatocellular carcinogenesis was induced by DEN (200 mg/kg body weight) and two-thirds partial hepactectomy was carried out 3 weeks later and were sacrificed 8 weeks after DEN initiation. Tuna oil group showed smaller area of placental glutathione S-transferase (GST-P) positive foci than corn oil group. Corn oil group of 25% (w/w) showed the widest area of GST-P positive foci, and tuna oil group showed significantly smaller area of GST-P positive foci than corn oil in 25% (w/w) level but had no differences between oil levels. Thiobarbituric acid reactive substances (TBARS) content was the highest in 25% (w/w) level of tuna oil group fed long chain and highly polyunsaturated fatty acids. Also serum gamma-glutamyltranspeptidase (GGT) activities in 25% level of tuna oil group were significantly higher than by other levels. As oil contents increased, glucose 6-phosphatase (G6Pase) seems to decrease in corn oil groups but remained the same in tuna oil groups. Glutathione reductase (GR) activities were significantly higher in tuna oil group, and the higher the level of tuna oil, the higher GR activities. But Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities didn't seem to be influenced by levels and kind of dietary fats. Therefore, as oil levels increased, corn oil rich in n-6 fatty acids promoted carcinogenesis but tuna oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of n-3 fatty acids suppressed. Although lipid peroxidation products were elevated in 25% (w/w) tuna oil group, GST-P positive foci didn't increase. Therefore preneoplastic lesions might be reduced through mediation of a lipid peroxidation process in tuna oil. As fat contents of tuna oil increased, elevated GR activities may give a rise to produce more reduced glutathione in order to protect against free radical attack, and high G6Pase activities remained the same and they contributed to membrane stability. So tuna oil diet seems to protect hepatocarcinogenesis.

Effect of diet on aflatoxin B1-DNA binding and aflatoxin B1-induced glutathione S-transferase placental form positive hepatic foci in the rat

Effects of diets on hepatic aflatoxin B1 (AFB1)- DNA binding and AFB1-induced glutathione S- transferase placental (GST-P) form positive hepatic foci have been examined in young male Fischer rats. Animals were fed either AIN-76A or Purina Chow (PC) diet for 1 wk before AFB1- DNA binding studies in vivo and in vitro. Animals were injected i.p. with AFB1 (1 mg/kg body wt) and 3 days later were given either AIN-76A or PC diet with or without 0.1% phenobarbital (PB) in their drinking water. All animals were sacrificed 10 wks after AFB1 dosing for analysis of AFB1-induced GST-P positive hepatic foci by immunochemistry. Two h after i.p. injection of AFB1, hepatic AFB1-DNA binding in AIN-76A fed rats was twice as much as those in PC fed animals without affecting GSH levels. There was no significant effect of diet on either cytochrome P-450 content, GSH levels or microsomal cytochrome P-450 mediated AFB1-DNA binding to exogenous DNA. There was a 40% increase in cytosolic GSH S-transferase activity with 1-chloro-2,4-dinitrobenzene as a substrate in PC fed animals compared to those given AIN- 76A diet. The number and area of AFB1-induced GST-P positive hepatic foci were twice and fivefold as much in AIN-76A fed compared to those in PC fed rats. The number of AFB1-induced GST-P positive foci was increased 5-10 fold in the presence of PB in both groups. In summary, the present data indicate that feeding of PC diet compared to AIN-76A diet inhibits the initiation phase whereas AIN-76A stimulates the promotion phase of AFB1 hepatocarcinogenesis in rats by inhibiting AFB1-DNA binding and increasing AFB1-induced hepatic foci respectively.

Different Sources of omega Fatty Acids at the Fixed Ratio of p/s Affect Glutathione Dependent Enzymes in Rat Hepatocarcinogenesis

This study is conducted to determine the effects of dietary source of omega3 fatty acids on preneoplastic foci and the glutathione dependent enzymes in rat hepatocarcinogenesis initiated by diethylnitrosamine (DEN). Male Sprague-Dawley rats were fed one of three diets containing 10% (w/w) fats fixed p/s = -1.0 and omega6/omega3 ratio = -0.4 or 4.0 ; fish oil-com oil blended (FC), com oil-beef tallow-fish oil blended (CF), com oil-beef tallow-perilla oil blended (CP), from gestation period. At 10 weeks, animals of experimental groups were injected intraperitoneally with DEN (200 mg/kg body weight) and two-thirds partial hepatectomy was carried out 3 weeks later and were sacrificed 8 weeks after DEN initiation. The area and number of glutathione S-transferase placenta (GST-P) positive foci were significantly decreased in rats fed diets containing fish oil (FC and CF) than those fed perilla oil diet (CP). Fish oil feeding significantly increased the activities of glutathione dependent enzymes. Rats fed diets containing fish oil (FC and CF) significantly increased the glutathione (GSH) content and the activities of glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST). Glutathione dependent enzymes had significantly negative correlation with GST-P positive foci. Glucose 6-phosphatase (G6Pase) was increased in rats feeding fish oil. Thiobarbituric acid reactive substances were not different among groups. Therefore, the preventive effect against hepatocarcinogenesis might be explained by induction of the glutathione dependent enzymes and G6Pase.

Expression of Glutathione S-Transferase, E-Cadherin, and Catenins during N,N-Diethylnitrosamine-Induced Hepatocarcinogenesis in Rat Liver

N,N-Diethylnitrosamine (DEN) has been proved to have carcinogenic potential in the initiation or promotion stage and the transformed cells proliferate to form preneoplastic nodules which are positive for placental form of glutathione S-transferase (GST-P). E-Cadherin, a member of the cadherin family, is expressed in epithelial cells. To evaluate the role of adhesion molecules (E-Cadherin, alpha-catenin, and beta-catenin), which have not been well understood in carcinogenesis, we investigated the changes of E-cadherin, alpha-Catenin and beta-Catenins by immunohistochemistry and immunoblotting in DEN-induced hepatocarcinogenesis of rat liver. In addition, the sequential analysis of histopathology and the expression of GST-P were also examined. Immunoreactive areas for GST-P were gradually increased from early period of carcinogenesis and strong GST-P positive foci were noted in various lesions, especially in the clear cell and eosinophilic cell nodules. Immunohistochemically, the E-Cadherin expression was increased in DEN-treated preneoplastic nodules in 4 and 10 weeks and hepatocellular carcinomas displayed relatively reduced expression compared with the hyperplastic nodules. But alpha- and beta-catenin expression was increased in hyperplastic nodules and hepatocellular carcinomas. Immunoblotting studies revealed that the level of alpha-catenin (cytosol and membranous fraction) was overexpressed in hyperplastic nodules as well as hepatocellular carcinomas, which showed markedly increased expression. The membranous fraction of beta-catenin was markedly increased in 10 weeks of DEN treatment and slightly reduced in hepatocellular carcinomas. These findings suggest that during DEN-induced hepatocarcinogenesis, the clear cell and eosinophilic cell nodules expressing GST-P in their cytoplasm are early transformed cell nodules. The altered expression of E-Cadherin and catenins is closely related with tumor propagation. Loss or reduced expression of E-cadherin may play a role in the progression of late hyperplastic nodule to hepatocellular carcinoma in DEN-induced rat hepato carcinogenesis.

The regulatory mechanism of phosphatidylinositol 3-kinase by insulin in 3T3 L1 fibroblasts: phosphorylation-independent activation of phosphatidylinositol 3-kinase

Phosphatidylinositol (PI) 3-kinase plays an important role in transducing the signals of various growth factor receptors. However, the regulatory mechanism of PI3-kinase activity by these growth factor receptors is not completely understood. Therefore, we attempted to clarify the regulatory mechanism of PI3-kinase using insulin and 3T3 L1 fibroblasts. Our results showed that insulin stimulated PI3-kinase activity seven-fold and concomitantly phosphorylated a p85 subunit at the tyrosine residue. However, this tyrosine phosphorylation was not significant in the activation of PI3-kinase as the PI3-kinase pulled down by the overexpressed GST-p85 fusion protein showed as high an activity as the immunoprecipitated one. The p110 subunit was phosphorylated at both serine and tyrosine residues without insulin treatment. Since the phosphorylation state was not changed by insulin. The results suggested that phosphorylation of the p110 subunit does not control PI3-kinase activity. Finally, it was shown that the insulin receptor substrate-1 (IRS-1) binding to PI3-kinase was not sufficient for full activation because the amount of IRS-1 pulled down by the GST-p85 fusion protein reached almost maximum, after incubation with insulin-treated cell lysates for 20 min, whereas PI3-kinase activity reached its maximum only after incubation for 5 h. All results suggest that the phosphorylation of p85 subunit at tyrosine residues and phosphorylation of p110 subunit at tyrosine or serine residues are not functionally significant in the regulation of PI3-kinase activity. They also suggest that P13-kinase is needed to bind to other protein(s) as well as the insulin receptor substrate-1 for full activation.

Promoting Effect of Aflatoxin B1 and D-Galactosamine on Development of Glutathione S-Transferase Positive Foci in Diethylnitrosamine-initiated Rat Liver

The enhancing potential of anatoxin a (AFB1) and D-galactosamine (DGA) on development of preneoplastic glutathione S-transferase placental form positive (GST-P+) hepatic foci was examined using an in vivo mid-term assay system based on two-stage concept of hepatocarci-nogenesis. Rats were initially given a single dose (200 mg/kg) of diethylnitrosamine (DEN) intraperi-toneally, and thereafter. with an interval of 2 weeks, AFBl at a graded concentration (0.06, 0.012, 0.0024, 0.00048, and 0.000096 mg/kg i.g.) and DGA (100 mg/kg i.p.) were administered for 6 weeks and then sacrificed. All rats were subjected to a two-thirds partial hepatectomy to induce a potent growth stimulus to DEN-altered hepatocytes at the week 3. The modifying potential was scored by comparing the number and the area (mm2) per cm2 of GST-P+ foci in the liver with those of the corresponding control group given DEN alone. AFBl (at a graded concentration between 96 ng/kg and 60 microgram/kg) exerted a strong promoting effect oil induction of GST-P+ foci with both the number and the area. The logarithmic dose of AFBl and the potency to promote hepatocarcinogenesis were in dose-dependent relationship. DGA, a known necrogenic chemical to cause periportal necrosis and stimulate hepatocellular proliferation. also revealed the increase in the area of GST-P+ foci. although its enhancing potentia1 was 1ess profound than that of AFBl. The results suggest that DGA is also a useful proliferative stimulus m improve the medium-termdetection of unknown carcinogens.

The Enhancing Effect of Ethanol on the Development of Glutatione S-Transferase Placental Form-Positive Foci Induced by Diethylnitrosamine in F344 Rat

The effects of ethyl alcohol and pig serum administration on the development of preneoplastic hepatic enzyme-altered foci were examined in an in vivo mid-term assay system. Rats were initially given a single dose (200 mg/Kg) intraperitoneal injection of diethylnitrosamine (DEN). Two weeks later, treatment was started with 10% ethanol + 10% sucrose solution, 10% sucrose solution, or tap water as drinking water for 6 weeks with or without intraperitoneal injection of porcine serum twice a week. All rats were subjected to a two-thirds partial hepatectomy at week 3. The modification potentials were evaluated by comparing the number and area per cm2 of glutathione S-transferase placental form-positive (GST-P+) foci in the liver of each group. As a result, ethanol significantly enhanced the development of GST-P+ foci. Unfortunately, the porcine serum injection produced no hepatic fibrosis and no significant alteration in GST-P+ foci.

Cancer promoting effects of N-nitrosodiethylamine (DEN) and 2-acetylaminofluorene (2-AAF) with medium term rat liver bioassay / 中国药理学通报

AIM To study the cancer promoting effects of N nitrosodiethylamine (DEN) and 2 Acetylaminofluorene (2 AAF). METHODS Medium Term Rat Liver Bioassay (MTRLB). Male SD rats were initially given a single dose (200 mg?kg -1 ) of DEN ip and starting 2 weeks later, were treated with 10, 33 and 100 ppm DEN in drinking water, or with 2 2, 6 6 and 22 mg?kg -1 2 AAF by gavage for 6 weeks. All rats were subjected to two thirds partial hepatectomy at week 3 and killed at the end of week 8. Carcinogenic potential was scored by comparing the numbers and areas in induced glutathione S transferase placental form (GST P) positive foci in the liver with those of corresponding control group given DEN alone. RESULTS Both DEN and 2 AAF caused the increases of the numbers and areas of GST P positive foci in the liver, and showed dose response relationship. CONCLUSION Both DEN and 2 AAF shows cancer promoting effects, and MTRLB was a convenient, economical and effective tool to study the cancer promoting effects of test chemicals.