Inhibitory effects of S-methylcysteine and cysteine on the promoting potential of sodium phenobarbital on rat liver carcinogenesis.

The effects of S-methylcysteine (SMC) and cysteine on the promotion stages of rodent hepatocarcinogenesis in a medium-term bioassay previously developed by Ito were examined. Initiation was induced by a single dose of diethylnitrosamine (DEN), followed by dietary administration of the promoter sodium phenobarbital (NaPB) 2 weeks later, for 6 weeks. Partial hepatectomy was conducted on all the animals at week 3. Inhibitory potential was evaluated by analyzing two markers of carcinogenesis, namely numbers of glutathione S-transferase placental form (GST-P)-positive foci, and proliferating cell nuclear antigen (PCNA). In addition, the level of ornithine decarboxylase (ODC), one of the rate-limiting enzymes of polyamine metabolism induced by promoters, was analyzed. SMC and cysteine induced significant reduction in the areas of GST-P-positive foci. A significant reduction in the PCNA index was observed in the entire liver as well as in GST-P-positive areas. SMC also induced down-regulation of the ODC enzyme activity. Thus, SMC and cysteine were found to inhibit the promotion stage of DEN-induced hepatocarcinogenesis. No cocarcinogenic effects were evident on administration of either of these chemicals with NaPB.     

Chemopreventive effects of S-methylcysteine on rat hepatocarcinogenesis induced by concurrent administration of sodium nitrite and morpholine

The aim of the present study was to examine the chemopreventive efficacy of S-methylcysteine (SMC) on rat hepatocarcinogenesis induced by concurrent administration of sodium nitrite (NaNO(2)) and morpholine (Mor) using a medium-term rat liver carcinogenesis bioassay (Ito test). Administration of SMC caused significant reduction in the areas of glutathione S-transferase placental form positive foci along with a significant decrease of hepatocyte 5-bromo-2'-deoxyuridine (BrdU) labeling indices. These results demonstrated potent chemopreventive effects of SMC against hepatocarcinogenesis due to concurrent administration of Mor and NaNO(2). SMC could thus be an effective chemopreventive agent for decreasing the risk of carcinogenicity from environmental precursors of N-nitroso compounds.     

Inhibitory Effects of S-Methylcysteine and Cysteine on the Promoting Potential of Sodium Phenobarbital on Rat Liver Carcinogenesis

The effects of S-methylcysteine (SMC) and eysteine on the promotion stages of rodent hepatocar-cinogenesis in a medium-term bioassay previously developed by Ito were examined. Initiation was induced by a single dose of diethylnitrosamine (DEN), followed by dietary administration of the promoter sodium phenobarbital (NaPB) 2 weeks later, for 6 weeks. Partial hepatectomy was conducted on all the animals at week 3. Inhibitory potential was evaluated by analyzing two markers of carcinogenesis, namely numbers of glutathione S-transferase placental form (GST-P)-positive foci, and proliferating cell nuclear antigen (PCNA). In addition, the level of ornithine decarboxylase (ODC), one of the rate-limiting enzymes of polyamine metabolism induced by promoters, was analyzed. SMC and cysteine induced significant reduction in the areas of GST-P-positive foci. A significant reduction in the PCNA index was observed in the entire liver as well as in GST-P-positive areas. SMC also induced down-regulation of the ODC enzyme activity. Thus, SMC and cysteine were found to inhibit the promotion stage of DEN-induced hepatocarcinogenesis. No cocarcinogenic effects were evident on administration of either of these chemicals with NaPB.     

N-acetylcysteine and S-methylcysteine inhibit MeIQx rat hepatocarcinogenesis in the post-initiation stage

N-acetylcysteine (NAC) and S-methylcysteine (SMC), water soluble organosulfur compounds contained in garlic, were evaluated for chemoprevention of hepatocarcinogenesis after 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) initiation in rats. Intergastric treatment with NAC or SMC five times a week resulted in decreased numbers and areas of preneoplastic, glutathione S-transferase placental form (GST-P) positive foci of the liver in a dose-dependent manner. Moreover, cell proliferation was reduced in GST-P positive foci by NAC and SMC. Insulin-like growth factor I (IGF-I) and inducible nitric oxide synthase (iNOS) mRNA expressions were found downregulated in the liver by NAC. The studies indicate that NAC can serve as a chemopreventive agent for rat hepatocarcinogenesis induced by MeIQx by reducing cell proliferation, which may involve IGF-I and iNOS downregulation.     

Chemopreventive Effects of Coumaperine from Pepper on the Initiation Stage of Chemical Hepatocarcinogenesis in the Rat

This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepatocarcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg/kg/day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg/kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg/kg/day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)- positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.     

Three-dimensional Analysis of Glutathione S-Transferase Placental Form-positive Lesion Development in Early Stages of Rat Hepatocarcinogenesis

The development of glutathione S-transferase placental form (GST-P)-positive lesions in the rat liver, from single cells to foci, and their locations within liver lobules were examined by a combined stereological approach for calculation of three-dimensional (3-D) data and by 3-D computer graphics for reconstruction of lesions. Two weeks after initiation with diethylnitrosamine, the rats were divided into two groups. Animals in group 1 were given 2-acetylaminofluorene, and animals in group 2 were given basal diet for 6 weeks. Partial hepatectomy (PH) was performed at week 3. The GST-P-positive single cells increased in number per liver after PH in group 1, but not in group 2, where a plateau level was maintained. The number of GST-P-positive foci per liver in group 2 also reached an almost constant low value after 4 weeks. In contrast, foci in group 1 increased greatly after PH. A 3-D reconstruction, performed with a computer graphics system using up to 180 sections at 10 μm intervals, revealed the single cells to be distributed at random. Those that grew into foci were also not preferentially localized in any particular zone of the hepatic lobule. When foci within the same lobule came into contact, they underwent fusion. The present results thus indicate that only a small proportion of GST-P-positive single cells develops into foci, and that their growth is independent of zonal factors within individual lobules in early stages of rat hepatocarcinogenesis.     

Induction of rat liver gamma-glutamyltranspeptidase-positive foci by oral administration of 1-nitropyrene

In the present study, the question of whether the ubiquitous environmental pollutant, 1-nitropyrene (1-NP), can induce gamma-glutamyltranspeptidase (GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, when given orally to F344 rats was examined. Significant induction of GGT-positive foci was observed with all the doses of 1-NP (1000, 500, 250 and 100 mg/kg body wt) used in the present experiment after 6 repeated intragastric (i.g.) intubations when accompanied by partial hepatectomy (PH) performed midway, but not after a single i.g. 1000 mg/kg body wt intubation plus PH. The potential for induction of foci by 1-NP, however, was far less than that by benzo[a]pyrene (B[a]P). The results thus suggested a weak but substantial initiation activity for 1-NP in the rat liver when given orally, particularly with repeated doses.     

Dietary Administration of Inositol and/or Inositol-6-phosphate Prevents Chemically-induced Rat Hepatocarcinogenesis

Chemoprevention is considered a rational strategy for dietary approaches to prevention of cancer. Multiple lines ‍of evidence suggest that many of our dietary principles are able to intervene in the multistage carcinogenesis process ‍and phytic acid (inositol hexaphosphate, IP6), a phytochemical present in a variety of plant species, has been shown ‍to prevent various cancers, including those of the mammary gland, colon and liver. However, the mechanism of ‍chemoprevention by IP6 has not been fully elucidated. In the present study, we examined the effects of inositol and/ ‍or IP6 supplementation on rat hepatocarcinogenesis initiated by diethylnitrosamine (DEN) and promoted by partial ‍hepatectomy (PH). Supplementation with either inositol or IP6, or their combination, starting one week prior to ‍administration of DEN, resulted in a significant decrease in both the area and the number of placental glutathione Stransferase ‍positive (GST-P+) foci, a preneoplastic marker for DEN-initiated hepatocarcinogenesis. The administration ‍of inositol and/or IP6 in drinking water caused marked enhancement in the glutathione S-transferase (GST) activity. ‍In addition, the production of thiobarbituric acid reactive substances and the catalase activity were significantly ‍reduced in rats supplemented with inositol and /or IP6. Based on these findings, it is likely that the chemopreventive ‍effects of inositol and/or IP6 on rat hepatocarcinogenesis initiated by DEN and promoted by PH are associated with ‍induction of GST activity and suppression of lipid peroxidation.     

INHIBITORY EFFECT OF BOSCHNIAKIA ROSSICA ON DEN-INDUCED PRECANCEROUS HEPATIC FOCI AND ITS ANTIOXIDATIVE ACTIVITIES IN RATS

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Effects of pinocembrin on the initiation and promotion stages of rat hepatocarcinogenesis

Pinocembrin (5, 7-dihydroxyflavanone) is a flavanone extracted from the rhizome of Boesenbergia pandurata. Our previous studies demonstrated that pinocembrin had no toxicity or mutagenicity in rats. We here evaluated its effects on the initiation and promotion stages in diethylnitrosamine-induced rat hepatocarcinogenesis, using short- and medium-term carcinogenicity tests. Micronucleated hepatocytes and liver glutathione-S-transferase placental form foci were used as end point markers. Pinocembrin was neither mutagenic nor carcinogenic in rat liver, and neither inhibited nor prevented micronucleus formation as well as GST-P positive foci formation induced by diethylnitrosamine. Interestingly, pinocembrin slightly increased the number of GST-P positive foci when given prior to diethylnitrosamine injection.     

Enhancing Effects of Organosulfur Compounds from Garlic and Onions on Hepatocarcinogenesis in Rats: Association with Increased Cell Proliferation and Elevated Ornithine Decarboxylase Activity

Four organosulfur compounds from garlic and onions were examined for modifying effects on diethylnitrosamine (DEN)-induced neoplasia of the liver in male F344 rats using the medium-term bioassay system based on the two-step model of hepatocarcinogenesis. Carcinogenic potential was scored by comparing the numbers and areas per cm² of induced glutathlone S-transfcrasc placental form-positive foci. Isothiocyanic acid isobutyl ester (IAIE), dipropyl trisulfide (DPT), and allyl mercapton (AM) exerted enhancing effects on their development, while dimethyl trisulfide also tended to increase them. To investigate possible mechanisms of the modifying influence, sequential changes in ornithine decarboxylase activity (ODC) over 24 h were measured in AM-treated liver tissue without prior DEN initiation. The activity started to increase by 4 h after AM-treatment, and reached maximum at 16 h, compared to controls. Spermidine/spermine N¹-acetyltransferase activity was not significantly changed. An increase in proliferating cell nuclear antigen-positive cells followed the elevation of ODC activity. These results suggest that IAIE, DPT, and AM promote rat hepatocarcinogenesis and their promoting effect might be caused by increased cell proliferation with increased poly-amine biosynthesis. In evaluating relationships between diet and cancer, it is thus appropriate to consider not only a possible protective role of garlic and onions, but also enhancing effects.     

Effect of vitamin E on the induction and evolution of enzyme-altered foci in the liver of rats treated with diethylnitrosamine

The effects of dietary vitamin E (VE) on the steps of hepatocarcinogenesis,the induction and growth of -glutamyltranspeptidase (GGT)-positivefoci and their evolution into persistent nodules, were analyzedin the liver of rats treated with diethylnitrosamine (DEN).The induction of GGT-positive foci was inhibited by a diet containing0.36–1.5% VE given after initiation with 200 mg/kg bodyweight (b.w.) DEN for 6 weeks with partial hepatectomy (PH)on week 3. The numbers and areas of GGT-positive foci were enhancedby diets containing 036 and 0.72% VE, given for 1 week afterinitiation with 10 mg/kg b.w. DEN and PH, followed by selectionby 0.02/ 2-acetylaminofluorene (AAF) and carbon tetrachloride(CCl⁴), but these were not enhanced by a diet containing 1.5%VE. Remodeling of hyperplastic nodules was not affected by thediet containing 0.72% VE given after initiation with DEN andselection for 12 weeks. The staining characteristics of GGTwere different between remodeling and persistent nodules, exceptfor those of the glutathione-S-transferase placental form (GST-P).The results obtained suggest that VE could prevent the veryearly events during hepatocarcinogenesis, the induction of phenotypicallyaltered foci, but could no longer affect the later stages, theevolution of foci into persistent nodules.     

Inhibitory effect of boschniakia rossica on den-induced precancerous hepatic foci and its antioxidative activities in rats

Objective: To investigate the inhibitory effect of Boschniakia rossica (BR) on rat precancerous hepatic foci induced by diethylnitrosamine (DEN) and its antioxidative activities. Methods: The expression of tumor marker—placental form glutathione S-transferase (GST-P), p53 and p21 protein were investigated by immunohistochemistry techniques using ABC method. TNF-α was measured by ELISA and antioxidative activities of SOD, MDA, GSH-Px, GST and CAT were investigated by colorimetric method in rat serum and mitochondria of liver cells. Results: The 500 mg/kg of BR-H₂O extract fraction from BR-methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver and the expression of mutant p53 and p21 protein was lower than that of hepatic precancerous lesions. The serum TNF-α was increased by the administration of BR extract in the early stage of chemical hepatocarcinogenesis in rat livers. The serum and liver cells mitochondria activities of SOD and GSH-Px rose again in rats administered with BR-H₂O extract and the increasing activity of GST and content of MDA in the hepatic precancerous were decreased by the BH-H₂O extract. Conclusion: These results indicated that BR-H₂O extract has inhibitory effect on DEN-induced precancerous hepatic foci in rats and induced TNF-α production in rats. The antioxidative action was exhibited by the administration of BR-H₂O extract in the early stage of chemical hepatocarcinogenesis in rat livers. This work was supported by a grant from the National Natural Science Foundation of China (No. 39660021).     

Resistance of Copenhagen rats to chemical induction of glutathione S- transferase 7-7-positive liver foci

Copenhagen (Cop) rats are completely resistant to the chemical induction of mammary adenocarcinomas, but their susceptibility to hepatocarcinogenesis is virtually unknown. Rat liver is a well- characterized and easily manipulated tissue in which to study carcinogenesis. Therefore, if Cop rats are resistant to hepatocarcinogenesis, studies into resistance mechanisms may be feasible. Male Cop and F344 rats, 7-8 weeks old, were initiated using either N-nitrosodiethylamine (DEN) (200 mg/kg, i.p.) or a two-thirds partial hepatectomy (PH) followed by N-methyl-N-nitrosourea (MNU) (60 mg/kg, i.p.). The rats were then promoted using a modified resistant hepatocyte (RH) protocol (a combination of four doses of 2- acetylaminofluorene (2-AAF) and a single dose of CCl4 that provides a selective mitotic stimulus for initiated cells). Six weeks after initiation the rats were killed and liver sections were stained for glutathione S-transferase 7-7 (GST 7-7), a marker for putative preneoplastic hepatocytes. Cop rats were found to be highly resistant, having a approximately 9- and approximately 27-fold smaller percentage of liver area occupied by GST 7-7-positive foci than susceptible F344 rats following initiation by DEN and MNU respectively. Furthermore, gross liver nodules did not form in any of the Cop rats, whereas all F344 rat livers contained nodules. Hepatic necrosis caused by DEN during initiation, and CCl4 during promotion is necessary to stimulate compensatory hepatocyte division. We demonstrated that these agents do indeed increase serum transaminase levels and produce histologic evidence of necrosis in Cop rats. In order for liver foci to grow rapidly in the RH protocol, the surrounding normal hepatocytes must be mito-inhibited by 2-AAF. We found that the degree of mito-inhibition of normal hepatocytes by 2-AAF is the same in Cop and F344 rats. These results show that the Cop rat is highly resistant to the chemical induction of putative preneoplastic liver foci and nodules.      

Chemopreventive effects of coumaperine from pepper on the initiation stage of chemical hepatocarcinogenesis in the rat.

This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepato-carcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg / kg / day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg / kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg / kg / day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)-positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.     

Revised rat multi-organ carcinogenesis bioassay for whole-body detection of chemopreventive agents: modifying potential of S-methylcysteine

The DMBDD rat multi-organ carcinogenesis model based on two-stage carcinogenesis theory was revised to make more suitable assay system for detecting chemopreventive effects of chemical substances by increasing the doses of two carcinogens, 1,2-dimethylhydrazine dihydrochloride (DMH) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). The revised bioassay resulted in increasing preneoplastic or neoplastic lesions in the colon, urinary bladder and liver. S-Methylcysteine (SMC), a water-soluble organosulfur compound, was used as a test chemical in the new initiation regimen. Though SMC did not express clear-cut inhibitory effects in tumor levels, it showed modifying effects on the development of lung hyperplastic and colon preneoplastic lesions. In conclusion, the present model featuring high yields of preneoplastic and neoplastic lesions with low mortality in a short period (30 weeks), might be suitable for testing the efficacy of possible chemopreventive chemicals at the whole-body level.     

Modulation of genetic resistance to hepatocarcinogenesis in DRH rats by partial hepatectomy

The DRH is an inbred rat strain highly resistant to chemically induced hepatocarcinogenesis. Two clusters of resistance loci Drh1 and Drh2 on chromosome 1 and 4 yield strong resistance to the formation of enzyme-altered foci and their progression. To evaluate the effect of enhanced cell proliferation in the progression stage, a partial hepatectomy (PH) was carried out in (DRH x F344)F1 rats 8 weeks after the start of 3'-Me-DAB administration. The incidence of liver cancer in the PH-F1 rats was equivalent to that in the F344 rats, although the number of tumors per rat was much lower. In contrast, the F1 rats without PH rarely developed cancers. Such modulation was not due to the loss of the resistance allele, since none of 18 hepatocellular carcinomas in PH-F1 rats showed allelic imbalance at Drhl and Drh2.     

Induced drug resistance inhibits selection of initiated cells and cancer development

Compounds exerting a mitoinhibitory effect on normal hepatocytes are potent promoters in the resistant hepatocyte model of chemical carcinogenesis in combination with stimulation of regenerative growth by partial hepatectomy or treatment with carbon tetrachloride. 2- Acetylaminofluorene (2-AAF) almost completely inhibits liver cell regeneration after partial hepatectomy, allowing only resistant cells to participate in regenerative growth. After initiation by diethylnitrosamine and promotion with 2-AAF and partial hepatectomy (PH), focal growth of initiated cells generates liver lesions which occupy 40% of the hepatic volume three weeks after PH. In this work the mechanism for the anti promoting effects of phenobarbital and 3- methylcholantrene were investigated as well as their effects on the development of malignant hepatocellular carcinoma in the resistant hepatocyte model. Treatment with phenobarbital or, especially, 3- methylcholanthrene rendered normal rat hepatocytes resistant to the mitoinhibitory effect of 2-AAF. In combination with 2-AAF/PH, 3- methylcholanthrene shortened the regenerative growth period to less than one week. In the Solt-Farber protocol for experimental hepatocarcinogenesis, treatment with phenobarbital or 3- methylcholanthrene during promotion with 2-AAF/PH permitted hepatocytes surrounding the focal lesions to respond with regenerative growth. The foci and surrounding liver grew until the liver/body mass index reached the control value. With phenobarbital treatment the total focal volume was 20% of the liver volume three weeks after PH, whereas the corresponding value in the case of 3-methylcholanthrene was only 1%. Labelling index data supported the conclusion that growth of the liver lesions in the resistant hepatocyte model was dependent on differential inhibition of normal hepatocyte growth by the promoter and that the size of the foci obtained was related to the length of time after PH required to complete liver regeneration. 3-methylcholanthrene induced 2- AAF resistance prevented the development of large persistent nodules and hepatocellular carcinoma while phenobarbital delayed cancer development with several month. The data thus supports the idea that the degree of clonal expansion during promotion determines the size of the population at risk for malignant transformation, as well as the final frequency of carcinomas.      

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.