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.     

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.     

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.     

Agaricus blazei (Himematsutake) does not alter the development of rat diethylnitrosamine-initiated hepatic preneoplastic foci

The modifying potential of crude extracts of the mushroom Agaricus blazei Murrill (Himematsutake) on the development and growth of glutathione S-transferase placental form (GST-P)-positive liver foci (liver preneoplastic lesion) was investigated in adult male Wistar rats. Six groups of animals were used. Groups 2 to 5 were given a single i.p. injection of 200 mg/kg b.w. of diethylnitrosamine (DEN) and groups 1 and 6 were treated with saline at the beginning of the experiment. After 2 weeks, animals of groups 3 to 6 were orally treated with three dose levels of aqueous extracts of the mushroom A. blazei (1.2, 5.6, 11.5, and 11.5 mg/ml of dry weight of solids) for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and sacrificed at week 8. Two hours before sacrifice, ten animals of each group were administered a single i.p injection of 100 mg/kg of bro-modeoxyuridine (BrdU). Apoptotic bodies and BrdU-positive hepatocyte nuclei were quantified in liver sections stained for hematoxylin and eosin (H&E) (eosinophilic foci) and simultaneously stained for GST-P expression (GST-P-positive foci), respectively. The 6-week treatment with A. blazei did not alter the development (number and size) of GST-P-positive foci and did not affect the growth kinetics of liver normal parenchyma or foci in DEN-initiated animals. Our results indicate that the treatment with aqueous extracts of the mushroom A. blazei during the post-initiation stage of rat liver carcinogenesis does not exert any protective effect against the development of GST-P-positive foci induced by DEN. (Cancer Sci 2003; 94: 188–192)     

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.     

Biphasic Modifying Effect of Indole-3-carbinol on Diethylnitrosamine-induced Preneoplastic Glutathione S-Transferase Placental Form-positive Liver Cell Foci in Sprague-Dawley Rats

The biphasic modifying effects of indole-3-carbinol (I3C), a naturally occurring constituent of edible cruciferous vegetables, on the development of glutathione S-transferase placental form (GST-P)-positive liver cell foci were investigated by using a medium-term liver bioassay system and a newborn rat hepatocarcinogenesis system. In Experiment 1, a total of 65 male Sprague-Dawley (SD) rats were divided into 5 groups. Animals were given a single intraperitoneal (i.p.) injection of 200 mg/kg diethylnitrosamine (DEN) dissolved in saline for groups 1, 2, and 3 or a single i.p. injection of saline for groups 4 and 5. Group 1 was given the diet containing 0.25% I3C for 2 weeks prior to DEN initiation and then basal diet for 8 weeks. Group 2 was given basal diet for 4 weeks prior to and after DEN initiation and then the diet containing 0.25% I3C for 6 weeks. The rats of group 3 were placed on basal diet during the experiment. Animals of groups 4 and 5 were treated in the same manner as those of groups 1 and 2 except for injection with saline instead of DEN solution. All rats were subjected to two-thirds partial hepatectomy at week 3 and were killed at week 8 after DEN or saline injection. In Experiment 2, a total of 45 female SD rats were dosed with DEN (100 mg/kg, i.p.) or saline at 24 h after birth. After weaning at week 3, the rats were fed diet containing 0.25% I3C for 9 weeks and then were killed at week 12. In Experiment 1, preinitiation exposure to 0.25% I3C caused a significant decrease in numbers of GST-P-positive liver cell foci (P<0.05), while postinitiation exposure to 0.25% I3C caused significant increases in both number (No./cm²) and area (mm²/cm²) of GST-P-positive liver cell foci (P<0.05 or 0.01). In Experiment 2, the relative liver weight in the DEN + I3C group was significantly increased (P<0.001). The numbers and areas of GST-P-positive liver cell foci in the DEN + I3C group were significantly increased as compared to the values of the DEN-alone group (P<0.001). These results clearly demonstrated that I3C exerts a promoting effect on the postinitiation stage as well as an inhibitory effect on the preinitiation stage in the medium-term liver bioassay.     

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.     

Dose-dependent effects of butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquin in induction of foci of rat liver cells containing the placental form of glutathione S-transferase

The dose-dependence effects of 3 antioxidants, butylated hydroxyanisole (BHA: 2.0%, 1.0% and 0.5%), butylated hydroxytoluene (BHT: 1.0%, 0.5% and 0.25%) and ethoxyquin (EQ: 0.5%, 0.25% and 0.125%) combined with partial hepatectomy on the development of preneoplastic lesions in the liver of diethylnitrosamine (DEN, 200 mg/kg body wt)-treated rats were investigated. Feeding of the antioxidants commenced 2 weeks after the single dose of DEN used to initiate the lesions. gamma-Glutamyl transpeptidase (gamma-GT) and the placental form of glutathione S-transferase (GST-P) were used for quantitation of altered focal populations. Results with both markers demonstrated a dose-dependent decrease of foci in BHA-treated rats relative to those in control rats. Morphometric analysis of gamma-GT-positive lesions also revealed decrease in both the number and area of foci in BHT- and EQ-treated groups. The discrepancy between results of quantitation of gamma-GT- and GST-P-positive foci was attributable to the induction of a background, periportal zone staining for gamma-GT, which made differentiation of smaller foci difficult. Comparison of results with the 2 markers suggested that GST-P is the more accurate marker for quantitative studies on enzyme altered foci in rat liver.     

Effects of tea on preneoplastic lesions and cell cycle regulators in rat liver.

The effects of tea polyphenols and tea pigments on rat liver precancerous lesions and some cell cycle regulators were studied. A modified Solt-Farber model in rats was established by multiple low-dosage of N-nitrosodiethylamine (NDEA) i.p. injections, followed by i.p. CCl(4) injection and partial hepatectomy. Sixty male Wistar rats were randomly divided into four groups: positive control group, two tea-treated groups, and negative control group. Rats in tea-treated groups were given tea polyphenols (0.1%) and tea pigments (0.1%) in drinking fluid during the whole experiment. The number and area of glutathione S-transferase P (GST-P)-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western blotting assay was carried out to detect the expression of cyclin D1, CDK4, and P21(WAF1/CIP1) on whole liver extract. At the end of the experiment (56 days), the number and area of GST-P-positive foci in liver increased significantly in carcinogen-administered positive control group, whereas no GST-P-positive foci were found in the negative control group in which animals did not receive carcinogen exposure. The number and area of GST-P-positive foci in tea-treated, carcinogen-exposed groups were significantly reduced as compared with the positive control group. It was also found that the expression of P21(WAF1/CIP1) was significantly induced and the expression of cyclin D1 and CDK4 was significantly inhibited in tea-treated groups. These results suggest that tea polyphenols and tea pigments are effective in preventing the precancerous liver lesions in rats, and modulation of cell cycle by regulating cell cycle regulators may be a possible mechanism.     

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

BoschnikiarossicaFedtsch.etFlerovisaparasiticplantgr0wingonther0otofAlnusplants(BetUlaceae).[']Itisoneofthevaluablemedicinalplantsgr0wingonChangbaiMountainareas,itgrowsgreatlyontheChangbaiMountainatl45orl8O0metersabovesealevel,Jilin,China.Itisals0gr0wsintheDemocraticPeople'sRepublicofKorea(DPRK),JaPanandRussian.Boschiakiarossicawasnamed"BuLaoCa0"(antisenilityplant),becauseithastheeffectsoftonifyingthekidneyandstrengtheningYang,andhasbeenusedasatonicinChina.Weis0latedfouririd0idc0mP…     

缺铁饲料对DEN诱发的鼠肝癌前病变的抑制作用研究

本实验采用肝癌发生的动物模型，进行了缺铁饲料对ＤＥＮ诱发的大鼠肝癌前病变即呈γ－谷胺酰转换酶（ＧＧＴ）阳性灶的定量分析及ＤＮＡ氧化损伤即ＤＮＡ中８－羟基－２－脱氧鸟苷（８－ＯＨｄＧ）产生量的测量。结果表明：缺铁组呈ＧＧＴ阳性的病灶数显著减少，但灶的大小无改变；ＤＮＡ中８－ＯＨｄＧ产生显著降低，提示出：铁缺乏对癌前病变的增生有抑制作用，其机理可能与ＤＮＡ的氧化损伤的减少有关。     

Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.     

In vitro induction of the anticarcinogenic marker enzyme, quinone reductase, in human hepatoma cells by food extracts

Summation of initiation activities of different carcinogens in the liver after partial hepatectomy (PH) was investigated with reference to induction of glutathione S-transferase placental form (GST-P) positive foci. Firstly, effects of repeated administration of 1,2-dimethylhydradine (DMH) were compared with the results of a single administration of the same total dose (Expt. I). Subsequently, we studied summation of initiation potential with serial administration of DMH with diethylnitrosamine (DEN) or N-bis (2-hydroxpropyl)-nitrosamine (DHPN). In Expt. I, induction of GST-P-positive foci by multiple low-dose administration was equal to that with the single large-dose treatment. In order to avoid toxicity in hepatectomized rats, the low repeated-dose approach appeared superior. In Expt. II, the numbers of GST-P-positive foci in the groups treated with DMH plus DHPN or DMH plus DEN were significantly higher than those in the groups receiving the carcinogens singly. It is concluded that there is summation of initiation potential with doses of a single or multiple carcinogens. These results suggest that the present initiation assay model is useful to investigate summation of initiation activities of various environmental chemicals.     

Expression of a 65 kda oncofetal phosphoprotein in the altered hepatic foci of rats fed 2-acetylaminofluorene followed by phenobarbital

The altered hepatic foci (AHF) developed in livers of Sprague-Dawley male rats,by acetylaminofluorene/phenobarbital protocol, were analyzed on paraffin sections for gamma-glutamyl-transpeptidase, glutathione S-transferase P and 65 kDa oncofetal protein (p65). 92% percent of the foci were GST-P-positive, 89% of those were also positive for GGT but only 10-12% were positive for p65. Comparison of tissues from different sources such as fetal and normal liver, foci, nodules, surrounding parenchyma and hepatocellular carcinomas revealed the presence of p65 protein in all tissues except normal rat liver and liver parenchyma surrounding the focal lesions.     

Anomalous elevation of glutathione S-transferase P-form (GST-P) in the elementary process of epigenetic initiation of chemical hepatocarcinogenesis in rats

The molecular mechanism of the specific expression of glutathione S-transferase P-form (GST-P) in the rat hepatic preneoplastic foci and "GST-P-positive" single cells requires elucidation. Immunochemical and stereological analyses revealed that the enzyme level in preneoplastic foci was 150-250-fold (6.7 +/- 2.4 mg/g liver and 0.29 +/- 0.1 mM subunits) higher than in normal cells. GST-P content in the single cells was higher than in preneoplastic foci, as determined by densitometry. In addition, the single cells were larger in cell diameter and area, corresponding to 2-3-fold increase in cell volume, relative to normal cells, but showed a significant shrinkage of their nuclei. Prior to the induction of single cells in the liver by diethylnitrosamine (DEN), microsomes were severely damaged as reflected by the low yield (approximately 60% that of untreated controls) after 2 h of DEN injection. Considering that GST-P is mainly a binding protein for GSH conjugates of endogenous carcinogens, together with our findings of morphological expansion, low viability of single cells and microsomal damage, our results suggest anomalous elevation of the ligand counterparts to lethal levels in preneoplastic cells, especially in single cells. We propose that the epigenetic mechanism rather than the genetic mechanism could account for GST-P induction in hepatocytes.     

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.     

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.     

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.     

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.