Effect of six edible plants on the development of AFB1-induced gamma-glutamyltranspeptidase-positive hepatocyte foci in rats

Six edible plants, green tea (GT), black tea (BT), Lentinus edodes (berk) Sing (LE), Hericium erinaceus (Bull. ex Fr.) Pers. (HE), Mixture of Ganoderma Lucidum (Ley ss ex Fr.) Karst et Ganoderma Japanium (Fr.) Lloyd (MGLJ) and mung bean (MB), were tested for the effect on the development of AFB1-induced gamma-glutamyltranspeptidase positive hepatocyte foci (gamma-GT foci) using an in vivo short-term test model in rats. The rats received intraperitoneally 12 doses of initiator AFB1, 400 micrograms/kg per dose for 2 successive weeks. Two weeks after the initiation, the rats were submitted to a modified "Solt-Farber promotion program", i.e., a two weeks' feeding of a diet containing 0.015% acetylaminofluorene plus a two-third partial hepatectomy (PH) on day 7. The rats were sacrificed 10 days after PH and the livers were processed to gamma-glutamyltranspeptidase staining. The tested substances were powdered and mixed with the basal diet at the concentration level of 30% for MB and 5% for the others. The rats were fed with the diet-containing tested substances from 10 days before the AFB1 initiation to 3 days after the AFB1 conclusion. Consequently, the liver of the rats which had consumed GT showed significantly less and smaller gamma-GT foci, and those which had consumed BT, HE and LE showed somewhat less and significantly smaller foci than the control groups. It is indicated that the four diets have an inhibiting effect on AFB1-induced gamma-GT foci in different degrees. MB and MGLJ show no significant influence on the foci.     

AFB1致肝癌作用短期体内实验模型的长期观察及绿茶和BHA对AFB1诱发大鼠肝癌的影响

 200只大鼠分为4组,各组均先施以AFB₁致肝癌作用短期体内实验模型的基本程序。在此基础上,3个实验组在AFB₁期间分别饲用0.5%BHA、0.2%BHA及5%绿茶饲料,对照组此时用正常饲料。短期模型结束后,再进行长期观察。结果发现,0.5%BHA组的r-GT灶最少、最小,肝癌发生率最低、对照组的r—GT灶最多、最大,肝癌发生率最高。这提示:（1）本短期模型对于AFB₁诱发肝癌方面的基础研究是适用的。（2）r-GT灶的数量及大小与肝癌发生率有平行关系,故其在本模型中是可靠的肝癌前病变的标志。(3)绿茶及BHA均有抑制AFB₁致肝癌的作用。     

Enhancing effect of ethanol on aflatoxin B1-induced hepatocarcinogenesis in male ACI/N rats

The modifying effect of ethanol (EtOH) on aflatoxin B1 (AFB1)-induced hepatocarcinogenesis was examined in male ACI/N rats by chronic treatment at the post-initiation phase. Rats received an ip injection of AFB1 (1.5 mg/kg) twice a week for 10 weeks (a total of 20 doses). Following a week of acclimation, they were given 10% EtOH as drinking water for 56 weeks. The effect of EtOH on the hepatocarcinogenesis was evaluated in terms of the incidence of altered hepatocellular foci and neoplasms at the end of the experiment. Exposure to AFB1 alone induced a substantial number of altered foci (6.98 iron-excluding foci/cm2) in rats. The number of altered liver cell foci in rats receiving AFB1 followed by EtOH was significantly increased (26.39 iron-excluding foci/cm2). In the rats given EtOH after AFB1, the total area and mean diameter of both iron-excluding foci and altered foci identified in hematoxylin and eosin-stained sections were significantly higher than in the rats exposed to AFB1 alone. The incidence of liver cell tumors of the group given AFB1 and EtOH (3/15, 20%) was higher than that of the group treated with AFB1 alone (0/14, 0%). Treatment with EtOH alone for 56 weeks did not induce either. These results indicate an enhancing effect of EtOH on AFB1-induced hepatocarcinogenesis when it is given in the promotion phase.     

Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry.

Previous studies have demonstrated that dietary administration of the schistosomicidal drug 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) ameliorates the hepatotoxicity of aflatoxin B1 (AFB1). Notably, mortality, altered hepatic function, hepatic AFB1-DNA adduct levels, and expression of hepatic enzyme-altered foci were markedly reduced in the rat by concurrent feeding of oltipraz during exposures to AFB1. Collectively, these studies prompted us to evaluate the chemoprotective properties of oltipraz against AFB1-induced liver cancer. In addition, preliminary molecular dosimetry studies were undertaken to determine the utility of measurements of urinary aflatoxin-N7-guanine excretion as a marker of relative risk for hepatocarcinogenesis in AFB1-exposed rats. For the carcinogenesis studies, 5-wk-old male F344 rats were randomly divided into two groups. One group (55 rats) received the AIN-76A diet, and the other group (56 rats) received the AIN-76A diet supplemented with 0.075% oltipraz. The oltipraz-supplemented diet was fed for 4 wk. Beginning 1 wk after starting the experimental diets, all rats in both groups received 25 micrograms of AFB1/rat/day by gavage for 5 days per wk over the next 2 wk. One wk following cessation of dosing with AFB1, oltipraz was removed from the diet, and all rats were fed the AIN-76A diet for the remainder of the experiment. At 3 mo after dosing, livers of ten sentinel rats from each group were analyzed for the burden of gamma-glutamyltranspeptidase-positive foci. In accord with previous findings, rats fed the oltipraz-supplemented diet exhibited substantial reductions in the focal burden (97% reduction; P less than 0.05) of these AFB1-induced lesions. The remaining rats were maintained for the cancer study until they became moribund or the termination of the experiment at 23 mo. Gross liver lesions were identified at autopsy and confirmed by microscopic evaluation. An 11% incidence of hepatocellular carcinoma was observed in the AFB1-treated, control diet-fed rats. An additional 9% of this group had hepatocellular adenomas. Oltipraz afforded complete protection against both AFB1-induced hepatocellular neoplasms. Using Kaplan-Meier survival analyses, rats in the oltipraz group had a significantly (P less than 0.02) longer life span and an increased survival free of liver tumors (P less than 0.0002). Molecular dosimetry studies used rats fed either the oltipraz-supplemented or control diet for 1 wk and then challenged with a single dose of AFB1 to examine the initial rates of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 excreted in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Lipid peroxidation of liver microsome membranes induced by choline-deficient diets and its relationship to the diet-induced promotion of the induction of gamma-glutamyltranspeptidase-positive foci

The effects of varying the type of dietary fat in the choline-deficient (CD) diet on the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the liver of carcinogen-treated rats were investigated, and the results were correlated with the extent of membrane lipid peroxidation induced by the diets. Male Sprague Dawley rats were initiated with a single dose of diethylnitrosamine. Thereafter, groups of rats were fed choline-supplemented or CD diets in which the amount of saturated fat was varied by using hydrogenated vegetable oil (Primex) and corn oil (CO), either alone or in combination. The number and size of GGT-positive foci induced by the CD diet with CO as the sole source of fat were larger than those induced by the diet containing mixtures of Primex and CO. The CD diet with Primex alone was the least effective in inducing GGT-positive foci. Peroxidation of liver microsomal membrane lipids in rats fed regular CD or CD:CO diets was examined by determining the formation of conjugated dienes. The generation of diene conjugate in rats fed a CD:CO diet was evident after 2 days of the diet feeding, and the levels increased at 1 and 2 weeks. No significant diene conjugate was demonstrated in rats fed a regular CD diet for 2 days. However, after 1 and 2 weeks, there was generation of diene conjugate, the levels of which were lower in rats fed the CD diet than those on a CD:CO diet. Addition of an antioxidant, 0.25% butylated hydroxytoluene, to both CD and CD:CO diets abolished the generation of diene conjugate in rat liver microsomal membranes and markedly inhibited the promotion of GGT-positive foci in the liver of diethylnitrosamine-initiated rats. The results suggest that membrane lipid peroxidation in the liver may be related to the promotion of the induction of GGT-positive foci by a CD diet. The enhanced promotion by the inclusion of a higher level of polyunsaturated fat in the diet may be, in part, due to its greater susceptibility to peroxidation.     

COMPARATIVE STUDY ON THE INHIBITORY EFFECT OF GREEN TEA, COFFEE AND LEVAMISOLE ON THE HEPATOCARCINOGENIC ACTION OF DIETHYLNITROSAMINE

The present study aimed at clarifying whether Chinese green tea, coffee and levamisole (LMS) have similar Inhibitory effect on hepatocarclnogenesis induced by diethylnltrosamine (DEN) as they had been proved in our previous aflatoxin B1 (AFB1) experiments. Male Wistar rates were divided into control (A), green tea (B), coffee (C) and levamisole (D) groups. All rats received the same basic DEN treatment according to the program originally designed by Solt and Farber. During the two weeks before and one week after i. p. injection of DEN, the group B, C and D were given 2. 5% green tea, 5% coffee and 0. 1% LMS diet, respectively. The results demonstrated that coffee, LMS and , in particular,green tea showed Inhibitory effect against DEN-induced hepatocarcinogenesis, indicating that green tea can be used as chemopreventive agent for DEN-, as well as for AFB1-induced hepatocarcinogenesis.     

Choline deficient diet enhances the initiating and promoting effects of methapyrilene hydrochloride in rat liver as assayed by the induction of gamma-glutamyltranspeptidase-positive hepatocyte foci

Earlier we demonstrated that short-term feeding of methapyrilene hydrochloride (MPH) and of a choline deficient (CD) diet to rats induced peroxidative damage of microsomal membrane lipids of liver cells. In the present study, we investigated whether a CD diet modifies the extent of MPH-induced lipid peroxidation and whether the modifications lead to changes in the initiating and promoting action of these agents using assays of the induction of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci. Addition of 0.1% MPH to a CD diet enhanced the extent of microsomal lipid peroxidation induced by a CD diet alone. Feeding a choline supplemented (CS) or a CD diet containing 0.1% MPH for 2 weeks followed by 7 weeks promotion by a CD diet plus phenobarbital was ineffective in inducing GGT-positive foci. Feeding MPH in a CS or a CD diet for 4 weeks, however, resulted in the development of substantial numbers of GGT-positive foci. There was a 3 fold increase in the number of foci in rats initiated with a CD + MPH diet over that in rats initiated with a CS + MPH diet. 0.1% MPH in a CS diet or a CD diet exerted significant promotional effects on the induction of GGT-positive foci in rats initiated with a single injection of diethylnitrosamine. Addition of MPH to a CD diet was additive in inducing GGT-positive foci. The results suggest that lipid peroxidation of the liver may be involved in the carcinogenic and/or promoting effects of MPH and a CD diet.     

Enhancing Effect of Ethanol on Aflatoxin B1-induced Hepatocarcinogenesis in Male ACI/N Rats

The modifying effect of ethanol (EtOH) on aflatoxin B₁ (AFB₁)-induced hepatocarcinogenesis was examined in male ACI/N rats by chronic treatment at the post-initiation phase. Rats received an ip injection of AFB₁ (1.5 mg/kg) twice a week for 10 weeks (a total of 20 doses). Following a week of acclimation, they were given 10% EtOH as drinking water for 56 weeks. The effect of EtOH on the hepatocarcinogenesis was evaluated in terms of the incidence of altered hepatocellular foci and neoplasms at the end of the experiment. Exposure to AFB₁ alone induced a substantial number of altered foci (6.98 iron-excluding foci/cm²) in rats. The number of altered liver cell foci in rats receiving AFB₁ followed by EtOH was significantly increased (26.39 iron-excluding foci/cm²). In the rats given EtOH after AFB₁ the total area and mean diameter of both iron-excluding foci and altered foci identified in hematoxylin and eosin-stained sections were significantly higher than in the rats exposed to AFB₁ alone. The incidence of liver cell tumors of the group given AFB, and EtOH (3/15, 20%) was higher than that of the group treated with AFB₁ alone (0/14, 0%). Treatment with EtOH alone for 56 weeks did not induce either. These results indicate an enhancing effect of EtOH on AFB₁-induced hepatocarcinogenesis when it is given in the promotion phase.     

Effects of 3-aminobenzamide on the induction of gamma-glutamyl-transpeptidase-positive foci by various chemicals in rat liver

The effects of 3-aminobenzamide (ABA), a representative inhibitor of poly(ADP-ribose)polymerase, on the induction of gamma-glutamyl-transpeptidase (GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, in rat liver by various chemicals were studied in Fischer 344 and Wistar rats. ABA exhibited an enhancing effect on the induction of GGT-positive foci by benzo[a]pyrene, but no effects on induction by a methylating agent, N-methyl-N-nitrosourea, in both rat strains. In contrast, the induction of GGT-positive foci by another methylating agent, 1,2-dimethylhydrazine, was enhanced by ABA in Wistar rats, but not affected in Fischer rats. There were no effects of ABA on the induction of GGT-positive foci by N-bis(2-hydroxypropyl) nitrosamine in Wistar rats. These results indicate a differential effect of ABA on the induction of GGT-positive foci by different chemicals in different rat strains.     

Effects of rat strain, diet composition, and phenobarbital on hepatic gamma-glutamyl transpeptidase histochemistry and on the induction of altered hepatocyte foci and hepatic tumors by diethylnitrosamine

To extend our ongoing characterization of modulatory influences on hepatic tumorigenesis, we examined effects of rat strain (Sprague-Dawley versus Fischer), diet composition (semipurified diet versus standard nonpurified laboratory chow), and dietary phenobarbital on the production of gamma-glutamyl transpeptidase (GGT)-positive hepatocyte foci and hepatic tumors initiated by diethylnitrosamine. In addition to GGT-positive foci, we observed, under certain conditions, the appearance of extensive hepatic GGT staining not associated with focal lesions. This elevated nonfocal GGT was found in rats of both strains fed the nonpurified rather than the purified diet, but the level of staining was higher in Fischer than in Sprague-Dawley rats. Enhancement of this nonfocal staining by dietary phenobarbital appeared insignificant. By comparison, frequencies of GGT-positive foci were generally higher in rats fed the semipurified rather than the nonpurified diet, and the frequencies of GGT-positive foci were invariably higher in Sprague-Dawley than in Fischer rats. Moreover, dietary phenobarbital generally enhanced focus production. Assessments of focus and tumor yields among these experimental groups showed that differences in focus frequencies did not correspond closely to differences in subsequent tumor formation. These results document the need to consider the influences of diet and rat strain on experimental end points in designing protocols for hepatocarcinogenesis studies, especially those involving GGT histochemistry. The data also raise questions about the mechanistic relevance of GGT induction to hepatocarcinogenesis and support our prior evidence against the putative lineal relationship between foci and tumors.     

Inhibitory Effects of Benzyl Isothiocyanate and Benzyl Thiocyanate on Diethylnitrosamine-induced Hepatocarcinogenesis in Rats

The effects of two aromatic thiocyanates, benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC), on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were examined in rats. A total of 108 male ACI/N rats, 5 weeks old, were divided into 6 groups (18 rats in each). Group 1 was given a single i.p, injection of DEN (200 mg/kg body weight) one week after the start of the experiment and then kept on the basal diet until the end of the experiment (1 year). Groups 2 and 3 were treated with DEN and received dietary BITC (100 ppm) or BTC (100 ppm), respectively, throughout the experimental duration. Groups 4 and 5 were not given the carcinogen and were fed the diet containing BITC or BTC, respectively. Group 6 was kept on the basal diet alone and served as a control. Liver neoplasms were seen in Groups 1, 2 and 3. Incidence and average number of liver neoplasms in Group 2 were significantly smaller than in Group 1 ( P <0.0005 and P <0.001, respectively). The incidence of liver neoplasms in Group 3 was slightly lower than in Group 1, although the difference was not statistically significant. The numbers of glutathione S-transferase placental form (GST-P)-positive foci in Group 2 and γ-glutarnyltranspepridase (GGT)-positive foci in Groups 2 and 3 were significantly smaller than those in Group 1 ( P <0.001). The average and unit areas of GST-P- or GGT-positive foci in Group 2 or 3 were also significantly smaller than those in Group 1 ( P <0.05). These results suggest that BITC and BTC are chemopreventive agents for DEN-induced liver tumorigenesis.     

Dietary carotenoids inhibit aflatoxin B1-induced liver preneoplastic foci and DNA damage in the rat: role of the modulation of aflatoxin B1 metabolism

To study the effects of carotenoids on the initiation of liver carcinogenesis by aflatoxin B1 (AFB1), male weanling rats were fed beta- carotene, beta-apo-8'-carotenal, canthaxanthin, astaxanthin or lycopene (300 mg/kg diet), or an excess of vitamin A (21000 RE/kg diet), or were injected i.p. with 3-methylcholanthrene (3-MC) (6 x 20 mg/kg body wt) before and during i.p. treatment with AFB1 (2 x 1 mg/kg body wt). The rats were later submitted to 2-acetylaminofluorene treatment and partial hepatectomy, and placental glutathione S-transferase-positive liver foci were detected and quantified. The in vivo effects of carotenoids or of 3-MC on AFB1-induced liver DNA damage were evaluated using different endpoints: liver DNA single-strand breaks (SSB) induced by AFB1, and in vivo binding of [3H]AFB1 to liver DNA and plasma albumin. Finally, the modulation of AFB1 metabolism by carotenoids or by 3-MC was investigated in vitro by incubating [14C]AFB1 with liver microsomes from rats that had been fed with carotenoids or treated by 3- MC, and the metabolites formed by HPLC were analyzed. In contrast to lycopene or to an excess of vitamin A, both of which had no effect, beta-carotene, beta-apo-8'carotenal, astaxanthin and canthaxanthin, as well as 3-MC, were very efficient in reducing the number and the size of liver preneoplastic foci. In a similar way as 3-MC, the P4501A- inducer carotenoids, beta-apo-8'-carotenal astaxanthin and canthaxanthin, decreased in vivo AFB1-induced DNA SSB and the binding of AFB1 to liver DNA and plasma albumin, and increased in vitro AFB1 metabolism to aflatoxin M1, a less genotoxic metabolite. It is concluded that these carotenoids exert their protective effect through the deviation of AFB1 metabolism towards detoxication pathways. In contrast, beta-carotene did not protect hepatic DNA from AFB1-induced alterations, and caused only minor changes of AFB1 metabolism: seemingly, its protective effect against the initiation of liver preneoplastic foci by AFB1 is mediated by other mechanisms.      

Hexachlorobenzene as a promoter of diethylnitrosamine-initiated hepatocarcinogenesis in rats and comparison with induction of porphyria

In rats, hexachlorobenzene (HCB) causes uroporphyria and livertumours predominantly in females. To investigate the promotionalproperties of HCB, male and female rats received diethylnitrosamine(DEN) in the drinking water (0.015%) for 3 weeks. After a 2-weekrecovery period rats were fed control diet or one containingHCB (0.02%) for 30 weeks. HCB was an efficient promoter of DEN-initiatedhepatocarcinogenesis in both sexes as hudged by the size andnumbers of visible tumours and by the percentage of liver sectionsthat stained strongly positive for -glutamyltranspeptidase (GGT)activity. Tumours were larger in males whereas regions of GGT-positivetumour and non-tumour tissue were greater in females. Inhibitionof the haem biosynthesis enzyme uropor-phyrinogen decarboxylase(UD) only occurred in the liver of females treated with HCBor DEN/HCB. In the latter group. UD was inhibited both in andoutside tumours whereas uroporphyrin only accumulated in non-canceroustissue. No significant inhibition of UD was observed in theliver of males. In another study, rats received one i.p. doseof DEN (20 mg/kg) and after 3 weeks were fed HCB for 30 weeks.Numbers of GGT-positive foci were greatly increased by HCB inboth sexes, but especially in males (1.4-fold greater than females).Thus HCB was shown to be a promoter of hepatocarcinogenesis.However, the lack of a consistent marked sex difference suggeststhat this is only a partial explanation for the induction oftumours which mainly occur in females when this chemical isadministered alone for prolonged periods.     

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.     

Interactive effects of methyl-deficiency and dietary restriction on liver cell proliferation and telomerase activity in Fischer 344 rats pretreated with aflatoxin B(1)

The effects of methyl-deficiency and dietary restriction (DR) on hepatic cell proliferation and telomerase activity was studied in male Fischer 344 rats pretreated with aflatoxin B(1) (AFB(1)). Five-week-old rats were gavaged 5 days per week for 3 weeks with AFB(1) (25 microg/rat per day) or solvent (100 microl 75% dimethylsulfoxide). Rats were then divided into four groups. Two groups were fed a methyl-sufficient (MS) diet either ab libitum (AL) or with DR. The other two groups were fed a methyl-deficient (MD) diet either AL or with DR. At 15, 20, and 32 weeks of age, hepatic cell proliferation, telomerase activity, and the number of glutathione S-transferase-P positive (GST-P(+)) foci were determined. DR reduced hepatic cell proliferation, while the MD diet and AFB(1) pretreatment increased cell proliferation. Telomerase activity was decreased by DR and increased by the MD diet and AFB(1) pretreatment. The same trend was observed with GST-P(+) foci: in AFB(1)-pretreated rats, methyl deficiency increased the number of foci, while DR decreased the number. These results are consistent with a role of telomerase in hepatocarcinogenesis.     

Effects of black tea, green tea and wine extracts on intestinal carcinogenesis induced by azoxymethane in F344 rats

We investigated whether polyphenolic extracts from black tea, green tea or red wine affect azoxymethane (AOM)-induced intestinal carcinogenesis. Male F344 rats were treated 10 times (1 week apart) with AOM (7.4 mg/kg, s.c.) and then allocated into groups receiving black tea, green tea or red wine extracts mixed in the diet at a dose of 50 mg/kg body weight for 16 weeks. In the rats treated with black tea or wine extracts, there were significantly fewer colorectal tumours than in controls (the mean +/- SE number of tumours/rat was 2.54 +/- 1.6 in controls, 1.54 +/- 1.4 in the black tea group, 3.2 +/- 1.9 in the green tea group and 1.63 +/- 1.6 in the wine extract group). Significantly fewer rats in the black tea and wine extract groups had adenomas than in controls (86%, 59%, 90% and 50% of rats in the control, black tea, green tea and wine extract groups, respectively, had adenomas). The tumours from the black tea group and, to a lesser extent, those from the wine group, had a significantly greater apoptotic index than tumours in controls (mean +/- SE apoptotic index: 2.92 +/- 0.25, 4.13 +/- 0.46, 2.88 +/- 0.30 and 3.72 +/- 0.46 in controls, black tea, green tea or wine extract groups, respectively). In contrast, the apoptotic index of the normal mucosa did not vary among groups. These data indicate that black tea and wine extracts, but not green tea extracts, can protect against AOM-induced colon carcinogenesis by a mechanism probably involving increased apoptosis in tumours.     

Effect of high and low dietary protein on the dosing and postdosing periods of aflatoxin B1-induced hepatic preneoplastic lesion development in the rat

Aflatoxin B1-induced liver lesion development is readily modified by dietary protein intake. Earlier work had shown that low-protein diets enhanced the acutely toxic lesion but depressed the carcinogenic lesion. This study examined the emergence of these lesions as a function of dietary protein intake, particularly with respect to whether the protein modification occurred during or after the aflatoxin B12 dosing period. High (20%) and low (5%) casein diets were fed to growing Fischer 344 rats during the dosing and postdosing periods of aflatoxin B2-induced hepatic preneoplastic lesion development. Focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transferase (GGT). Animals fed low casein diets during the dosing period displayed a characteristic spectrum of lesions including hepatomegaly, severe bile duct proliferation, cholangiofibrosis, and a tendency for developing large remodeling GGT-positive foci. These lesions were regarded as symptomatic of acute hepatoxicity. Animals fed high-protein diets during the dosing period had small, densely stained, GGT-positive foci, with only mild bile duct proliferation and no cholangiofibrosis, hepatomegaly, or large, remodeling GGT-positive foci. During the postdosing period, protein modulation markedly influenced the total number of foci. Animals fed high casein diets during this period exhibited an approximate 6-fold increase in the number of foci, regardless of the level of protein fed during the earlier dosing period. The marked increase in foci number (as well as area of liver occupied) in high casein diet animals during the postdosing period is regarded as an increased tendency for tumor development.     

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.