Enhancing effect of malachite green on the development of hepatic pre-neoplastic lesions induced by N-nitrosodiethylamine in rats.

The effects of malachite green (MG) and phenobarbitone (PB) were compared on the development of pre-neoplastic lesions during N-nitrosodiethylamine(DEN)-induced hepatocarcinogenesis in male Wistar rats. Rats were administered 200 p.p.m. DEN in drinking water for a period of 1 month. After an interval of 2 weeks the animals were given either MG (25 p.p.m.) or PB (500 p.p.m.) in drinking water for 2.5 months. The effects were monitored on the basis of the morphological appearance of the liver, histological pattern, gamma-glutamyltranspeptidase (GGT)-positive foci, total GGT activity and the induction of glycogen-deficient islands. Both MG and PB were found to enhance liver carcinogenesis to a significant extent when compared with either their corresponding controls or animals given DEN alone. The enhancing effect of MG at 25 p.p.m. is comparable with PB at 500 p.p.m. An enhancing effect of MG on DEN-induced hepatocarcinogenesis in the rats was demonstrated.     

Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F1 mice at 100 or 200 mumol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 mumol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7-3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.     

Progressive atypia in spontaneous and N-nitrosodiethylamine-induced hepatocellular adenomas of C3H/HeNCr mice.

The progression of hepatocellular adenomas to carcinomas has been less well documented in mice than in rats. We studied progression of spontaneous and chemically induced hepatocellular adenomas in male C3H/HeNCr mice by image analysis. Spontaneous lesions in 15, 18 and 21 month old untreated male C3H/HeNCr mice and experimentally induced lesions were examined. Experimental group 1 received a single i.p. injection of N-nitrosodiethylamine (DEN) (5 mg/kg body wt) at 15 days of age. Groups 2 and 3 were injected a second time with DEN at 15 or 20 weeks of age (75 mg/kg body wt), with interim sacrifices at 11, 16 and 34 weeks after the second DEN injection. Atypia in adenomas were classified into four grades according to cell size, tinctorial changes, cellular pleomorphism and trabecular pattern. At earlier stages of the neoplastic process (11 or 16 weeks after the second DEN dose), most adenomas were well-differentiated lesions with no atypia or focal grade 1 or 2 atypia. At later stages (34 weeks after the second DEN dose), a large proportion of hepatocellular tumors were classified as adenoma with grade 3 atypia or carcinoma. The proportion of carcinomas in mice treated with a second dose of DEN at 20 weeks of age was significantly higher than in mice treated with a single dose of DEN or in mice given a second dose of DEN at 15 weeks. A positive correlation was found between increase in the size of lesions and increased atypia in both spontaneous and DEN-induced lesions and with age for spontaneous tumors. These results support the hypothesis that mouse hepatocellular adenomas are truly neoplastic lesions in different stages of progression toward malignancy.     

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

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

Modulating Effects of Diets High in ω-3 and ω-6 Fatty Acids in Initiation and Postinitiation Stages of Diethylnitrosamine-induced Hepatocarcinogenesis in Rats

The effects of sardine fish oil or corn oil on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were investigated in male F344 rats. Starting at 5 weeks of age, animals were divided into 11 groups and fed 23.5% corn oil (HCO) (groups 1 and 7) or 5% corn oil (LCO) (groups 2 and 8), 22.5% sardine oil + 1% corn oil (FO) semipurified diet (groups 3 and 9) or basal diet (CE-2) (groups 4–6, 10 and 11). At 6 weeks of age, all animals except the vehicle-treated groups were given DEN (200 mg/kg body weight, i.p. once weekly for 3 weeks). One week after the final exposure to DEN, groups 1–3 were changed to the basal diet, and groups 4–6 were switched to the HCO, LCO or FO diet, respectively. Animals in groups 1–3 and 10 were given drinking water containing 0.05% phenobarbital (PB). Liver sections from the animals at the termination of the experiment (24 weeks) were doubly stained for glutathione S-transferase placental form (GST-P) and silver-stained nucleolar organizer regions (AgNORs). The multiplicity of hepatocellular neoplasms of group 1 was significantly larger than that of group 2 or 3. The number of GST-P-positive foci of group 2 or 3 was significantly smaller than that of group 1. Among the groups fed the experimental diets in the post initiation phase (groups 4–6), no significant difference was found in the incidence of liver tumors. AgNORs values of the enzyme-altered foci in rats of the HCO diet groups were larger than those of the other diet groups. These results indicate that the enhancing effect of a high dose of corn oil in hepatocarcinogenesis is mainly present during the initiation phase but not during post initiation phase, and fish oil rich in polyunsaturated ω-3 fatty acids could inhibit DEN-induced hepatocarcinogenesis in rats.     

Cell Proliferation and regulation of negative growth factors in mouse liver foci

Foci of altered hepatocytes are preneoplastic lesions capableof progressing to hepatocellular carcinomas. To Characterizethe growth of preneoplastic hepatic lesions, size of hepaticfoci was analyzed with regard to growth factor regulation andhepatocyte proliferation in focal and non-focal hepatocytes.Twelve-day-old female B6C3F1 mice were initiated with a singledose of the potent mutagen N-nitrosodiethylamine (DEN) (5 mg/kgbody weight). Beginning at 6 weeks of age, mice were exposedfor 16 weeks to 2038 p.p.m. unleaded gasoline (UG) vapor or1 p.p.m. ethinyl estradiol (EE) in the diet. Analysis of hepaticfoci demonstrated that UG significantly increased, but EE significantlydecreased the size of DEN-initiated foci. Hepatic labeling index(LI), as measured by the incorporation of 5-bromo-2'-deoxyuridine,was similar in non-focal hepatocytes at 16 weeks in all groups(0.4–0.8%) and greatly increased in hepatic foci. HepatocyteLI was significantly increased in DEN/UG foci (29%, n = 41)and significantly decreased in DEN/EE foci (6% n=23) relativeto DEN/control focal hepatocytes(18% n=25). The mean LI of focicorrelated with the focal size differences observed in the treatmentgroups. Immunohistochemical analysis with antibodies directedto the negative growth regulator transforming growth factorbetal (TGF-ß1) demonstrated a consistent decreaseof TGF-ß1 in DEN/Ct and DEN/UG hepatic foci relativeto non-lesion hepatocytes. Similar results were seen with mannose6-phosphate/insulin-like growth factor-11 receptor (M6P/IGF-IIR), which facilitates activation of latent TGF-ß1.In contrast, only 50% of DEN/EE foci had decreased levels ofTGF-ß1 and M6P/IGF-II R relative to non-focal hepatocytes.These data suggest that proliferative responses observed inhepatic foci may be correlated with foci size. In contrast,chemically induced proliferative responses in non-focal hepatocytesafter subchronic exposure cannot necessarily be used to predictproliferative effects in preneoplastic cell populations. Furthermore,these studies suggest that hepatic foci may occur by M6P/IGF-IIR enhancing activation of latent TGF-ß1 in non-focalhepatocytes but not in the focal hepatocytes, thereby affordingfocal hepatocytes a selective growth advantage.     

Inhibition of tumor promotion and hepatocellular growth by dietary restriction in mice

The effects of dietary restriction on the growth of hepaticfocal lesions in phenobarbital (PB) promoted mice were examined.Dietary restriction which can inhibit many age-related diseasesin rodents including hepatic cancer also decreases cell proliferationand increases apoptosis in the liver. In contrast, PB, a non-genotoxicrodent hepatocarcinogen, enhances the growth of hepatic focallesions in mice and rats by increasing cell proliferation andinhibiting apoptosis. The present study examined the impactof dietary restriction on PB-induced hepatic tumor promotion.Focal lesions were produced by diethylnitrosamine (DEN) treatment(35 mg DEN/kg body weight injections, twice per week for 8 weeks).After lesions were produced, mice were placed into one of thefollowing four groups: NIH-07 control diet/no PB (group 1);NIH-07 diet/500 mg PB per liter of drinking water (group 2);dietary restricted NIH-07 diet/no PB (group 3); and dietaryrestricted NIH-7 diet/500 mg PB per liter of drinking water(group 4). In this study, PB (500 mg/1) treatment to ad libitum-fedmice (group 2) enhanced focal lesion volume, number, and labelingindex compared with group 1. In addition, PB treatment (group2) inhibited apoptosis in normal and focal hepatocytes comparedwith untreated control mice (group 1). In contrast, in dietaryrestricted mice treated with PB (group 4) a significantly lowerfocal lesion volume, number and labeling index were seen comparedwith the ad libitumfed/PB treatment group (group 2). PB treatmentin dietary restricted mice (group 4) did not inhibit focal apoptosis,in fact, the incidence of focal apoptosis was increased in thesemice compared with ad libitum and PB-treated mice (group 2).In dietary restricted mice treated with PB (group 4), the abilityof PB to promote the growth of preneoplastic focal lesions wasinhibited. These results show that dietary restriction can ablatethe tumor promotional effects of PB in hepatic focal lesionsand suggest that inhibition of focal lesion DNA synthesis andenhancement of apoptosis may be a mechanism for this effect.     

Threshold dose dependence in phenobarbital promotion of rat hepatocarcinogenesis initiated by diethylnitrosamine.

The dose-response of phenobarbital (PB) promotion of hepatocarcinogenesis in rats was investigated. Male F344 rats were given 1, 4, 16, 75, 300 or 1200 p.p.m. PB solutions given ad libitum as their drinking water for 39 weeks following initiation with a single i.p. injection of diethylnitrosamine (DEN) (100 mg/kg). At week 40, the incidence of hepatic tumors was increased clearly in the DEN + PB groups given 300 p.p.m. PB or above, as compared to that in the group given DEN only. Linear dose-response curves for numbers and sizes of enzyme-altered hepatic foci (gamma-glutamyl-transpeptidase or placental glutathione S-transferase positive foci) were obtained in the dose range 16-1200 p.p.m. PB. The minimum promoting dose level of PB for enzyme-altered foci, estimated from dose-response curves by the Logit model, was calculated to be 15-23 p.p.m. Thus while dose dependence was demonstrated over a large range, a threshold was evident at low doses.     

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.     

Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3F1 mice

Potentially preneoplastic hepatocellular hyperplastic foci andhepatocellular neoplasms were studied in weanling male B6C3F1mice that received a single i.p. injection (80 mg/kg) of diethylnitrosamine(DEN) at 4 weeks of age, followed by oral administration ofphenobarbital (PB) or di(2-ethylhexyl)- phthalate (DEHP) thatbegan 2 weeks after DEN injection and continued for up to 6months. PB was administered in drinking water at 500 p.p.m.and DEHP in the feed at 3000, 6000 or 12 000 p.p.m. Groups ofmice were sacrificed at 2, 4 and 6 months after DEN exposure;formalin-fixed liver samples were evaluated histologically.Hepatocellular neoplasms and foci of hyperplasia were quantifiedwith the aid of an image analysis computer. Few foci were seenat 2, 4 or 6 months in mice exposed to DEN, PB or DEHP alone,while numerous foci and neoplasms were seen in mice given DEHPor PB after DEN. Area-perimeter measurements for each hepatocellularfocus or neoplasm transection revealed that foci and neoplasmsin PB-exposed mice increased both in size (area and volume)and in number throughout the study. In DEHP-exposed mice thepattern of response was different in that the numbers of focidid not increase between 4 and 6 months, but the foci increasedin mean diameter and volume throughout the experiment. Fociand tumors appeared earlier in mice given higher dietary levelsof DEHP than in those given lower doses. By the end of the studythe number of foci per unit volume of liver was similar in micegiven any dose of DEHP, but their volume was dose-related. Hepatocellularfoci and neoplasms in PB-exposed mice were composed predominantlyof eosinophilic hepatocytes, while in DEHP exposed mice, basophilicfoci and neoplasms predominated; the latter were more malignantin appearance than neoplasms in PB-exposed mice. At 6 months,the neoplasms in high dose DEHP-exposed mice were significantlylarger than those in PB exposed mice. Histochemistry, however,revealed similarities between lesions in mice exposed to PBor DEHP. PB given continuously for 6 months revealed no initiatingactivity of DEHP given once by gavage and followed by PB indrinking water. Both morphology and biology of hepatocellularfoci and neoplasms, which develop in mice after a single exposureto a carcinogen with initiating activity, thus depend, in part,on the subsequent promoting agent. More than one process oftumor promotion, as characterized by a specific sequence ofmorphologic and biochemical changes, is possible for the mousehepatocyle.     

Interstrain differences in susceptibility to liver carcinogenesis initiated by N-nitrosodiethylamine and its promotion by phenobarbital in C57BL/6NCr, C3H/HeNCrMTV- and DBA/2NCr mice

Selected inbred strains of mice were compared with respect totheir susceptibility to two-stage liver carcinogenesis. Five-week-oldmale mice of strains C57BL/6NCr (C57), C3H/HeNCr^MTV- (C3H) andDBA/2NCr (DBA) were given a single i.p. injection of N-nitrosodiethylamine(DEN, 90 mg/kg body weight) or the solvent tricaprylin (10 ml/kg).Beginning 2 weeks later, half of the DEN-treated and half ofthe control mice were given drinking water containing 0.05%phenobarbital (PB). Ten mice from each treatment group werekilled at 12, 24, 36 and 52 weeks of age (5, 17, 29 and 45 weeksexposure to PB). PB significantly increased both the numberof hepatocellular foci/cm² and the incidence of hepatocellulartumors after 17 weeks of treatment in 24-week-old DEN-initiatedmice of strains C3H (0.11 ± 0.07 versus 2.9 ±0.3 foci/cm² and 20 versus 70% incidence of hepatocellular tumors)and DBA (0.09 ± 0.09 versus 3.72 ± 0.6 foci/cm²and 0 versus 90% incidence of hepatocellular tumors) but wasineffective in C57 mice (0.04 ± 0.04 versus 0.07 ±0.07 foci/cm²). At 36 weeks of age the incidence of liver celltumors in mice given DEN but not PB was 10 (DBA), 10 (C57) and50% (C3H); the incidence was increased by PB to 90% in DBA and100% in C3H mice, but there was no increase in C57 mice. Evenat 52 weeks, the low incidence of hepatocellular tumors in C57mice given DEN only (20%) was not significantly increased bysubsequent exposure to PB. Serum PB levels observed at 12, 24and 36 weeks of age were significantly higher in DBA mice thanin C57 or C3H mice. Similar results were observed in a separatestudy in which PB was administered in drinking water to 7-week-oldmale mice of these three strains for 20 days, during which periodserum PB levels were measured at shorter intervals. DBA micethus appear to be unable to metabolize PB, which itself ratherthan its metabolites is probably responsible for tumor-promotingeffects. DBA mice were especially sensitive, while C57 micewere refractory to promotion of hepatocar-cinogenesis by PB.These two strains, which differ with respect to other significantparameters for chemical carcinogenesis including inducibilityfor aryl hydrocarbon hydroxylase and susceptibility to promotionof hydrocarbon-initiated skin tumors by 12-O-tetradecanoyl-phorbol-13-acetate,thus also provide a means for analysis of the pharmacogeneticsof susceptibility to hepatocellular tumor promotion.     

Lack of Promoting Effects of Phenobarbital at Low Dose on Diethyl-nitrosamine-induced Hepatocarcinogenesis in TGF-a Transgenic Mice

Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat livercarcinogenesis. To test the hypothesis that the hormesis earlier observed for PB induced hepatocarcinogenesis mightalso exist in the TGF-α transgenic mice model, one which is highly susceptible to carcinogenesis, the carcinogenic orpromotion effects of a wide range of phenobarbital (PB) concentrations were investigated. Two weeks after a singlei.p. dose of 5 mg /kg bw of diethylnitrosamine (DEN) to 15 day old mice, animals were treated with diet containingPB at doses of 0, 2, 15 or 500 ppm. The incidence and multiplicity of tumors, including hepatocellular adenomas andcarcinomas, were significantly increased by the high dose of PB, but no significant difference among the groupsreceiving 2 and 15 ppm for liver tumors when compared to DEN alone group. The proliferating cell nuclear antigenindices for liver tumors and surrounding hepatocytes in high dose PB treated mice were significantly increased, butno change was noted at the lower doses. The total cytochrome P450 content in the liver was also elevated by 500 ppmof PB, while hepatic 8-OHdG levels demonstrated no significant change. In conclusion, PB at high dose enhancesDEN-induced hepatocarcinogenesis in TGF-α transgenic mice, but low doses lack any significant effects. One possiblemechanism of phenobarbital carcinogenicity might be influenced by cytochrome P450 system exhibiting a strongpromoting activity for liver of mice.     

High sensitivity of fatty liver Shionogi (FLS) mice to diethylnitrosamine hepatocarcinogenesis: comparison to C3H and C57 mice

The fatty liver Shionogi (FLS) mouse is a new inbred strain that spontaneously develops fatty liver with infiltration of mononuclear cells. Moreover, this mouse is known to frequently develop spontaneous hepatic cancers. Recently, human non-alcholic steatohepatitis (NASH) has been focused of attention regarding hepatocellular carcinoma. Therefore, this mouse has potential as a model for human hepatic cancer due to steatosis. It is of interest therefore, whether it exhibits elevated susceptibility not only regarding spontaneous tumor development but also to chemical hepatocarcinogens. To examine this concern, we examined diethylnitrosamine (DEN) hepatocarcinogenesis in FLS mice with 30ppm in drinking water for 26 weeks in comparison to two other strains of mice, C3H and C57. The induction of spontaneous and DEN-induced hepatic tumors was clearly increased in the FLS case, along with levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, as compared to the other strains, with or without DEN treatment. These results indicate that the oxidative DNA stress is intimately involved in hepatocarcinogenesis in FLS mice and provide further support for use of this mouse as a useful model for investigating hepatocarcinogenesis due to human hepatic steatosis.     

Prevention of dual promoting effects of pentachlorophenol,an environmental pollutant,on diethylnitrosamine-induced hepato- and cholangiocarcinogenesis in mice by green tea infusion

In order to explore a possibility that the custom of drinking green tea infusion is efficacious for reducing the carcinogenic risk of environmental exposure to pentachlorophenol (PCP), we examined the effects in a hepato- and cholangiocarcinogenesis model in mice exposed to diethylnitrosamine (DEN). In the first experiment, groups of 15 male mice were initially treated with DEN at a dose of 20 p.p.m. in the drinking water for the first 8 weeks followed by a 4 week recovery interval by PCP at concentrations of 0 (basal diet), 300 or 600 p.p.m. in the diet for 23 weeks. Further groups of animals were treated with DEN and PCP in the same manner and received 2% green tea infusion (GT) instead of the drinking water from week 10 until death. PCP exposure at the high dose promoted DEN-induced hepatocarcinogenesis, and also caused progression of cystic hyperplasias of the intrahepatic bile ducts to cholangiocellular tumors. Co-administration of GT was able to prevent the increases of incidences and multiplicities of DEN-induced hepatocellular tumors and also arrest the progression of cholangiocellular tumors. In the second experiment, co-treatment with GT in the drinking water from 1 week before 300 or 600 p.p.m. PCP treatment in the diet to the end of the experiment at week 3 in B6C3F1 male mice suppressed increases of serum ALT activities, 8-oxodeoxyguanosine levels in liver DNA and bromodeoxyuridine labeling indices of hepatocytes and intrahepatic biliary epithelial cells induced by PCP. These findings suggest that regular intake of green tea may reduce the carcinogenic risk posed by an environmental pollutant, PCP, presumably due to effects on oxidative stress.     

Dietary supplementation of the citrus antioxidant auraptene inhibits N,N-diethylnitrosamine-induced rat hepatocarcinogenesis

OBJECTIVES: We have previously reported that an antioxidant, auraptene (AUR), isolated from citrus fruit effectively inhibits chemically induced carcinogenesis in digestive tracts, such as the oral cavity, esophagus and large bowel. In this study, we investigated the modifying effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in male F344 rats in two different experiments to determine whether the compound exerts a cancer-chemopreventive action in other organs. METHODS: In the first experiment, animals were fed diets containing AUR at dose levels of 100 and 500 ppm for 7 weeks 1 week before, during, and 1 week after the start of liver carcinogenesis induced by DEN (40 ppm in drinking water for 5 weeks) to predict the modulatory effect on hepatocarcinogenesis. After 7 weeks, the numbers of hepatocellular enzyme-altered foci (EAF; cm(2)) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-alpha were determined on immunohistochemically stained sections. In the second experiment conducted to confirm the findings, animals subjected to DEN treatment were fed AUR-containing diets (100 and 500 ppm) during either the initiation stage ('initiation' feeding for 7 weeks) or post-initiation phase ('post-initiation' feeding for 25 weeks) of DEN-induced hepatocarcinogenesis. RESULTS: In the first experiment, feeding with AUR at both doses during DEN exposure decreased the mean numbers of GST-P-positive and TGF-alpha-positive EAF/cm(2), and the reduction in the number of TGF-alpha-positive EAF by feeding 500 ppm AUR was statistically significant (p < 0.005). In the second experiment, the 'initiation' feeding with 500 ppm AUR significantly inhibited the incidence (33 vs. 83%, p = 0.000511) and multiplicity (0.67 +/- 1.09 vs. 1.96 +/- 1.85, p < 0.005) of liver cell carcinoma. Also, the 'post-initiation' feeding with AUR at both doses significantly reduced the development of hepatocellular carcinoma (100 ppm: incidence, 15%, p = 0.000006; multiplicity: 0.25 +/- 0.64, p < 0.001; 500 ppm: incidence, 11%, p = 0.000002; multiplicity, 0.26 +/- 0.81, p < 0.001). In addition, AUR feeding reduced cell proliferation and the apoptotic index in liver cell neoplasms. CONCLUSIONS: The results suggest that the citrus antioxidant AUR is a potential chemopreventive agent against DEN-induced hepatocarcinogenesis in rats.     

Hepatocyte expression of tumor associated aldehyde dehydrogenase (ALDH-3) and p21 Ras following diethylnitrosamine (DEN) initiation and chronic exposure to di(2-ethylhexyl)phthalate (DHEP)

Phthalate ester such as di(2-ethylhexyl)phthalate (DEHP) eitherpromote or inhibit rat liver tumorigenesis depending on thecarcinogenesis protocol. In this study, we examined the expressionof two histochemical markers, the tumor associated isozyme ofaldehyde dehydrogenase (ALDH-3) and the oncoprotein p21 Ras,in the livers of male F344 rats. The rats were initiated withDEN and further treated with either DEHP (a known inhibitorof hepatocarcinogenesis), phenobarbital (PB, a known promoterof hepatocarcinogenesis), or a combination of DEHP and PB. Thestudies were designed to examine the expression of these markersin both normal appearing liver and hepatic hyperplastic andneoplastic lesions andto correlate the early expression of themarkers at 26 weeks in the normal appearing liver to later tumorincidence at 52 weeks. The expression of each marker was detectedby immunohistochemical methods on formalin-fixed paraffin embeddedsections of normal appearing liver or liver lesions. We foundthat ALDH-3 and p21 expression were significantly enhanced inrats receiving PB after DEN initiation at 26 weeks and thatthe incidence of hepatocellular carcinomas was likewise increasedcompared to control or DEN only treated animals. DEN initiationfollowed by a combination of PB and either 0.1 or 0.5% DEHPsignificantly reduced ALDH-3 but not p21 Ras expression at 26weeks compared to DEN plus PB only. These treatment regimensalso reduced the incidence of hepatocellular carcinomas at 52weeks. DEN followed by any of the three doses of DEHP withoutPB resulted in ALDH-3 expression similar to DEN alone. However,p21 Ras expression was significantly increased after these treatments.For all treatment groups, both the early (26 weeks) expressionof p21 Ras and ALDH-3 correlated with hepatocellular carcinomaincidence at 52 weeks. However, the correlation between hepatocellularcarcinoma and ALDH-3 expression was better than p21 Ras or theother markers we have studied. We concluded that ALDH-3 expressionis significantly downregulated after DEHP treatment, and thatexpression of the isozyme correlated with later hepatocarcinomaincidence and may indicate a significant relationship betweenALDH-3 expression and epatocarcinogenesis during DEHP treatment.     

Modulating effects of diets high in omega-3 and omega-6 fatty acids in initiation and postinitiation stages of diethylnitrosamine-induced hepatocarcinogenesis in rats.

The effects of sardine fish oil or corn oil on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were investigated in male F344 rats. Starting at 5 weeks of age, animals were divided into 11 groups and fed 23.5% corn oil (HCO) (groups 1 and 7) or 5% corn oil (LCO) (groups 2 and 8), 22.5% sardine oil + 1% corn oil (FO) semipurified diet (groups 3 and 9) or basal diet (CE-2) (groups 4-6, 10 and 11). At 6 weeks of age, all animals except the vehicle-treated groups were given DEN (200 mg/kg body weight, i.p. once weekly for 3 weeks). One week after the final exposure to DEN, groups 1-3 were changed to the basal diet, and groups 4-6 were switched to the HCO, LCO or FO diet, respectively. Animals in groups 1-3 and 10 were given drinking water containing 0.05% phenobarbital (PB). Liver sections from the animals at the termination of the experiment (24 weeks) were doubly stained for glutathione S-transferase placental form (GST-P) and silver-stained nucleolar organizer regions (AgNORs). The multiplicity of hepatocellular neoplasms of group 1 was significantly larger than that of group 2 or 3. The number of GST-P-positive foci of group 2 or 3 was significantly smaller than that of group 1. Among the groups fed the experimental diets in the postinitiation phase (groups 4-6), no significant difference was found in the incidence of liver tumors. AgNORs values of the enzyme-altered foci in rats of the HCO diet groups were larger than those of the other diet groups. These results indicate that the enhancing effect of a high dose of corn oil in hepatocarcinogenesis is mainly present during the initiation phase but not during postinitiation phase, and fish oil rich in polyunsaturated omega-3 fatty acids could inhibit DEN-induced hepatocarcinogenesis in rats.     

突变型p53蛋白在二乙基亚硝胺诱发大鼠肝癌前病变中的表达

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

Curcumin-containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis

Curcumin has been widely used as a spice and coloring agent in foods. Recently, curcumin was found to possess chemopreventive effects against skin cancer, forestomach cancer, colon cancer and oral cancer in mice. Clinical trials of curcumin for prevention of human cancers are currently ongoing. In this study, we examine the chemopreventive effect of curcumin on murine hepatocarcinogenesis. C3H/HeN mice were injected i.p. with N-diethylnitrosamine (DEN) at the age of 5 weeks. The curcumin group started eating 0.2% curcumin-containing diet 4 days before DEN injection until death. The mice were then serially killed at the scheduled times to examine the development of hepatocellular carcinoma (HCC) and changes in intermediate biological markers. At the age of 42 weeks, the curcumin group, as compared with the control group (DEN alone), had an 81% reduction in multiplicity (0.5 versus 2.57) and a 62% reduction in incidence (38 versus 100%) of development of HCC. A series of intermediate biological markers were examined by western blot. While hepatic tissues obtained from the DEN-treated mice showed a remarkable increase in the levels of p21(ras), PCNA and CDC2 proteins, eating a curcumin-containing diet reversed the levels to normal values. These results indicate that curcumin effectively inhibits DEN-induced hepatocarcinogenesis in the mouse. The underlying mechanisms of the phenomenon and the feasibility of using curcumin in the chemoprevention of human HCC should be further explored.