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…     

珍珠梅提取物对二乙基亚硝胺所致大鼠肝脏癌前病变的抑制作用及抗氧化活力的影响

目的研究珍珠梅对二乙基亚硝胺致大鼠肝脏癌前病变灶及抗氧化活力的抑制作用.方法75只大鼠随机分为A、B、C 3组对照组、模型组和治疗组,每组25只.按略改良的Solt-Farber氏方法制作大鼠肝脏癌前病变模型,用珍珠梅提取物饲养大鼠6周后处死,通过免疫组织化学检测癌前病变组织谷胱甘肽S-转移酶(GST-P)和p53蛋白的表达,放射免疫法检测血清肿瘤坏死因子(TNF-α)的含量,比色分析法检测血清、肝线粒体超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)的含量.结果B和C组GST-P的表达率为86.7%和28.6%,p53的表达率为56%和36%,差异均有显著意义,P<0.05;B组血清TNF-α的含量与C组比较,差异有显著意义,P<0.05;B组血清、肝线粒体SOD、GSHPx活性及MDA含量与C组比较,差异均有显著意义,P<0.05.结论珍珠梅提取物对DEN所致大鼠癌前病变灶有抑制作用,可诱发TNF-α的生成,并有抗氧化作用.     

Inhibition by phenobarbital and lack of effect of amobarbital on the development of liver tumors induced by N-nitrosodiethylamine in juvenile B6C3F1 mice

The effects of phenobarbital (PB) and amobarbital (AB) on the rate of development of hepatocarcinogenesis induced by N-nitrosodiethylamine (DEN) were studied in mice. Groups of 40 B6C3F1 male mice were injected i.p. at 15 days of age with 5 micrograms DEN/g body wt. Beginning at 4 weeks of age, DEN treated groups were given either normal drinking water or water containing either 0.05% PB or AB for up to 36 weeks. DEN alone induced multiple focal hepatic lesions including hepatocellular foci, hepatocellular adenomas and trabecular carcinomas. Subsequent exposure to PB had a suppressing effect on DEN-induced hepatocarcinogenesis. Hepatocellular foci in PB-exposed mice were significantly smaller in size (area) and fewer in number throughout the study. Also, PB treatment either prolonged the latency period or significantly slowed the rate at which hepatocellular tumors developed in these mice. No such effects were seen in AB-exposed mice; AB neither inhibited nor promoted the development of focal hepatic lesions in DEN-pretreated mice. Possible mechanisms responsible for the inhibition of DEN-induced hepatocarcinogenesis include the feminizing effects of perinatal administration of PB.     

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.     

Differential expression and role of p21<Superscript>cip/waf1</Superscript> and p27<Superscript>kip1</Superscript> in TNF-α-induced inhibition of proliferation in human glioma cells

Background  

The role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) – p21^cip/waf1 and p27^kip1 in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) as in vitro models.     

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。     

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.     

Correlation of sera TNF-alpha with percentage of bone marrow plasma cells,LDH, beta2-microglobulin,and clinical stage in multiple myeloma

Tumor necrosis factor-α (TNF-α) is important for function, differentiation, and transformation of B-lymphocytes in multiple myeloma (MM) but can also induce apoptosis of myeloma cells. Based on this opposite effect, it is very crucial to analyze the correlation of the serum level of TNF-α with clinical parameters of the patients. In this article, we analyzed 18 MM patients, 48% male and 52% female, with a mean age of 52 yr (range: 35–81 yr), clinical stage I in 21.4%, stage II in 26.4%, and stage III in 52.2% of patients. Patients with advanced clinical stage, presence of osteolysis, and elevated lactate dehydrogenase (LDH) had a significant difference (Mann-Whitney U-test, p<0.05) in the serum level of TNF-α in comparison with those in the early stage, without osteolysis, and normal LDH. The correlation of individual values of TNF-α with the percentage of plasma cells in the bone marrow, LDH, β₂-microglobulin, fibrinogen, and sedimentation rate was significant (p<0.05). However, we have not found a significant correlation between TNF-α and concentration of hemoglobin, the number of white blood cells or platelets (p>0.05). We concluded that our data indicate determination of TNF-α as a good parameter for estimation of tumor mass presence, among individual patients with MM, and may by used for monitoring during application of different therapy protocols.     

Different inhibition of DNA synthesis by transforming growth factor {beta} and phenobarbital on GST-P-positive and GST-P-negative hepatocytes

Hepatocyte resistance against inhibition of DNA synthesis bytransforming growth factor ß₁ (TGF-ß₁) wasstudied in vitro. Hepatocytes were isolated from rats that hadreceived diethylnitrosamine (DEN) for 6 weeks. The effect ofTGF-ß₁ and phenobarbital (PB) on DNA synthesis indifferent cell populations was studied using bromodeoxyuridine(BrdU) incorporation and placental glutathione S-transferase(GST-P) as markers. It was found that GST-P-positive cells wereresistant to the growth inhibitory effect of TGF-ß₁and PB, whereas GST-P-negative cells were inhibited. It is concludedthat resistance to TGF-ß₁-dependent growth controlmay develop early during DEN-induced hepatocarcinogenesis.     

Inhibitory effect of sinigrin and indole-3-carbinol on diethytnitrosamine-induced hepatocarcinogenesis in male ACI/N rats

The modifying effects of sinigrin (Sin) and indole-3-carbinol(I3C) on the hepatocarcinogenesis induced by diethyl-nitrosamine(DEN) were investigated in male ACI/N rats. Rats were dividedinto six groups: group 1 was given DEN (40 p.p.m.) in the drinkingwater for 5 weeks, starting at 7 weeks of age; group 2 was treatedwith DEN and diet containing 1200 p.p.m. Sin; group 3 receivedDEN and diet containing 1000 p.p.m. I3C; group 4 was given Sindiet alone; group 5 was given I3C diet alone; and group 6 servedas controls. The diet containing Sin or I3C was fed to the ratsstarting at 6 weeks of age until 1 week after the carcinogenexposure. At termination of the experiment (week 29), the incidencesof iron-excluding altered foci (11.22±3.22/cm²) and livercell tumors (6/12, 50%) and the tumor multiplicity (0.9/rat)in rats of group 2 were significantly smaller than those ofgroup 1 (foci incidence, 48.33±6.34/cm², tumor incidence,10/10, 100% multiplicity, 9.5/rat) (P < 0.02). Similarly,the incidence of iron-excluding hepatocellular foci (17.65±4.67/cm2)and tumor multiplicity (2.4/rat) with a slight reduction oftumor incidence (9/12, 75%) in rats of group 3 were significantlylower than those of group 1 (P < 0.001). There were no livercell neoplasms in rats of groups 4, 5 and 6. Thus, Sin and I3Cinhibited the hepatocarcinogenesis induced by DEN when theywere administered concurrently with the carcinogen.     

靶动脉灌注NaHCO3提高部分抗肿瘤药物疗效的基础及临床研究

Objective: To observe the influence of pH value on the proliferation of LAK cells and on the killing effect of rIL-2,IFN-α2b, TNF-α, LAK cells and doxorubicin on malignant tumor cells, and investigate the possibility of increasing the efficacy of rIL-2 or IFN-α2b and doxorubicin by infusing sodium bicarbonate (NaHCO3) through target arteries. Methods: Separating single nucleus cells from peripheral blood of healthy men, and observing the influence of pH on the activation of single nucleus cells by rIL-2. MTT assay was used to measure the killing effect of rIL-2, IFN-α2b and TNF-α on 7404 cells and the increased effect of doxorubicin on rIL-2 and IFN-α2b, the cytotoxity of LAK cells in different pH. Forty-two patients with advanced primary liver cancer were obtained by stratified random, NaHCO3, rIL-2/IFN-α2b and doxorubicin were infused through target arteries. The efficacy was estimated after two cycles. Results: The conditions of pH 7.3 and pH 7.6 in vitro helped the proliferation of LAK cells and the killing effect of rIL-2, IFN-α2b and LAK cells on 7404 cells. In the condition of pH 6.8 there was almost no killing effect for LAK cells. In the condition of pH 7.0, 7.2, 7.4 and 7.6, the killing rate of TNF-α to 7404 cells increased by degrees, and in pH 7.4 the killing effect was the optimum. After two cycles treatments in the 42 patients with advanced primary liver cancer,the response rate (CR PR) was 88% (37/42). The median overall response and median overall survival were increased, and no complication associated with infusing sodium bicarbonate was observed. Conclusion: The killing effect of rIL-2, IFN-α2b, TNF-αand doxorubicin on malignant tumor cells was enhanced by increasing the pH value.     

肿瘤坏死因子α致肝细胞凋亡效应及其机制的研究

Objective: To investigate the apoptosis effect of TNF-a on HepG2 cells and its mechanism in vitro.Methods: HepG2 cells were treated with TNF-α(400 U/mL).HepG2 cells treated with TNF-αfor 24 h and apoptosis was proved by DNA fragments on gel electrophoresis.Fluorescent quantitative real-time PCR was used to detect Fas and FasL expression.HepG2 cells treated by TNF-αwere co-cultivated with normal HepG2 cells.Apoptosis of HepG2 cells was determined by the method of FACS.Results: After 24 h TNF-αtreatment, DNA was collapsed into fragments to form DNA ladder in gel electrophoresis; FasL expression increases and induces HepG2 cells apoptosis.By FACS, 98.4% of the co-cultivated cells were apoptosis, but 16.5% cells in control group were apoptosis.Conclusion: TNF-αcan induce apoptosis of HepG2 cells in vitro.FasL expression on TNF-αpre-treated HepG2 cells increased and these cells can lead normal HepG2 cells to apoptosis.This may attribute to the cure of virus hepatitis and hepatoma.     

The protective effects of ambroxol on radiation lung injury and influence on production of transforming growth factor β<Subscript>1</Subscript> and tumor necrosis factor α

The aim of this article was to investigate the effect of ambroxol on radiation lung injury and the expression of transforming growth factor β₁ (TGF-β₁), as well as tumor necrosis factor α (TNF-α) in plasma. Totally, 120 patients with locally advanced lung cancer in radiotherapy were randomized into treatment and control groups. Patients in the treatment group took ambroxol orally at a dosage of 90 mg, three times per day for 3 months from the beginning of radiotherapy. The expression of TGF-β₁ and TNF-α in plasma was analyzed. The clinical symptoms and lung diffusing capacity were monitored using high resolving power computed tomography. The level of TGF-β₁ in the control group was increased (11.8 ± 5.5 ng/ml), whereas in ambroxol-treated patients, the increase was not significant (5.6 ± 2.6 ng/ml, P < 0.001). Radiotherapy-induced elevation of TNF-α levels, seen in control patients, was also abolished after treatment with ambroxol (5.1 ± 1.0 vs. 2.4 ± 0.8 ng/ml, P < 0.001). In the treatment group, carbon monoxide diffusion capacity was not significantly decreased at 6, 12, and 18 months post-radiotherapy, compared with the control group (P < 0.05). Ambroxol decreased the expression of TGF-β₁ and TNF-α, and minimized the diminishment of lung diffusion capacity after radiotherapy.     

Inhibitory effect of celery seeds extract on chemically induced hepatocarcinogenesis: modulation of cell proliferation, metabolism and altered hepatic foci development

The chemopreventive activity of methanolic extract of Apium graveolens seeds (celery seeds) has been investigated against Solt Farber protocol of hepatocarcinogenesis, oxidative stress and induction of positive foci of gamma-GT in the liver of Wistar rats. The prophylactic treatment of celery seeds extract protected dose dependently against diethylnitrosoamine (DEN)+2-acetylaminofluorine (AAF)+partial hepatectomy (PH) induced hepatocarcinogenesis and other related events such as induction of gamma-GT positive foci (P<0.001). 2-AAF administration in diet with PH in rats resulted in increased hepatic ornithine decarboxylase (ODC) activity and a consequent increase in the rate of DNA synthesis when compared to saline treated control group while pretreatment of rats with celery seeds extract resulted in inhibition of aforementioned parameters dose dependently. The augmentation of quinone reductase (QR), glutathione-S-transferase (GST) and serum gamma-glutamyl transpeptidase (GGT) activities; and depletion of the tissue GSH content after 2-AAF (i.p. injection) for five consecutive days was prevented with the administration of celery seed extract. On the basis of the above results it can be said that A. graveolens is a potent plant against experimentally induced hepatocarcinogenesis in Wistar rats.     

Expression of TNF-α leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-α

Transmembrane TNF-α (tmTNF-α) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-α (tmTNF-α) at high level was resistant to soluble TNF-α (sTNF-α)-induced cytotoxicity, accompanied by constitutive NF-κB activation. In contrast, MCF-7 cells expressing tmTNF-α at very low level were sensitive to sTNF-α-induced cell death and had no detectable NF-κB activation. Consistently, siRNA-mediated tmTNF-α knockdown blocked NF-κB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-α, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-α containing LS constitutively activated NF-κB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-α lacking the cytoplasmic segment of LS neither activated NF-κB nor affected the sensitivity. However, NF-κB inhibitor PDTC suppressed NF-κB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-κB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)₂-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-κB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-κB and resistance to sTNF-α-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-α-induced resistance in the breast cancer cells. D. Yan and N. Qin have contributed equally to this work.     

Enhancement of dimethylnitrosamine-initiated hepatocarcinogenesis in hamsters by subsequent administration of carbon tetrachloride but not phenobarbital or p,p'-dichlorodiphenyltrichloroethane

The effect of phenobarbital (PB), p,p'-dichlorodiphenyl-trichloethane(DDT) or carbon tetrachloride (CCl₄) on dimethylnitrosamine(DMN)-induced hepatocarcinogenesis in Syrian golden hamsterswas examined. Hamsters were given a single injection of DMN(6 mg/kg body wt) followed by either PB or DDT in the diet orrepeated CCl₄ gavage for 30 weeks. The numbers of both alteredliver cell foci and hepatocellular neoplasms in the hamstersgiven 0.1 ml CCl₄, i.g. once every 2 weeks after the DMN weresignificantly higher than in the animals given DMN alone. PBor DDT (500 p.p.m. in the diet) after DMN did not produce asignificantly higher incidence of altered foci or hepatocellularneoplasms compared to DMN alone. Thus, an enhancing effect ofCCl₄ on DMN-induced hepatocarcinogenesis in the hamster wasdemonstrated, but neither PB or DDT—both liver neoplasmpromoters in rats and mice—displayed promoting activityunder the conditions of study.     

The monocyte tumor necrosis factor-alpha production in patients with acute leukemia in complete remission

Tumor necrosis factor-α (TNF-α) production by unstimulated and lipopolysaccharide (LPS)-stimulated peripheral monocytes has been studied in 17 acute myeioid leukemia (AML) patients, 54 AML patients in complete remission (AML-CR), 9 acute lymphoblastic leukemia (ALL) patients and 13 ALL patients in complete remission (ALL-CR). TNF-α production by the unstimulated monocytes in ALL patients (n - 6, mean: 6.6 ± 4.9 u/ml) was higher than that of normal controls (n = 13, 0.9 ± 0.7 u/ml), AML patients (n = 14, 2.0 ± 2.1 u/ml) and AMLCR patients (n = 21,1.4 ± 1.2 u/ml). TNF-α production by the LPS-stimulated monocytes of the AML-CR patients (n = 54,12.4 ± 13.4 u/ml) was significantly higher than that of the normal controls (n = 21, 3.5 ± 2.5 u/ml) and the AML patients (n = 17, 2.6 ± 2.4 u/ml),p < 0.01, but there were not any significant differences among the AML-CR patients and the ALL patients or the ALL-CR patients. We separated the AML-CR patients into 3 groups, depending on the length of their remission, and found that AML-CR patients with longer than 6 months (M) but less than 60 M (n = 21,15.7 ± 16.9 u/ml) and the patients with a remission longer than 60 M (n = 11,18.2 ± 15.9 u/ml) had significantly higher TNF-α production than that of the controls.     

Biochemical microanalysis of pyruvate kinase activity in preneoplastic and neoplastic liver lesions induced in rats by N-nitrosomorpholine

Fundamental aberrations in carbohydrate metabolism have beenpreviously demonstrated in focal hepatlc lesions emerging earlyduring hepatocarcinogenesis induced in rat liver by limitedoral administration (stop model) of N-nltrosomorpholine. Usingthis experimental approach, we have now investigated quantitativelythe activity of pyruvate kinase (PK), a key enzyme of glycolysisand gluconeogenesis, in individual preneoplastic and neoplastichepatic lesions, particularly in glycogen storage foci, mixedcell foci, basophilic cell foci, hepatocellular adenomas andcarcinomas and in a specific type of preneoplastic hepatic lesiondesignated as an enzymatically hyperactive focus (EHF). Thefocal lesions were dissected from freeze-dried tissue sectionswith a laser microdissection device. This permits the excisionof very small foci and the measurement of enzyme activitiesin serial sections of the same focus with different substrateconcentrations, thus enabling possible changes in the isoenzymepattern to be detected. On average, PK activity was increasedin glycogen storage foci. Mixed cell foci showed a nearly normalor slightly decreased enzyme activity. However, a pronouncedreduction in PK activity was observed in low glycogen basophilicfoci and in basophilic hepatic tumors. An exceptionally highPK activity was found in one glycogenotic adenoma. An increasedactivity was also observed in EHF. The results suggest thata reduction in PK activity is a relatively late event duringthe sequence of cellular changes leading from glycogenotic focito hepatocellular carcinomas. A drastic decrease in the enzymeactivity occurs only when low glycogen basophilic cell populationsdevelop from the glycogenotic foci in later stages of hepatocarcinogenesis.     

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.