DEN Treatment Research Articles

Characterization of N-diethylnitrosamine-initiated and ferric nitrilotriacetate-promoted renal cell carcinoma experimental model and effect of a tamarind seed extract against acute nephrotoxicity and carcinogenesis

Renal cell carcinoma (RCC), the commonest malignancy in adult kidney, lacks of early signs, resulting often in metastasis at first diagnosis. N-Diethylnitrosamine (DEN)-initiated and ferric nitrilotriacetate (FeNTA)-promoted RCC may be a useful experimental model, but it is not well characterized. In this study, histological alterations and oxidative stress markers were analyzed at different times throughout RCC development, histological subtype was re-evaluated in the light of current classification, and a tamarind seed extract (TSE) effect was examined. Male Wistar rats experimental groups were control, TSE, DEN, DEN+FeNTA, and TSE+DEN+FeNTA. TSE was given 2 weeks before DEN administration (200 mg/kg) and throughout the experiment. Fourteen days after DEN treatment, two FeNTA doses (9 mg Fe/kg) for acute nephrotoxicity study, and increasing FeNTA doses (3-9 mg Fe/kg) twice a week for 16 weeks for carcinogenesis protocol, were administered. In acute study, necrosis and renal failure were observed and TSE ameliorated them. Throughout carcinogenesis protocol, preneoplastic lesions were observed since 1 month of FeNTA treatment, which were more evident at 2 months, when also renal cysts and RCC were already detected. RCC tumors were obtained without changes in renal function, and clear cell histological subtype was identified in all cases. 4-Hydroxy-2-nonenal and 3-nitro-L: -tyrosine levels increased progressively throughout protocol. TSE decreased both oxidative stress markers and, although there was no statistical difference, it delayed RCC progress and decreased its incidence (21 %). This study brings an insight of the time course events in this carcinogenesis model, identifies clear cell subtype and establishes TSE renoprotective effects.

Elevation of KL-6 serum levels in clinical trials of tumor necrosis factor inhibitors in patients with rheumatoid arthritis: a report from the Japan College of Rheumatology Ad Hoc Committee for Safety of Biological DMARDs

The associations between elevated levels of serum Krebs von den Lungen-6 (KL-6) and treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF) inhibitors were investigated in five Japanese clinical trials. Percentages and incidence rates were calculated for elevated serum KL-6 levels. Adverse events associated with elevated levels of serum KL-6 were investigated. In RISING, a clinical trial for infliximab, 15.6 % of the enrolled patients met criterion B (KL-6 ≥500 U/ml and >1.5-fold increase over the baseline value) by week 54. In HIKARI, 7.8 % of the certolizumab pegol (CZP) group and 0 % of the placebo group met criterion B during the double-blind (DB) period (p = 0.003). In J-RAPID, 8.4 % of the methotrexate (MTX) + CZP and 3.9 % of the MTX + placebo groups met criterion B during the DB period. In GO-MONO, 1.8 % of the golimumab (GLM) and 1.3 % of the placebo groups met criterion B during the DB period. In GO-FORTH, 7.1 % of the MTX + GLM and 0 % of the MTX + placebo groups met criteron B during the DB period (p = 0.017). No adverse events accompanied the elevation of serum KL-6 levels in 95.7 % of these patients. Serum KL-6 levels may increase during anti-TNF therapy without significant clinical events. In these patients, continuing treatment with TNF inhibitors under careful observation is a reasonable option.

Autologous bone marrow cell infusions suppress tumor initiation in hepatocarcinogenic mice with liver cirrhosis

We have previously reported the efficacy and safety of autologous bone marrow cell infusion (ABMi) therapy for liver cirrhosis patients without hepatocellular carcinoma in a multicenter clinical trial. However, since liver cirrhosis is highly oncogenic, evaluation of the effects of ABMi on the mechanisms of hepatocarcinogenesis is of great importance. Therefore, frequent ABMi was performed in hepatocarcinogenic mice, and its effects on hepatocarcinogenesis were analyzed. The N-nitrosodiethylamine (DEN)/green fluorescent protein (GFP)-carbon tetrachloride (CCl(4) ) model was developed by administering DEN once, followed by repeated administration of CCl(4) intraperitoneally as for the control group. In the administration (ABMi) group, GFP-positive bone marrow cells were infused through a tail vein. The kinetics of hepatocarcinogenesis were evaluated histologically 4.5 months after DEN treatment. At 4.5 months, there was significantly lower incidence of foci and tumors in the ABMi group, and they were smaller in number, while their size was almost equal. No GFP-positive tumors were found in ABMi livers. Moreover, ABMi livers showed significantly reduced liver fibrosis, consistent with significantly lower 8-hydroxy-2'-deoxyguanosine levels, higher superoxide dismutase activity, and increased nuclear translocation of nuclear factor-erythroid 2 p45-related factor 2. These results demonstrate that frequent ABMi might contribute to suppressed tumor initiation during stages of hepatocarcinogenesis, consistent with improvements in liver fibrosis and stabilization of redox homeostasis.

Liver tumor promoting effect of etofenprox in rats and its possible mechanism of action

To investigate the liver tumor-promoting effects of etofenprox (ETF), a pyrethroid-like insecticide, 6 week-old male F344 rats were given an intraperitoneal injection of N-diethylnitrosamine (DEN). After 2 weeks from the DEN treatment, 12 rats per group received a powdered diet containing 0, 0.25, 0.50, or 1.0% ETF for 8 weeks. At the time of 2nd week of ETF administration, all animals were subjected to two-thirds partial hepatectomy (PH). One rat per group except for the 0.25% ETF group died due to surgical operation of PH. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of DEN-initiated rats given 0.50% and 1.0% ETF compared with the DEN-alone group. Quantitative real-time RT-PCR analysis revealed that the mRNA expression of phase I enzymes Cyp2b1/2, phase II enzymes such as Akr7a3, Gsta5, Ugt1a6, Nqo1 significantly increased in the DEN+ETF groups. The immunohistochemistry showed the translocation of CAR from the cytoplasm to the nuclei of hepatocytes in the ETF-treated groups. Reactive oxygen species (ROS) production increased in microsomes isolated from the livers of ETF-treated rats, and thiobarbituric acid-reactive substances (TBARS) levels and 8- hydroxy-2-deoxyguanosine (8-OHdG) content significantly increased in all of the ETF-treated groups and DEN+1.0% ETF group, respectively. The results of the present study indicate that ETF has a liver tumor-promoting activity in rats, and suggest that ETF activates the constitutive active/androstane receptor (CAR) and enhances microsomal ROS production, resulting in the upregulation of Nrf2 gene batteries; such an oxidative stress subsequently induces liver tumor-promoting effects by increased cellular proliferation.

Mechanistic study on liver tumor promoting effects of flutamide in rats

Flutamide (FLU), a nonsteroidal anti-androgen, is used for the treatment of prostate cancer but is also a cytochrome P450 (CYP) 1A inducer. Some CYP1A inducers are known to exert hepatocellular tumor-promoting activities in rodents, and reactive oxygen species (ROS) produced by CYP1A1 induction via a metabolism of FLU is probably involved in the liver tumor promotion. In the present study, to clarify the possible liver tumor promoting effect of FLU, a two-stage liver carcinogenesis assay was performed using male F344 rats. Rats received an intraperitoneal (ip) injection of 200 mg/kg body weight of N-diethylnitrosamine (DEN) and fed a diet containing 0, 0.1 or 0.2% FLU for 6 weeks. After 2 weeks of DEN treatment, all rats were subjected to two-thirds partial hepatectomy. Animals were killed 8 weeks after ip injection of DEN. Immunohistochemically, the number and area of glutathione S-transferase placental form (GST-P)-positive foci significantly increased in the liver of rats given 0.2% FLU as compared with the control. Ki-67-positive cell ratio also increased in rats given FLU at both concentrations. ROS generation in the microsomal fraction and production of thiobarbituric acid-reactive substance [TBARS] and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content in the liver did not increase in any of the FLU-treated groups. The results of microarray and real-time RT-PCR revealed that phase 1 drug-metabolizing enzymes such as CYP1A1, Ugt1a61 and Nqo1 and phase II drug-metabolizing enzymes such as Yc2, Akr1b7, Akr1b8, Akr1b10, Aldh1a1, Gpx2 and Me1 were up-regulated in rats treated with FLU. In addition, the MAPK pathway family-related genes such as Prkcα, Mek1, Rafb, Myc, Mek2, Raf1 and Egfr were also up-regulated in FLU-treated groups. The results of the present study indicate that FLU is a CYP1A inducer but does not cause any production of microsomal ROS in the liver and suggest that microsomal ROS is not involved in the liver tumor promoting effect of FLU.

Modulatory Effects of Capsaicin on N-diethylnitrosamine (DEN)-induced Mutagenesis in Salmonella typhimurium YG7108 and DEN-induced Hepatocarcinogenesis in gpt Delta Transgenic Rats

Capsaicin from the red chili pepper is a prospective chemopreventive agent. To explore the possible antigenotoxic effects of capsaicin on N-diethylnitrosamine (DEN)-induced mutagenesis in vitro, we conducted bacterial mutation assays with Salmonella typhimurium YG7108, a sensitive strain to mutagenic alkylating agents. Capsaicin was not mutagenic either with or without S9 activation. Unexpectedly,it enhanced the mutagenicity of DEN in the presence of S9 activation significantly. Capsaicin also enhanced the mutagenicity of 2-aminoanthracene and benzo[a]pyrene in the presence of S9 activation and benzo[a]pyrene diolepoxide in the absence of S9 activation. However, it reduced the mutagenicity of ethylnitrosourea in the absence of S9 activation. To examine whether capsaicin modulates DEN-induced mutagenesis and hepatocarcinogenesis in vivo, we took advantage of gpt delta rats, transgenic rodents that carry reporter genes for mutations. Female gpt delta rats were given drinking water containing 40 ppm DEN for five weeks. They were fed diets containing capsaicin at doses of 0, 100 or 500 ppm for seven weeks, starting one week before the DEN treatment. Samples were collected at weeks 7 and 32, respectively, for mutagenicity and carcinogenicity assays. DEN enhanced gpt mutant frequency more than 200 fold in the liver. However, capsaicin displayed no modulating effects on the mutagenesis. Rather, it reduced the number of liver neoplasms, especially liver cell adenomas, in a dose-dependent manner although the reduction in hepatocellular carcinoma was statistically insignificant. These results suggest that chemopreventive effect of capsaicin against DEN-induced hepatocarcinogenesis is slight and that the effect is not due to antimutagenesis. The results also caution that chemopreventive effects of chemicals should be examined not only in vitro but also in vivo with multiple indexes, e.g., in vitro and in vivo mutations and pathological examinations.

Atividade hepatoprotetora do extrato alcoólico da Camellia sinensis (L.) Kuntze (chá-verde) em ratos Wistar tratados com dietilnitrosamina

Green tea (Camellia sinensis (L.) Kuntze) is used for its properties: antioxidant, chemoprotector and anti-inflammatory in several pathological situations, mainly those facing carcinogen chemical substances. This work has evaluated the hepatic protection effect of the green tea extract (GTE) on lipoperoxidation and necrosis, induced by the carcinogenic DEN in rats´ liver. Adult male Wistar rats were used and exposed to one only intra peritoneal dose of 200 mg/kg of DEN, and orally 120 mg/kg of ECV in different experimental moments. After 24 h of a DEN treatment, the animals were sacrificed and the following aspects were evaluated: the AST/ALT levels in plasma, lipoperoxidation of TBARS and FOX in the liver. Necrosis and Hepatic hemorrhage were observed through a histological examination. The chemoprotector action and the decrease in lipoperoxidation were detected after a decrease of AST/ALT, TBARS, FOX and hepatic necrosis. The evaluation of these results confirmed the importance of the use of the green tea as a chemoprotector agent, particularly as a preventive method.

Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats

The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.

Regulation of ser‐45 mutated beta‐catenin in transgenic mouse livers

Wnt/β‐catenin activation is frequently observed in hepatocellular carcinomas (HCC) due to multiple mechanisms. A common event is mutation at serine‐45 in β‐catenin rendering a stable protein. S45‐mutated β‐catenin was utilized to generate liver‐specific transgenic mice (Hep‐S45D‐Tg). Two independent lines were characterized and had a similar phenotype. Increased liver weight to body weight ratio from 10‐20% was observed in Hep‐S45D‐Tg (TG) relative to FVB wild‐type (WT). Increased levels of β‐catenin in the nucleus were observed at 1 month, which coincided with increased cyclin‐D1 and significantly higher Ki‐67‐positive hepatocytes. Interestingly, elevated β‐catenin was observed only at the hepatocyte membrane at 2 and 6 months with no change in proliferation but with an increase in average hepatocyte size. Decreased apoptosis was evident at 6 months only. No spontaneous tumors were observed in TG mice up to 12 months. DEN‐induced hepatocarcinogenesis was investigated in WT and TG mice. Females were protected up to 9 months. Male TG mice showed hepatic adenomas (HA) and HCC at 9 months, whereas WT mice showed HA only. Thus, TG mice demonstrated nuclear β‐catenin and activation that led to increased liver size at 1 month that was later regulated by sequestration of ‐catenin at the hepatocyte membrane. Also, TG mice showed higher incidence of HCC after DEN treatment.

β-Naphthoflavone enhances oxidative stress responses and the induction of preneoplastic lesions in a diethylnitrosamine-initiated hepatocarcinogenesis model in partially hepatectomized rats

The tumour-promoting effects of β-naphthoflavone (BNF), a novel aryl hydrocarbon receptor (AhR) agonist, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) at a dose of 200 mg/kg body weight and were fed a diet containing 0% (basal diet), 0.5% or 1% BNF for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after the BNF treatment. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of rats treated with BNF with concomitantly increased cell proliferation compared to those in the livers of the DEN alone group. Global gene expression analysis and subsequent quantitative real-time reverse transcription-polymerase chain reaction revealed that BNF induced not only the ‘AhR gene battery’ Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Aldh3a1 and Ugt1a6 but also the transcription factor NF-E2-related factor 2 (Nrf2)-regulated genes such as Gstm1, Gpx2, Akr7a3 and Yc2 (and also Nqo1), presumably due to the adaptive response against BNF-triggered oxidative stress responses. Reactive oxygen species production increased in microsomes isolated from the livers of BNF-treated rats, and this enhancement was suppressed by the P450 inhibitor SKF-525A. Furthermore, BNF enhanced oxidative DNA damage and lipid peroxidation, estimated by the levels of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances. These results suggest that the administration of BNF at a high dose and over a long-term enhance oxidative stress responses which may contribute to its hepatocarcinogenic potential in rats.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10, 25, and 50 microM, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage.

Microcystin-LR is not Mutagenic in vivo in the .LAMBDA./lacZ Transgenic Mouse (Muta Mouse)

The water pollution of toxic cyanobacteria (blue-green algae) is causing a serious public health problem in many parts of the world. Microcystin-LR (MCLR) is a potent cyclic heptapeptidic hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. MCLR presents acute and chronic hazards to human health and has been linked to primary liver cancer in humans chronically exposed to this peptide toxin through drinking water. To assess the in vivo mutagenecity of MCLR, the λ/lacZ transgenic mice (MutaTMMouse) were treated with MCLR (1 mg/kg per week x 4) and examined for mutant frequencies (MFs) in the lacZ and cII genes of liver and lungs. Micronucleus induction in peripheral blood cells was also assessed. Co-mutagenic effect of MCLR was studied in combination with N-nitrosodiethylamine (DEN). MCLR did not increase either MFs of the target genes in liver and lungs or micronucleus frequency in the peripheral blood cells of the λ/lacZ transgenic mouse. While DEN treatment increased MFs significantly, the co-administration of MCLR did not potentiate its mutagenicity. We conclude that pure MCLR has no in vivo mutagenicity as it failed to induce gene mutation and micronucleus in transgenic mouse. Its tumor promoting effect is independent of its interaction to DNA.

Essential contribution of a chemokine, CCL3, and its receptor, CCR1, to hepatocellular carcinoma progression

We previously observed that a chemokine, macrophage inflammatory protein-1 alpha/CCL3, and its receptor, CCR1, were aberrantly expressed in human hepatocellular carcinoma (HCC) tissues. Here, we show that CCL3 and CCR1 are also expressed in 2 different models of this cancer; N-nitrosodiethylamine (DEN)-induced HCC and HCC induced by hepatitis B virus surface (HBs) antigen-primed splenocyte transfer to myelo-ablated syngeneic HBs antigen transgenic mice. At 10 months after DEN treatment, foci number and sizes were remarkably reduced in CCR1- and CCL3-deficient mice, compared with those of wild-type (WT) mice, although tumor incidence were marginally, but significantly, higher in CCR1- and CCL3-deficient mice than in WT mice. Of note is that tumor angiogenesis was also markedly diminished in CCL3- and CCR1-deficient mice, with a concomitant reduction in the number of intratumoral Kupffer cells, a rich source of growth factors and matrix metalloproteinases (MMPs). Among growth factors and MMPs that we examined, only MMP9 and MMP13 gene expression was augmented progressively in liver of WT mice after DEN treatment. Moreover, MMP9, but not MMP13, gene expression was attenuated in CCR1- and CCL3-deficient mice, compared with that of WT mice. Furthermore, MMP9 was expressed mainly by mononuclear cells but not hepatoma cells, and MMP9-expressing cell numbers were decreased in CCR1- or CCL3-deficient mice, compared with WT mice. These observations suggest the contribution of the CCR1-CCL3 axis to HCC progression.

Modulation of liver tumorigenesis in Connexin32-deficient mouse

Connexin32 (Cx32) is the major gap junction forming protein in liver. Mice deficient in Cx32 demonstrate enhanced liver tumor formation, but are resistant to promotion of hepatocarcinogenesis by the model tumor promoter phenobarbital (PB). Here, we re-evaluate data on the number and sizes of glucose-6-phosphatase (G6Pase)-deficient liver lesions, both in Cx32-wildtype (WT) and Cx32-null male mice, obtained from two earlier experiments with similar protocols but paradoxical outcomes. In these experiments, enzyme-altered lesions were induced in mice of both strains by a single injection of N-nitrosodiethylamine (DEN) at age 6 weeks with a dose of 90 μg/g body weight (experiment 1) or at age 2 weeks with 10 μg/g body weight (experiment 2). Three weeks after DEN treatment groups of mice (sub-divided by Cx32 status) were also started on a PB-containing (0.05%) diet to test the responsiveness of the lesions to the tumor promoter. Additionally, for experiment 1, tumors were analyzed for the presence of Ha-ras and β-catenin mutations. Based on the mutational analysis and the mathematical analysis of the G6Pase-deficient lesions, the two studies are consistent with the hypothesis of two types of lesions, ‘late-type’ lesions which are mainly characterized by β-catenin mutations, and ‘early-type’ lesions that are frequently (but not exclusively) Ha-ras mutated. This concept affords an explanation as to the differential response seen in the two experiments with regard to Cx32 status and the role of PB as a tumor promoter (experiment 1) or inhibitor (as in experiment 2). Our findings also underscore the importance of the timing (6 weeks versus 2 weeks) of the genotoxic insult in relation to the developmental stage of the liver and the importance of clonal selection during tumor promotion.

Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay

Annatto (Bixa orellana L.) is a natural food colorant extensively used in many processed foods, especially dairy products. The lower cost of production and the low toxicity, make annatto a very attractive and convenient pigment in substitution to the many synthetic colorants. In the present study we investigate the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione S-transferase (GST-P) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after DEN treatment (200 mg/kg body wt, i.p.), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both GST-P foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver.

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; cm²) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-α 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-α-positive EAF/cm², and the reduction in the number of TGF-α-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.

Alterations of the M6p/Igf2 receptor gene in hepatocellular carcinomas induced by N-nitrosodiethylamine and a choline-deficient L-amino acid-defined diet in rats.

To elucidate whether the M6p/Igf2 receptor (M6p/Igf2r) gene might be involved in exogenous and endogenous liver carcinogenesis, we investigated its alteration in hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) and by a choline-deficient L-amino acid-defined (CDAA) diet in rats. Male F344 rats, 6 wk old, received a single intraperitoneal (i.p.) injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell cycle disturbance, and a selection procedure regimen, HCCs being obtained after 42 wk. With continuous CDAA diet feeding, tumors were sampled after 75 wk. Total RNA was extracted from individual HCCs for assessment of mutations within exons 27, 28, 31, 33, and 34, and aberrant transcript of the M6p/Igf2r gene by reverse transcription (RT)-polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and RT-PCR analyses, respectively. Mutations were detected in three of 15 HCCs (20%) induced by the CDAA diet, a TTT to TTG (Phe to Leu) transversion at codon 1516 and two AAG to AGG (Lys to Arg) transitions at codon 1620, but in none of those caused by DEN. Aberrant transcripts were found in seven of 15 HCCs after DEN treatment (46.7%) and in two of 15 HCCs induced by the CDAA diet (13.3%). These results suggest that alterations of the M6p/Igf2r gene may be involved in both exogenous and endogenous liver carcinogenesis with the different patterns and frequencies.

Effect of dietary selenite on N-nitrosodiethylamine-induced and phenobarbital promoted multistage hepatocarcinogenesis in rat: reflection in some minerals

Selenium (Se), a dietary micronutrient, plays a vital role in cancer chemotherapy in many organs including the liver. We have studied the relationship between some minerals, which are essential in normal functioning of cells and anticancer effect of Se in N-nitrosodiethylamine (DEN) induced and phenobarbital (PB) promoted multistage hepatocarcinogenesis. Se (4 ppm through drinking water; as sodium selenite) was given to animals throughout the study, before initiation and during promotion phase of hepatocarcinogenesis, in a defined experimental protocol. Se, sodium, potassium, calcium and iron were measured either in hepatoma, or surrounding liver tissue or whole liver tissue and serum of experimental animals. DEN and PB treatment significantly ( P < 0.001) increased potassium, calcium and iron levels in serum, while it decreased ( P < 0.001) the Se and sodium levels when compared with control rats. We have also observed significantly increased ( P < 0.001) sodium, calcium and iron levels in hepatoma and surrounding liver tissue, whereas, Se, and potassium level was found to be decreased ( P < 0.001) when compared with control rats. Supplementation of selenite throughout the study, before initiation and during promotion stage significantly alters the above mineral content. Results showed that the most significant beneficial effect of selenium during hepatocarcinogenesis was exerted potentially in long-term continuous and/or before the initiation phase of carcinogenicity, rather than in the promotion phase. The present and previous results from our laboratory suggest that sub-optimal intake of a single trace mineral can have broad effects on chemotherapy, providing a framework for understanding the multiple beneficial effects of selenium in cancer chemoprevention.

WY-14,643-mediated promotion of hepatocarcinogenesis in connexin32-wild-type and connexin32-null mice

Connexin32 (Cx32) is the major gap junction forming protein in liver and lack of functional Cx32 enhances hepatocarcinogenesis. Many tumour-promoting agents block gap junctional intercellular communication, which may favour clonal expansion of neoplastic cells. We recently demonstrated that liver tumourigenesis is accelerated in Cx32-wild-type but not in Cx32-null mice by the model tumour promoter phenobarbital (PB). In the present study, male Cx32-wild-type and Cx32-null mice were treated with a single injection of 90 micro g/g body wt of N-nitrosodiethylamine (DEN) at 6 weeks of age and were subsequently kept on a diet containing the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) or on control diet. Thirty-eight weeks after DEN treatment, mice were killed, frozen liver sections were prepared and (pre)-neoplastic lesions were identified by alterations in glucose-6-phosphatase (G-6-Pase) and glutamine synthetase (GS) staining. G-6-Pase-deficient lesions were generally small in size and were observed in all groups of mice. Large focal pre-neoplastic and benign neoplastic lesions, however, which demonstrated increased rather than decreased activity in G-6-Pase were exclusively present in DEN/Wy-14,643-treated mice. G-6-Pase-positive lesions were strongly promoted by Wy-14,643, both in Cx32-wild-type and Cx32-null mice without significant difference in response between mice of the two genotypes. This contrasts G-6-Pase-negative lesions and lesions over-expressing GS, which were both increased by WY-14,643 treatment in number and size in Cx32-wild-type but not in Cx32-null mice. GS-positive lesions from WY-14,643-treated mice harboured beta-catenin mutations, a hallmark of lesions selected during promotion by PB, while G-6-Pase-positive lesions, which displayed negative or diffuse GS staining, did not show beta-catenin mutations. Our results demonstrate significant differences between mouse liver lesions of differing phenotype and genotype in their response towards selection by Wy-14,643 during the promotional phase of hepatocarcinogenesis.

Tumor promotion by metanil yellow and malachite green during rat hepatocarcinogenesis is associated with dysregulated expression of cell cycle regulatory proteins.

Metanil yellow (MY) and malachite green (MG) are textile dyes, which, despite a ban, are used as food-coloring agents. MY and MG have promoter effects on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine (DEN). Tumor-promoting agents are not mutagenic but may alter the expression of genes whose products are associated with hyper-proliferation, tissue remodeling, and inflammation. Cell cycle controls normally function to ensure the integrity of the genome and arrest of cells at G1/S or G2/M checkpoints until all the prerequisite events are completed. In order to understand the mechanism(s) of tumor promotion by MY and MG, we have studied the levels of PCNA, a marker of cell proliferation and cell cycle regulatory proteins, cyclin D1, and its associated kinase, cdk4, cyclin B1, and associated kinase, cdc2. Immunohistochemical staining showed an elevated level of PCNA in animals administered MY and MG subsequent to DEN treatment. Western and Northern blot hybridization showed an increased expression of both cyclin D1 and its associated kinase cdk4, and cyclin B1 and its associated kinase cdc2, in livers of rats administered MY and MG after administration of DEN as compared to untreated or DEN controls. The increased level of mRNA was due to the increased rate of transcription of these genes as studied by run-on transcription assay. These data obtained by the in vivo model of liver tumor development provide strong evidence for a link between dysregulation of the two critical checkpoints of the cell cycle as one of the possible mechanism(s) during tumor promotion by malachite green and metanil yellow.