Glutathione S-transferase Placental Form Research Articles

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.

Decreased Diethylnitrosamine-induced Liver Preneoplastic Lesions by Estradiol-3-benzoate Treatment

To clarify whether inhibitory effect of estrogen on liver tumor is associated with cell proliferation, we investigated its role in diethylnitrosamine (DEN)-induced rat preneoplastic lesions, with time sequenced manners. F344 male rats (n = 90) were divided into three groups at 5 weeks of age. The mini-osmotic pumps providing a continuous infusion of DEN was implanted into the abdominal cavity of each animal in group 1, 2 and 3 at 6 weeks of age. To see the effect of estrogen, pellet containing 1 or 10 μg of estradiol- 3-benzoate (EB) was implanted subcutaneously in the animals of groups 2 or 3, respectively, one week prior to DEN treatment. Ten animals of each group were euthanized at 10, 14 and 18 weeks after DEN treatment. Liver tissues at each time point were fixed in 10% phosphate-buffered formalin and were processed and embedded in paraffin and 5 μm sections mounted on a silanized slide. Glutathione S-transferase placental form (GST-P) positive foci and 5-bromo-2-deoxyuridine (BrdU) labeling cells were detected at each time point. Area of GST-P positive foci in DEN+EB 1 or 10 μg group was significantly decreased compared to DEN alone at 14 weeks (p < 0.01 or p < 0.05, respectively) an at 18 weeks (p < 0.05 or p < 0.01, respectively). BrdU index in DEN+EB 1 or 10 μg groups was significantly decreased compared to DEN alone at 14 weeks and at 18 weeks (p < 0.01). Taken together, we conclude that EB treatment decrease the DEN-induced liver preneoplastic lesions and this may be associated with decrease of 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.

Comprehensive toxicity study of safrole using a medium-term animal model with gpt delta rats

In order to investigate a medium-term animal model using reporter gene transgenic rodents in which general toxicity, genotoxicity and carcinogenicity are evaluated, F344 gpt delta rats were given a diet containing 0.1% and 0.5% (a carcinogenic dose) safrole for 13 weeks. Serum biochemistry and histopathological examinations revealed overt hepatotoxicity of safrole, in line with previous reports. In the current study, safrole treatment possibly resulted in renal toxicity in male rats. In the in vivo mutation assays, an increase or a tendency to increase of the gpt mutant frequencies (MFs) was observed in both sexes at the carcinogenic dose. The number and area of foci of glutathione S-transferase placental form (GST-P) positive hepatocytes, ratio of proliferating cell nuclear antigen (PCNA)-positive hepatocytes and 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA were significantly increased in both sexes of the 0.5% group. The overall data suggested that the present model might be a promising candidate for investigating comprehensive toxicities of the agents. In addition, data demonstrating the base modification and cell proliferation due to exposure to safrole could contribute to understanding safrole-induced hepatocarcinogenesis, which imply expanding in application of this model.

Medium-term multi-organ carcinogenesis bioassay of ethyl tertiary-butyl ether in rats

The modifying potential of ethyl tertiary-butyl ether (ETBE) on tumor development was investigated in a medium-term multi-organ carcinogenesis bioassay using male F344 rats. Animals were sequentially given 5 carcinogens with different target sites in the first 4 weeks for multi-organ initiation. After one week they received ETBE by gavage at dose levels of 0 (control), 300 or 1000 mg/kg/day until experimental week 28. Further groups were also given ETBE at doses of 0 or 1000 mg/kg/day without prior carcinogen application. Incidences and multiplicities of follicular cell hyperplasias and neoplasms in the thyroid were significantly increased at dose levels of more than 300 mg/kg/day. Combined incidences of squamous cell hyperplasias and papillomas of the forestomach were also significantly increased at 300 and 1000 mg/kg/day. Incidences and multiplicities of adenocarcinomas in the colon were increased at 1000 mg/kg/day. The numbers and areas of glutathione S-transferase placental form (GST-P) positive foci per unit area of the liver sections, and the incidence of hepatocellular adenomas were also significantly increased at 1000 mg/kg/day, along with multiplicities of atypical hyperplasias of renal tubules of the kidney and the incidence of papillomatosis of the urinary bladder. This latter lesion was also seen at low incidence at 1000 mg/kg/day without initiation. Thus, the current results indicate that ETBE has tumor promoting potential for the thyroid and forestomach at dose levels of 300 mg/kg/day and more, and for the colon, liver, kidney and urinary bladder at 1000 mg/kg/day, under the present experimental conditions.

Abstract 3684: Saffron protects against liver cancer in animal model: Molecular and imaging insights

Abstract Saffron is a natural product that is a promising chemo-preventive agent against different cancer. The aim of the present work was to investigate the chemo-protective effect of saffron ethanol extract on chemically-induced liver cancer in rats. Hepatocarcinogenesis was induced by a single intraperitoneal injection of diethyl nitrosamine (DEN) (200 mg/kg body weight) and 2 weeks later, the carcinogenic effect was promoted by 2-acetylaminofluorene (2-AAF) (0.05%). 42 Wister rats were divided into seven groups: negative control, positive control (DEN+2-AAF), saffron-alone group (saffron for 22 weeks, no DEN or 2-AAF) and 3 preventive groups (where saffron was administered at 300, 150 and 75 mg/kg for 2 weeks before and 20 weeks after inducing cancer). The incidence and multiplicity of liver tumors as well as the expression of cancer marker, glutathione S-transferase placental form, were significantly reduced in preventive groups. This hepatopreventive effect was confirmed by the decreasing in cancer marker, α feto protein in plasma. Immunohistochemical analyses showed a decrease in cell proliferation marker (Ki67) and an increase of TUNEL-and caspase 3-positive apoptotic cells along with strong expressions of p53 in liver of preventive groups. These protective effect found to be mediated by anti-inflammatory and antioxidant effects. The anti-inflammatory activity was confirmed by saffron-based inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. The effect of saffron on the expression of nuclear factor nuclear factor-Kappa B (NF-kappa B) was also assessed. The expression of NF-kappa B in the preventive groups was significantly low compared to positive control. The in vitro antioxidant capacity of saffron (FRAP Assay, DPPH Scavenging activity and ABST+ Assay) was significantly high in ethanol-based extract. In addition, NF-kappa B transcription factor seems to regulate the saffron chemo-protective effects. Finally, texture analysis was applied to analyze in vivo MRI images and results were consistent with that of histopathology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2011-3684

Abstract 1340: Examination of in vivo mutagenicity and carcinogenicity in the gpt delta rat with Dammar resin

Abstract Dammar resin is a food additive as a thickening agent. The purposes of this study are to evaluate carcinogenicity and in vivo mutagenicity of Dammar resin in gpt delta rats. 6 week-old male gpt delta rats were divided into 5 groups. Groups 1-3 were treated with five carcinogen (DMBDD) during the first 4 weeks of experiment as follows: diethylnitrosamine (100 mg/kg b.w. i.p.), N-methyl-N-nitrosourea (20mg/kg b.w. i.p.), 1,2-dimethylhydrazine (40 mg/kg b.w. s.c.), 0.05% N-butyl-N-(4-hydroxypropyl) nitrosamine in the drinking water from week 1 to 2, and 0.1% dihydroxybutyl-di-N-propylnitrosamine in the drinking water from week 3 to 4. Groups 4-5 were given vehicle. From experimental week 5, groups 1-3 were treated with Dammar resin in diet at doses of 0, 0.03, and 2% for 13 weeks. Groups 4-5 were treated with Dammar resin in diet at doses of 0, and 2%, respectively. At 18 weeks after starting the experiment, quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci, which are preneoplastic lesions in the rat liver and mutation assays (gpt and Spi- mutation assays) were performed. Both numbers and area of GST-P positive foci were significantly increased in rats administered DMBDD with 2% Dammar resin (group 3) compared to rats administered DMBDD alone (group 1). 0.03% Dammar resin (group 2) had no effects on the development of GST-P positive foci. There were no significant differences in mutant frequencies of gpt and red/gam genes in the livers between rats administered basal diet (group 4) and 2% Dammar resin (group 5). These findings indicated that Dammar resin exerts promotion effect on liver carcinogenesis but lack of in vivo mutagenicity in the livers of gpt delta rats. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1340. doi:10.1158/1538-7445.AM2011-1340

Indole-3-carbinol enhances oxidative stress responses resulting in the induction of preneoplastic liver cell lesions in partially hepatectomized rats initiated with diethylnitrosamine

The liver tumor-promoting effects of indole-3-carbinol (I3C), a cytochrome P450 (CYP) 1A inducer found in cruciferous vegetables, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing 0 (DEN-alone), 0.25, 0.50 or 1.0% of I3C for 8 weeks from 2 weeks after DEN-initiation. The number and area of liver cell foci positive for glutathione S-transferase placental form (GST-P) significantly increased in the livers of rats given 0.5% I3C or more, compared to those in the DEN-alone group. The number of GST-P positive foci also increased in the 0.25% I3C group. The number of liver cells positive for proliferating cell nuclear antigen (PCNA) significantly increased in all I3C groups compared to that in the DEN-alone group. Real-time RT-PCR analysis showed that I3C increased transcript levels of not only Cyp1a1 but also aryl hydrocarbon receptor (AhR) and/or nuclear factor (erythroid-derived 2)-like 2 (Nrf2) gene batteries, such as Cyp1a2, Cyp1b1, Ugt1a6, Nrf2, Nqo1, Gsta5, Gstm2, Ggt1and Gpx2. Reactive oxygen species (ROS) in the microsomal fraction significantly increased in all I3C-treated groups compared to the DEN-alone group, and thiobarbituric acid-reactive substances (TBARS) levels and 8-hydroxy-2′-deoxyguanosine (8-OHdG) content significantly increased in all of the I3C-treated groups and 1.0% I3C group, respectively. These results suggest that I3C is an AhR activator and enhances microsomal ROS production resulting in the upregulation of Nrf2 gene batteries, but the oxidative stress generated overcomes the antioxidant effect of Nrf2-related genes. Such ‘a redox imbalance’ subsequently induces liver tumor-promoting effects by enhancing cellular proliferation in rats.

Quercetin Inhibits Diethylnitrosamine-Induced Hepatic Preneoplastic Lesions in Rats

Quercetin is an antioxidant flavonoid, found ubiquitously in nature and extensively used in herbal medicines and food additives. This study aimed to investigate the effect of quercetin on diethylnitrosamine-induced preneoplastic lesions, using the medium-term rat liver bioassay. The γ-benzene hexachloride was used as promoter at the doses of 0.1, 1.0, and 10.0 mg/kg against a single dose of diethylnitrosamine (200 mg/kg) in male Sprague-Dawley rats. All the rats were subjected to 70% partial hepatectomy at Week 4. The protective effect of quercetin (5 and 25 mg/kg) was examined against the highest dose of γ-benzene hexachloride (10 mg/kg). A significant increase in the number as well as the mean area of glutathione S-transferase placental form (GST-P) positive foci, p53 positive hepatocytes, and the percentage of apoptotic cells were observed in the diethylnitrosamine-treated group. In the present investigation, both doses of QC (5 and 25 mg/kg) led to a significant decrease in the number as well as the mean area of GST-P positive foci, TUNEL positive apoptotic cells, p53 positive hepatocytes, and restoration of cellular morphology. These results clearly indicate that quercetin inhibits diethylnitrosamine-induced hepatic preneoplastic lesions in medium-term rat liver bioassay.

Concomitant apoptosis and regeneration of liver cells as a mechanism of liver-tumor promotion by β-naphthoflavone involving TNFα-signaling due to oxidative cellular stress in rats

β-Naphthoflavone (BNF) is a strong inducer of cytochrome P450 1A enzymes, and exerts liver tumor-promoting activity through enhancement of oxidative stress responses in rats. This study investigated the role of the tissue environment surrounding hepatocellular preneoplastic lesions in the early tumor-promotion stage by BNF, using enzymatically modified isoquercitrin (EMIQ) as an anti-oxidative chemopreventive agent. Male F344 rats were fed a diet containing BNF (0.5%) for 6 weeks, with or without EMIQ (0.2%) in the drinking water, 2 weeks after initiation with N-diethylnitrosamine, and were subjected to two-thirds partial hepatectomy 1 week after starting BNF-promotion. BNF-treatment increased concentrations of liver thiobarbituric acid-reactive substances, single liver cells expressing glutathione S-transferase placental form or heme oxygenase (HO)-1, and concomitant apoptosis and proliferation of liver cells. Transcript upregulation of anti-oxidative enzymes ( Aldh1a1 and Nqo1), cell cycle-related molecules ( Cdc20 and Cdkn2b) and inflammation-related molecules including proinflammatory cytokines ( Ccl2, Col1a1, Il6, Nos2 and Serpine1) was also evident. Furthermore, BNF increased HO-1-expressing Kupffer cells and liver cells expressing tumor necrosis factor receptor 1 (TNFR1) and the TNFR1-associated death domain. Most of these BNF-induced fluctuations disappeared or were suppressed by EMIQ in conjunction with suppression of tumor-promotion. Tnf transcript levels with BNF were also suppressed by EMIQ. These results suggest that BNF-induced oxidative stress causes single liver cell toxicity, allowing subsequent concomitant apoptosis and regeneration involving inflammatory responses including TNFα-signaling, contributing to tumor promotion. Kupffer cells may act to protect against inflammatory stimuli induced as a result of oxidative cellular stress by BNF, causing proinflammatory cytokine level fluctuations.

Histone Methyltransferase PR-Set7 and Histone Variant H2A.Z, Induced during Hepatocarcinogenesis, Repress the Promoter Activity of the Tumor Marker Gene and the Ras-Induced Colony Formation Activity

Genetic and epigenetic studies are required to understand molecular mechanisms of carcinogenesis and tumorigenesis. Although alterations of DNA methylation and histone modification profiles are observed in cancer cells, the knowledge of the epigenetic regulatory factors involved in carcinogenesis is insufficient. In this study, we showed that the histone variant, H2a.z, and the histone methyltransferase, Pr-set7/Set8/KMT5a, were up-regulated during chemically induced hepatocarcinogenesis. During this process, the glutathione S-transferase placental form (GST-P), which is completely repressed in normal liver, is strongly induced and is therefore an excellent tumor marker. Reporter analysis performed using the regulatory region of the Gst-p gene revealed that H2A.Z and PRSET7 repressed Gst-p promoter activity. The enhancer element responsible for hepatocarcinogenic-specific gene expression was required for repression by H2A.Z, and the negative regulation by PR-SET7 mediated the regulatory element but not the enhancer. Furthermore, we examined the effects of overexpression of H2A.Z and PR-SET7 on the colony formation activity of mouse NIH-3T3 cells grown on a soft agarose medium. Colony formation based on anchoring independent cell growth is a feature of malignant transformed cells. These factors did not exhibit colony formation activity but repression of the ras oncogene induced this activity. PR-SET7 suppressed RAS val12 -mediated colony formation through methyltransferase activity. These results suggest that H2a.z and Pr-set7 that are induced during hepatocarcinogenesis may function as carcinogenesis suppressors.

Protective effects of fermented rice vinegar sediment (Kurozu moromimatsu) in a diethylnitrosamine-induced hepatocellular carcinoma animal model

Kurozu moromimatsu is the sediment of Kurozu, a jar-fermented Japanese black vinegar produced from unpolished rice. Here, we examined the protective effects of Kurozu moromimatsu in a diethylnitrosamine-induced model of hepatocellular carcinoma. Thirty-two F344 rats were divided into two groups; the control group received basal CE-2 diet, and the Kurozu moromimatsu group received CE-2 diet containing Kurozu moromimatsu. At 16 weeks after initial intraperitoneal administration of diethylnitrosamine (150 mg/kg/week), serum was collected from half the rats. These rats were sacrificed and the liver was resected for histological examination of hematoxylin-eosin-stained sections and assay of matrix metalloproteinase-2 and matrix metalloproteinase-9 levels in tumor tissues. Glutathione S-transferase placental form-positive foci were evaluated by immunostaining for glutathione S-transferase placental form. The remaining rats were maintained for evaluation of survival. There were no significant differences of serum transaminases, tumor necrosis factor-alpha, and also no marked hepatic histological differences, between the two groups. However, the size of hepatocellular carcinomas was greatly decreased and the levels of activated matrix metalloproteinase-2 and -9 were significantly reduced in the Kurozu moromimatsu group. Further, survival was significantly prolonged in the Kurozu moromimatsu group compared with the control. These results indicate that Kurozu moromimatsu inhibited the growth of hepatocellular carcinoma.

Evaluation of in vivo genotoxic potential of fenofibrate in rats subjected to two-week repeated oral administration

Fenofibrate (FF), a peroxisome proliferator-activated receptor-alpha agonist, has been used as one of the hypolipidemic drugs in man and induces oxidative stress and promotes hepatocarcinogenesis in the liver of rodents. This chemical belongs to a class of non-genotoxic carcinogens, but DNA damage secondary to oxidative stress resulting from reactive oxygen species (ROS) generation is suspected in rodents given this chemical. To examine whether FF has genotoxic potential, partially hepatectomized F344 male rats were treated orally with 0, 1,000 or 2,000 mg/kg of FF for 2 weeks, followed by diet containing 0.15% 2 acetyl aminofluorene (2 AAF) for enhancement the tumor-promoting effect for 10 days and a single oral dose of carbon tetrachloride (CCl4) as the first experiment (liver initiation assay). As the second experiment, the in vivo liver comet assay was performed in hepatectomized rats, and the expression of some DNA repair genes was examined. In the liver initiation assay, the number and area of glutathione S-transferase placental form (GST-P)-positive single cells and foci did not increase in the FF treated groups. In the comet assay, positive results were obtained after 3 h of the last treatment of FF, and the expression of some DNA repair genes such as Apex1, Ogg1 and Mlh1 were upregulated in rats given the high dose of FF at 3 h after the treatment but not in 24 h after the treatment. The results of the present study suggest that FF causes some DNA damage in livers of rats, but is not a strong genotoxic substance leading to a DNA mutation since such DNA damage was repaired by the increased activity of some DNA repair genes.

Non-genotoxic mode of action and possible threshold for hepatocarcinogenicity of Kojic acid in F344 rats

Kojic acid (KA), a naturally occurring compound, is contained in traditional Japanese fermented foods and is used as a food additive, preservative and a dermatological skin-lightening agent. In the present experiment, initiation (experiment 1) and promotion (experiment 2) effects of KA-induced hepatocarcinogenesis were studied by rat medium-term bioassay for carcinogenicity. Male F344 rats were administered a diet containing 0–2% KA. Experiment 1 demonstrated that KA had no effect on induction of liver preneoplastic lesions or glutathione S-transferase placental form (GST-P) positive foci, in either number or area. In experiment 2, 2% KA treatment significantly increased the number and area of GST-P positive foci, but concentrations less than 0.5% did not. Moreover, 2% KA treatment significantly increased 8-OHdG levels and PCNA positive hepatocytes. The results indicated that low concentrations of KA do not have initiation effects on rat hepatocarcinogenesis, while higher concentrations of KA do promote hepatocarcinogenesis in rats. Thus, the results indicate that KA is a non-genotoxic hepatocarcinogen, showing the possible existence of a perfect threshold.

Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non-genotoxic hepatocarcinogens with cytochrome P450-inducible potency

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP-inducible potency. In the present study, we investigated 8-hydroxydeoxyguanosine (8-OHdG) levels, in vivo mutagenicity and glutathione S-transferase placental form (GST-P)-positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time-dependent and statistically significant increases in 8-OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)-positive hepatocytes at 4 weeks, while no increase in 8-OHdG levels was found in PhB-treated rats. No changes in mutant frequencies of gpt and red/gam (Spi(-)) genes in liver DNA from PBO- or PhB-treated rats were observed at 4 or 13 weeks. A 13-week exposure to either PBO or PhB did not affect the number and area of GST-P-positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8-OHdG in PBO-treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8-OHdG-related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation.

2-Amino-3,8-Dimethylimidazo[4,5-f ]Quinoxaline (MeIQx)–Induced Hepatocarcinogenesis Is Not Enhanced by CYP1A Inducers, Alpha- and Beta-Naphthoflavone

Interaction of more than two chemicals from foods is a very important factor for carcinogenic risk assessment and management. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic heterocyclic amines in cooked foods, is speculated to be a human liver carcinogen. MeIQx is metabolically activated by CYP1A2 and then N-acetyltransferase (NAT), findings that suggest that its carcinogenic potential might be enhanced by simultaneous exposure to chemical(s) inducing CYP1A2. Therefore, we here investigated the effects of alpha- and beta-naphthoflavone as CYP1A2 inducers on MeIQx-induced rat hepatocarcinogenesis in a medium-term rat liver bioassay. Unexpectedly, no modifying influence of naphthoflavones on MeIQx-induced hepatocarcinogenesis was demonstrated with reference to glutathione S-transferase placental form (GST-P) positive foci in the liver, although up-regulation of CYP1A2 was detected on Western blot analysis. Activity of NAT was not affected. In MeIQx-treated rats, CYP1A expression was mainly detected in zone 3 of the liver where GST-P positive foci were preferentially located, while naphthoflavones alone or combinations of naphthoflavones and MeIQx induced CYP1A expression in zone 1. This difference in intralobular distribution of CYP1A might be related to the fact that MeIQx hepatocarcinogenesis was not modified by the two CYP1A inducers.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

To clarify the involvement of signaling of transforming growth factor (TGF)-β during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6weeks after starting promotion) and late-stage promotion (57weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 — identified as Smad3 by later immunoblot analysis — increased in the subpopulation of GST-P+ foci, while Smad4, a nuclear transporter of Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P+ lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P+ foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P− foci induced by promotion with agonists of peroxisome proliferator-activated receptor-α did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P+ proliferative lesions.

Dietary lycopene and tomato extract supplementations inhibit nonalcoholic steatohepatitis‐promoted hepatocarcinogenesis in rats

Epidemiological and experimental studies provide supportive evidence that lycopene (LY), a major carotenoid from tomatoes and tomato products, may act as a chemopreventive agent against certain types of cancers. We recently showed that high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) promoted diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in a rat model. Using this model, we investigated the efficacy of an equivalent dosage of dietary LY from either a pure compound or a tomato extract (TE) against NASH-promoted hepatocarcinogenesis. Six groups of rats were injected with DEN and then fed either Lieber-DeCarli control diet or HFD with or without LY or TE for 6 weeks. Results showed that both LY and TE supplementations significantly decreased the number of altered hepatic foci expressing the placental form of glutathione S-transferase in the livers of HFD-fed rats. This was associated with significantly lower proliferating cell nuclear antigen positive hepatocytes and cyclinD1 protein, as well as decreased activation of extracellular signal-regulated kinase and nuclear NF-kappaB. Although both LY and TE supplementations reduced HFD-induced lipid peroxidation in the livers, we observed significantly decreased cytochrome P450 2E1, inflammatory foci and mRNA expression of proinflammatory cytokines (TNF-alpha, IL-1beta and IL-12) in the HFD+TE fed group but increased nuclear NF-E2-related factor-2 and heme oxygenase-1 proteins in the HFD+LY fed group, relative to HFD feeding alone. These data indicate that LY and TE can inhibit NASH-promoted hepatocarcinogenesis mainly as a result of reduced oxidative stress, which could be fulfilled through different mechanisms.

Detection of Initiation Activity of 1,2-Dimethylhydrazine in in vivo Medium-Term Liver Initiation Assay Ssytem using 4-Week-Old Rats without Hepatocellular Proliferative Stimuli during the Test Chemical Treatment Period

We have developed an in vivo medium-term liver initiation assay system to detect initiation activities of chemicals on multi-organ carcinogenesis. However, cell proliferation stimuli during the test chemical treatment period, required in the previously used assay models using adult rats, are laborious; moreover, those cause decrease of hepatic metabolic enzymes and psychological and physical discomfort to animals resulting in inaccurate interpretation. Therefore, we investigated the utility of another in vivo medium-term liver initiation assay model using 4-week-old rats without the cell proliferation stimuli. In this study, we confirmed that 4-week-old and 4.5-week-old male rats have high hepatocyte proliferation activity and similar enzyme activities of hepatic Cytochrome P450 subtypes as compared with 8-week-old male rats. Next, the in vivo medium-term liver initiation assay model using 4-week-old rats without cell proliferation stimuli was evaluated for the detection of the initiation activity of 1,2-dimethylhydrazine (DMH), which is a well-known genotoxic carcinogen. Four-week-old rats were orally administered DMH (single dose, 4 or 16 mg/kg; or 4-day repeat, 1 or 4 mg/kg); subsequently, these rats were treated promotion treatment consisted of administration of 2-acetylaminofluorene and carbon tetrachloride. Four weeks after the first DMH administration, the glutathione S-transferase placental form (GST-P)-positive foci induced by DMH in the liver was measured immunohistochemically. The inductions of GST-P-positive foci in all DMH-treated groups were dose-dependent, duration-dependent and significantly higher than that in non-DMH-treated group. From these results, our assay model was detected the initiation activity of DMH simply, and would be useful to evaluate the carcinogenicity of chemicals.

A Medium-Term, Rapid Rat Bioassay Model for the Detection of Carcinogenic Potential of Chemicals

The Ito Liver Model and the Ito Multi-organ Model are used in conjunction and constitute an efficient and rapid bioassay for the identification of both genotoxic and nongenotoxic carcinogenic chemicals. The Ito Liver Model is an 8-week bioassay system that uses the number and size of foci of hepatocytes positive for glutathione S-transferase placental form (GST-P) as the end-point marker. One hundred fifty-nine compounds were tested using the Ito Liver Model: 61 of 66 hepatocarcinogens tested positive, and 10 of 43 nonliver carcinogens were also positive. The false-positive detection of noncarcinogens was low; a single false-positive result was obtained from the 50 noncarcinogens tested. Since more than half of all known carcinogens are hepatocarcinogens in rodents, the initial 8-week bioassay is able to detect most carcinogens. The Ito Multi-organ Model is a 28-week bioassay system for the detection of carcinogens that were not identified by the Ito Liver Model. Results are evaluated by preneoplastic and neoplastic lesions in major organs. Forty-four compounds were tested using the Ito Multi-organ Model: 17 out of 17 liver carcinogens were positive, and 19 out of 22 (86%) nonliver carcinogens were positive. None of the five noncarcinogens tested positive.