Global DNA methylation screening of liver in piperonyl butoxide-treated mice in a two-stage hepatocarcinogenesis model.

Disruptive epigenetic gene control has been shown to be involved in carcinogenesis. To identify key molecules in piperonyl butoxide (PBO)-induced hepatocarcinogenesis, we searched hypermethylated genes using CpG island (CGI) microarrays in non-neoplastic liver cells as a source of proliferative lesions at 25 weeks after tumor promotion with PBO using mice. We further performed methylation-specific polymerase chain reaction (PCR), real-time reverse transcription PCR, and immunohistochemical analysis in PBO-promoted liver tissues. Ebp4.1, Wdr6 and Cmtm6 increased methylation levels in the promoter region by PBO promotion, although Cmtm6 levels were statistically non-significant. These results suggest that PBO promotion may cause altered epigenetic gene regulation in non-neoplastic liver cells surrounding proliferative lesions to allow the facilitation of hepatocarcinogenesis. Both Wdr6 and Cmtm6 showed decreased expression in non-neoplastic liver cells in contrast to positive immunoreactivity in the majority of proliferative lesions produced by PBO promotion. These results suggest that both Wdr6 and Cmtm6 were spared from epigenetic gene modification in proliferative lesions by PBO promotion in contrast to the hypermethylation-mediated downregulation in surrounding liver cells. Considering the effective detection of proliferative lesions, these molecules could be used as detection markers of hepatocellular proliferative lesions and played an important role in hepatocarcinogenesis.

Rapid deposition of glomerular IgA in BALB/c mice by nivalenol and its modifying effect on high IgA strain (HIGA) mice.

To clarify the underlying mechanisms of IgA nephropathy (IgAN) induced by nivalenol (NIV), a trichothecene mycotoxin, we examined the time and dose relationships of glomerular deposition of IgA by NIV in BALB/c mice (Experiment 1), and also evaluated the modification of NIV on spontaneous IgAN in an inbred murine model, a high IgA strain (HIGA), during its early stage of pathogenesis (Experiment 2). In Experiment 1, female BALB/c mice were given a diet containing 0, 12, or 24 ppm concentration of NIV for 4 or 8 weeks. An increase in serum IgA levels was found at 24 ppm from 4 weeks. At week 8 of treatment, dose-dependent increases in serum IgA levels and glomerular deposition of IgA and IgG were observed without accompanying histopathological glomerular changes. On the other hand, in Experiment 2, control HIGA mice exhibited rather high levels of serum IgA as compared with BALB/c mice from 4 weeks of experiment as well as glomerular deposition of IgA and IgG and mesangial proliferation as revealed at week 8. NIV at 24ppm further increased serum IgA in this strain; however, it did not enhance glomerular immunoglobulin deposition or histopathological lesion. These results suggest that NIV-induced increase of serum IgA levels may be primarily responsible for glomerular immunoglobulin deposition; however, NIV does not enhance glomerular IgA deposition that may lead to exacerbation of predisposed IgAN in the short term, irrespective of the further elevation of serum IgA from the high basal levels.

Immunohistochemical analyses at the late stage of tumor promotion by oxfendazole in a rat hepatocarcinogenesis model.

The present study was performed to characterize immunohistochemically the expression levels of molecules related to not only xenobiotic and antioxidant functions but also cell proliferation and apoptosis in neoplastic lesions induced by the benzimidazole anthelmintic, oxfendazole (OX), at the late stage of its tumor promotion in a rat hepatocarcinogenesis model. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and 2 weeks later they were fed a diet containing 0% (basal diet) or 0.05% OX for 26 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and killed at week 28. Histopathologically, OX increased the incidence and multiplicity of altered foci (4.0- and 3.6-fold, respectively) and hepatocellular adenomas (HCAs) (3.0- and 5.5-fold, respectively). OX treatment induced 5.2- and 5.6-fold increases in the number of proliferating cell nuclear antigen (PCNA)-positive cells and single-stranded DNA (ssDNA)-positive cells in HCAs compared with the surrounding tissue, respectively. Staining for the cell cycle regulators P21 and C/EBPα and the AhR-regulated CYP1A1 molecules decreased but increased reactivity of the Nrf2-regulated, detoxifing/antioxidant molecules aldo-keto reductase 7 (AKR7) and glutathione peroxidase 2 (GPX2) were also seen in HCAs compared with the surrounding hepatocytes. These results suggest that dysregulation of cell proliferation and apoptosis and escape from oxidative stress elicited by OX treatment play an important role in OX-induced hepatocarcinogenesis in rats.

The threshold dose for liver tumor promoting effects of dicyclanil in ICR mice.

To determine the threshold dose of dicyclanil (DC) that induces hepatocellular tumor-promoting effects associated with reactive oxygen species (ROS) generation via their metabolic pathways, partial hepatectomized ICR male mice were fed diets containing 0, 187.5, 375 or 750 ppm DC after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Immunohistochemically, the proliferating cell nuclear antigen (PCNA)-positive cell ratio was significantly increased in the DEN + 750 ppm DC group compared with the DEN alone group. However, significant increases in the number of gamma-glutamyltranspeptidase (GGT)-positive cells and formation of microsomal ROS were not observed in the DEN + DC groups compared with the DEN alone group. Real-time polymerase chain reaction (RT-PCR) showed that the expression of Cyp1a1, Cyp1a2, and OGG1genes was significantly up-regulated in mice given diets containing 375 ppm DC or more, 187.5 ppm DC or more, and 750 ppm DC, respectively. These results suggest that the threshold dose of DC that induces ROS-mediated liver tumor promotion in mice is more than 750 ppm, although expression of the Cyp1a2 gene, which is related to ROS generation, was up-regulated in the liver of mice, even at a DC dose of 187.5 ppm.

Elevation of cell proliferation via generation of reactive oxygen species by piperonyl butoxide contributes to its liver tumor-promoting effects in mice.

Piperonyl butoxide (PBO) is a pesticide synergist used with pyrethroids as a domestic insecticide, and it acts as a non-genotoxic hepatocarcinogen in rats and mice. To clarify whether oxidative stress is involved in the liver tumor-promoting effect of PBO in mice, male mice were subjected to two-thirds partial hepatectomy, followed by N-diethylnitrosamine (DEN) treatment, and given a diet containing 0.6% PBO for 25 weeks. The incidences of cytokeratin (CK) 8/18-positive foci, adenomas, and carcinomas significantly increased in the DEN + PBO group compared with the DEN-alone group. The PCNA-positive ratio significantly increased in non-tumor hepatocytes, CK8/18-positive foci and adenomas in the DEN + PBO group compared with the DEN-alone group. PBO increased reactive oxygen species (ROS) production in microsomes but did not change oxidative DNA damage as assessed by 8-hydroxydeoxyguanosine (8-OHdG). In real-time RT-PCR, PBO upregulated the expression of genes related to metabolism, such as Cytochrome P450 1a1, 2a5, and 2b10, and metabolic stress, such as Por and Nqo1, but downregulated Egfr and Ogg1. PBO also increased early response genes downstream of mitogen-activated protein kinase (MAPK), such as c-Myc that is induced by excessive ROS production, and G1/S transition-related genes, such as E2f1 and Ccnd1. Thus, PBO can generate ROS via the metabolic pathway without any induction of oxidative DNA damage, activate cell growth, increase c-Myc- and E2F1-related pathways, and act as a liver tumor promoter of DEN-induced hepatocarcinogenesis in mice.

Promoting Effects of Streptozotocin-induced Diabetes on Induction of Hepatic Preneoplastic Lesions by Diethylnitrosamine in Rats.

The effects of streptozotocin (STZ)-induced diabetes on induction of hepatic preneoplastic lesions by diethylnitrosamine (DEN) were investigated in male Fischer rats. A single dose of STZ was injected intravenously either 2 weeks before or after initiation with DEN. The blood glucose levels were significantly elevated from 1 week after STZ-injection until autopsy. The numbers of GST-P positive foci at 1 week after DEN administration in the STZ-injected rats were similar to those in the non-diabetic rats. In contrast, both the numbers and areas of GST-P positive foci > 2 mm in diameter 8 weeks after DEN administration were increased significantly in the rats treated with STZ after DEN exposure compared with the non-diabetic control rats. The results suggest that hepatic preneoplastic lesions initiated with DEN are promoted by STZ treatment-inducing diabetes.

Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats.

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor-promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds of partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT-PCR analysis revealed decreases in mRNA expression levels of cytochrome P450, family 2, subfamily b, polypeptide 2 (Cyp2b2) and malic enzyme 1 (Me1) in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and aldo-keto reductase family 7, member A3 (Akr7a3) in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by the activation of CYPs.

Cytokeratin 8/18 is a useful immunohistochemical marker for hepatocellular proliferative lesions in mice.

In order to clarify whether cytokeratin (CK) 8/18 is a useful immunohistochemical marker for hepatocellular proliferative lesions in mice, partially hepatectomized male ICR mice were given 0.6% piperonyl butoxide (PBO) for 8 (Experiment I) or 25 weeks (Experiment II) after N-diethylnitrosamine (DEN) initiation treatment, and the livers were subjected to histological examinations on hematoxylin and eosin (HE) stained sections, CK8/18 immunohistochemistry and gamma-glutamyl transpeptidase (GGT) histochemistry. In Experiment I, the multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive for CK8/18 was 10.17 and 18.50, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 6.17 and 8.17, respectively. In Experiment II, the total multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive/negative for CK8/18 was 4.47 and 23.17, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 2.50 and 3.50, respectively. Most of the hepatocellular adenomas and carcinomas observed in HE-stained sections were positive for CK8/18, but some of the adenomas were negative for CK8/18. These findings indicate that more hepatocellular proliferative lesions can be detected in CK8/18 immunohistochemistry in addition to those observed in HE-stained sections, and suggest that CK8/18 may become a useful immunohistochemical marker for detecting hepatocellular proliferative lesions in mice.

Involvement of glycogen synthase kinase-3beta signaling and aberrant nucleocytoplasmic localization of retinoblastoma protein in tumor promotion in a rat two-stage thyroid carcinogenesis model.

To investigate the cell cycle kinetics during the tumor promotion process induced by hypothyroidism in a rat model of thyroid follicular cell carcinogenesis, immunohistochemical analysis of cell cycle molecules and related signaling molecules was performed in conjunction with analysis of cell proliferation activity in an initiation-promotion model. Male F344 rats were injected with N-bis(2-hydroxypropyl)nitrosamine, and one week later treated with 6-propyl-2-thiouracil (PTU) at 12ppm in the drinking water for 4, 10 or 15 weeks. At each time point, proliferative lesions increased the expression of cyclin A, cyclin D, cyclin E and cyclin-dependent kinase (Cdk)-2, in association with the development of lesion stages from the early focal hyperplasia to the late carcinoma, while a subpopulation of proliferative lesions showed decreased numbers of both cell division cycle-2- and Ki-67-positive cells at week 15 compared with that at week 10, suggesting a reduced promoting effect of serum thyroid-stimulating hormone in the sensitive cellular population after long-term exposure to PTU. On the other hand, increased immunolocalization of phosphorylated and inactive glycogen synthase kinase (GSK)-3beta was observed in a subpopulation of proliferative lesions, in parallel with the cyclins and Cdk2. Nuclear immunoreactivity of phosphorylated and inactive retinoblastoma (Rb) protein was also increased in association with lesion development, with carcinomas showing increased cytoplasmic localization. The results suggest that proliferative lesions activate the cell cycle machinery following tumor promotion via a regulatory mechanism involving inactivation of GSK3beta and Rb protein, the latter signaling mechanism involving its aberrant nucleocytoplasmic transport for the acquisition of a malignant phenotype.

Mechanistic study on hepatocarcinogenesis of piperonyl butoxide in mice.

To clarify the mechanism of piperonyl butoxide (PBO)-induced hepatocarcinogenesis in mice, male mice were subjected to a two-thirds partial hepatectomy, N-diethylnitrosamine (DEN) initiation, and a diet containing 0.6% PBO for eight weeks. The incidence of gamma-glutamyl transpeptidase (GGT)-positive foci and PCNA-positive cells was significantly increased in the DEN + PBO group compared with the DEN-alone group. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis showed up-regulation of genes related to metabolism, such as cytochrome P450 1A1 and 2B10, and metabolic stress, such as Por, Nqo1, Nrf2, abcc3, and abcc4. Early responsive genes downstream of mitogen-activated protein kinase (MAPK), such as c-fos, c-jun, c-myc, and activating transcription factor 3 (ATF3), were also up-regulated in this group. Positive immunohistochemical staining for ATF3 was diffusely observed in nonproliferating hepatocytes of the DEN + PBO group, but altered foci were negative or weakly positive for ATF3. The nuclei of hepatocytes within ATF3-negative foci were positive for cyclin D. Thus PBO can induce oxidative stress, activate the MAPK pathway, and increase ATF3 transcript levels in hepatocytes outside the altered foci during the early stage of PBO-induced hepatocarcinogenesis in mice.

Induction of liver preneoplastic foci in F344 rats subjected to 28-day oral administration of diheptyl phthalate and its in vivo genotoxic potential.

To investigate possible potential inducing preneoplastic lesions in liver and in vivo genotoxic potential of diheptyl phthalate (DHP), male F344 rats were subjected to repeated oral administration of DHP at 0, 2.5 or 5 g/kg/day for 28 days. In addition, F344 rats were subjected to once or 14 repeated oral administrations of 5 g/kg/day of DHP, and their livers were subjected to analysis in an alkaline single-cell gel electrophoresis (comet) assay. Furthermore, based on the results of these studies, partial hepatectomized male F344 rats given once, three times, and 14 repeated oral administration of 0, 2.5 or 5 g/kg body weight of DHP were examined by an in vivo liver initiation assay. In a 28-day repeated dose toxicity study, the number and area of glutathione-S-transferase placental form (GST-P) positive foci, a marker of hepatocellular preneoplastic lesions in rats, were significantly increased in DHP-treated groups compared with controls. At 24h after the 14 repeated administrations of DHP, DNA migration, a marker of DNA damage in the comet assay, was significantly induced in DHP-treated rat livers, whereas single treatment did not show such an alteration. In an in vivo liver initiation assay, a significant increase in the number and area of GST-P positive foci was observed in DHP-treated groups subjected to 14 repeated oral administrations of DHP as compared with the control group. These results indicate that DHP may induce altered hepatocellular foci in liver of rats which suggests that DHP is a genotoxic carcinogen in the liver of rats.

Modification of dietary copper levels on the early stage of tumor-promotion with propylthiouracil in a rat two-stage thyroid carcinogenesis model.

To investigate the role of copper (Cu)-related cellular responses on thyroid carcinogenesis, the expression of ceruloplasmin (Cp) and metallothionein (MT)-1/2 were examined in relation to the activities of cell proliferation/apoptosis in the thyroid of rats at an early stage of tumor promotion under different dietary Cu levels. Male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine by single subcutaneous injection at 2800 mg/kg body weight, and 1 week later promoted with 6-propyl-2-thiouracil at 12 ppm in the drinking water for 4 weeks. Animals were fed a diet containing Cu at 0.6, 6 or 60 ppm from the time point of initiator-treatment to create marginally deficient, normal, or non-toxic supplementary levels of Cu. At both 0.6 and 60 pm, the multiplicity of preneoplastic focal follicular cell hyperplasias (FFCHs) was decreased as compared with 6 ppm Cu, while adenomas also decreased at 0. 6 ppm Cu. Both 0.6 and 60 ppm Cu levels revealed decreased Ki-67-immunoreactive proliferating cells in both FFCHs and surrounding follicles accompanied by mRNA downregulation of Cdc2a and Ccnb1, while TUNEL-positive apoptotic cells were unaltered with change of dietary Cu. Both Cp and MT-1/2 were immunolocalized in FFCHs and adenomas, with higher distribution in the latter. At both 0.6 and 60 ppm, the immunoreactivities and/or thyroidal mRNA levels of Cp and MT-1/2 were also decreased. Transcript levels of several antioxidant enzymes were up- or downregulated in the same direction at both Cu levels. Serum levels of thyroid-related hormones were unaltered at both Cu levels, except for non-significant reduction of thyroid-stimulating hormone at 0.6 ppm. These results suggest an involvement of Cp and MT-1/2 on the thyroid tumor promotion that can be suppressed by dietary Cu level through inhibition of cell proliferation associated with altered redox balance.

Molecular expression analysis of beta-naphthoflavone-induced hepatocellular tumors in rats.

The present study was performed to characterize molecular expression levels of preneoplastic and neoplastic lesions induced by beta-naphthoflavone (BNF), an aryl hydrocarbon receptor (AhR) agonist in rat hepatocarcinogenesis. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and two weeks later, they were fed a diet containing 0% or 1% BNF for twenty-eight weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and sacrificed at week 30. Histopathologically, BNF increased the incidence and multiplicity of altered foci (1.7-fold and 3.3-fold) and hepatocellular adenomas (HCAs) (4.0-fold and 4.7-fold). Immunohistochemically, BNF increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in altered foci (2.3-fold) and HCAs (6.7-fold) compared with the surrounding tissue and decreased the staining of cell cycle regulators (P21, C/EBPalpha). In addition, loss of reactivity for AhR-regulated (CYP1A1, CYP1B1) molecules and increased reactivity of Nrf-2-regulated (AKR7, GPX2) molecules were also observed in proliferative lesions. Furthermore, increased staining of histone deacetylase (HDAC1) in the nucleus was prominent in HCAs. The differential expression patterns were confirmed at mRNA levels by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results suggest that enhanced cell proliferation and protection against oxidative stress play an important role in BNF-induced hepatocarcinogenesis in rats.

Crosstalk between PTEN/Akt2 and TGFbeta signaling involving EGF receptor down-regulation during the tumor promotion process from the early stage in a rat two-stage hepatocarcinogenesis model.

The present study investigated the involvement of signaling of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) and transforming growth factor-beta (TGFbeta) as well as receptor tyrosine kinases in the tumor promotion processes in a two-stage hepatocarcinogenesis model using male F344 rats. The cellular localization of related molecules was examined in liver cell foci expressing glutathione S-transferase placental form (GST-P) at the early stage of tumor promotion by fenbendazole (FB), piperonyl butoxide, or thioacetamide. Distribution in the liver cell foci and neoplastic lesions positive for GST-P was also examined at the later stage of FB promotion. In contrast to the initiation-alone cases, subpopulations of GST-P-positive foci induced by promotion for 6 weeks, regardless of the promoting chemicals used, enhanced down-regulation of PTEN and up-regulation of phosphorylated (active) Akt2 and phosphorylated substrate(s) of Akt-kinase activity. Also, up-regulation of TGFbeta receptor I and down-regulation of epidermal growth factor receptor (EGFR) were enhanced in the subpopulation of GST-P-positive foci in all promoted cases. A similar pattern of cellular distribution of these molecules was also observed in the neoplastic lesions at the late stage. These results suggest a crosstalk between Akt2 and TGFbeta signaling that involves a mechanism requiring EGFR down-regulation during the entire tumor promotion process starting from the early stage. In particular, a shift in subcellular localization of phosphorylated substrate(s) of Akt from the cell membrane in liver cell foci to the cytoplasm in carcinomas was observed, suggesting an alteration of the function or activity of the corresponding molecule(s).

Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats.

To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.

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.

No Modifying Effect of Antioxidant N-Acetyl-L-Cysteine on Fenofibrate-induced Hepatocarcinogenesis in Rats.

To clarify the modifying effect of N-Acetyl-L-Cysteine (NAC), which has antioxidative ability, on hepatocarcinogenesis promoted by fenofibrate (FF), a peroxisome proliferator-activated receptor (PPAR) alpha agonist , male F344/N rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) as an initiator followed by administration of a diet containing 3,000 ppm of FF for 16 weeks. Two-thirds partial hepatectomy was performed 1 week after the FF treatment. Additionally, NAC treatments for 14 weeks from 2 weeks after the FF treatment were performed. Although the expression level of tumor protein p53 (Tp53) mRNA decreased in the DEN+FF+NAC group as compared with that in the DEN+FF group, no significant differences between the DEN+FF and DEN+FF+NAC groups were observed in the number of hepatocellular altered foci and activities of hepatocellular proliferation. In addition, the results of an antioxidant enzyme assay and measurement of the amounts of total glutathione in the liver revealed no significant difference between the DEN+FF and DEN+FF+NAC groups; although no significant differences were observed in many genes between the DEN+FF and DEN+FF+NAC groups, only glutathione peroxidase 2 (Gpx2) mRNA increased in the DEN+FF+NAC group as compared with the DEN+FF group. The results under the present experimental conditions indicate no obvious modifying effect of NAC on liver tumor promotion by FF in rats.

Hepatocarcinogenic susceptibility of fenofibrate and its possible mechanism of carcinogenicity in a two-stage hepatocarcinogenesis model of rasH2 mice.

Fenofibrate (FF) has previously been shown to induce hepatocellular neoplasia in a conventional mouse bioassay (NDA 1993), but there has been no report to examine the carcinogenic susceptibility of rasH2 mice to this chemical. In the present study, male rasH2 mice were subjected to a two-thirds partial hepatectomy (PH), followed by an N-diethylnitrosamine (DEN) initiation twenty-four hours after PH, and given a diet containing 0, 1200, or 2400 ppm FF for seven weeks. The incidences of preneoplastic foci were significantly increased in mice from the FF-treated groups. Immunohistochemistry revealed that significant increases in proliferating cell nuclear antigen (PCNA)-positive cells and cytokeratin 8/18 positive foci were observed in FF-treated groups. In addition, the transgene and several downstream molecules such as c-myc, c-jun, activating transcription factor 3 (ATF3), and cyclin D1 were overexpressed in these groups. These results suggest that the hepatocarcinogenic activity of rasH2 mice to FF can be detected in this hepatocarcinogenesis model and that up-regulation of genes for the ras/MAPK pathway and cell cycle was probably involved in the hepatocarcinogenic mechanism of rasH2 mice.

Role of Nrf2 and oxidative stress on fenofibrate-induced hepatocarcinogenesis in rats.

Regional specific relationships between oxidative stress and the development of glutathione S-transferase placental form (GST-P)-positive or GST-P-negative lesions in rats, induced by fenofibrate (FF), a peroxisome proliferator, were examined using a two-stage hepatocarcinogenesis model in F344 rats. Animals were initiated with a single ip injection of 200 mg/kg N-diethylnitrosamine (DEN) and from 2 weeks later were fed a diet containing 3000 or 0 ppm FF for 28 weeks. Animals were subjected to a two-third partial hepatectomy at week 3 and sacrificed at week 28. The development of hepatocellular proliferative lesions, which were mainly attributed to GST-P-negative lesions, was significantly increased in the FF-treated groups. Immunohistochemically, GST-P-positive lesions were devoid of intracytoplasmic nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, whereas GST-P-negative lesions expressed higher levels of cytoplasmic Nrf2. On the other hand, nuclear accumulation of Nrf2 was observed in some cells of GST-P-positive lesions that were negative for Nrf2 in the cytoplasm and in GST-P-negative lesions of the DEN-FF group that were positive for Nrf2 in the cytoplasm. The mRNA expression levels of Gpx2 or Gsta2, Nrf2-inducible enzymes, were increased in GST-P-positive tumors or GST-P-positive lesions, respectively. These results suggest that the activation of Nrf2, due to nuclear translocation, occurs in the GST-P-positive lesions. In addition, the development of continuous oxidative stress was identified by mRNA expression analyses as well as by measurements of GST activity and 8-hydroxydeoxyguanosine. These results suggest that the relative inhibition of nuclear translocation of Nrf2 in GST-P-negative lesions aggravated the condition of oxidative stress in the liver of rats given FF, resulting in enhanced tumor promotion in FF-induced hepatocarcinogenesis.