Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

The carbonic anhydrase inhibitor acetazolamide inhibits urinary bladder cancers via suppression of ß-catenin signaling.

Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and progression of urothelial carcinoma (UC) in humans. The purpose of the present study was to evaluate the effects of the CA inhibitor acetazolamide (Ace) on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder carcinogenesis in mice and explore the function of CA2 in muscle invasion by UC. Male mice were treated with 0.025% (experiment 1) or 0.05% BBN (experiment 2) in their drinking water for 10 weeks, then treated with cisplatin (Cis), Ace, or Cis plus Ace for 12 weeks. In experiment 1, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Ace and BBN→Cis+Ace groups. In experiment 2, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Cis+Ace group, and the incidence of muscle invasive UC was significantly decreased in both the BBN→Ace and the BBN→Cis+Ace groups. We also show that overexpression of CA2 by human UC cells T24 and UMUC3 significantly increased their migration and invasion capabilities, and that Ace significantly inhibited migration and invasion by CA2-overexpressing T24 and UMUC3 cells. These data demonstrate a functional association of CA2 with UC development and progression, confirming the association of CA2 with UC that we had shown previously by immunohistochemical analysis of human UC specimens and proteome analysis of BBN-induced UC in rats. Our finding that inhibition of CA2 inhibits UC development and muscle invasion also directly confirms that CA2 is a potential therapeutic target for bladder cancers.

Response biomarkers of inhalation exposure to cigarette smoke in the mouse lung.

Cigarette smoking is known to increase the risk of cancer and chronic obstructive pulmonary disease (COPD). In this study, we evaluated the effects of short-term nose-only inhalation exposure to cigarette smoke in mice. Male 10-week-old C57BL mice were exposed to clean air (control) or mainstream cigarette smoke for 1 h/day, 5 days/week, for 2 or 4 weeks. Exposure to cigarette smoke increased the number of inflammatory cells, especially neutrophils, in the bronchoalveolar lavage fluid, increased inflammatory cell infiltration foci, and caused an increase in the thickness of the peripheral bronchial epithelium. Microarray gene expression analysis indicated that smoke exposure induced inflammatory responses, including leukocyte migration and activation of phagocytes and myeloid cells, as early as two weeks after the initiation of exposure. Importantly, chemokine (C-C motif) ligand 17, resistin-like alpha, and lipocalin 2 were upregulated and may serve as useful markers of the toxic effects of exposure to cigarette smoke before pulmonary histological changes become evident.

Expression of thrombospondin-1 in conjunctival squamous cell carcinoma is correlated to the Ki67 index and associated with progression-free survival.

PURPOSE: Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to investigate thrombospondin-1 expression and its association with prognosis. METHODS: In this retrospective study, a gene expression array along with immunohistochemistry were performed for the evaluation of thrombospondin-1 expression, localization, as well as Ki67 labeling cell indices in carcinoma in situ (Tis) and advanced conjunctival SCC (Tadv). The presence or absence and intensity of cytoplasmic and nuclear staining in tumor cells were also divided into groups with a score of 0-3 and semi-quantitatively analyzed to investigate intracellular staining patterns. The association between thrombospondin-1 expression and tumor progression in a series of 31 conjunctival SCCs was further investigated. RESULTS: All 31 patients in the cohort (100%) were East Asian. A simple comparison between Tis and Tadv demonstrated significant differences in expressions of 45 genes, including thrombospondin-1 (p < 0.01). In this cohort, 30/31 tumors were positive (96%) for thrombospondin-1. Furthermore, thrombospondin-1 intracellular staining pattern analysis scores were 2.12 and 0.96 for nuclear and cytoplasmic staining, respectively, with a significant difference observed between Tis and Tadv (p < 0.01). Alteration of the Ki67 labeling index was significantly correlated with that of the thrombospondin-1 cytoplasmic score (p = 0.030). Furthermore, univariate Cox regression analysis showed a significant correlation between thrombospondin-1 staining and progression-free survival (p = 0.026) and final orbital exenteration (p = 0.019). CONCLUSIONS: The present results demonstrated that thrombospondin-1 is a potential molecular target in the pathology of conjunctival SCC, in addition to serving as a prognostic factor.

Dimethylarsinic acid (DMA) enhanced lung carcinogenesis via histone H3K9 modification in a transplacental mouse model.

Pregnant CD-1 mice received 200 ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8-18, and tumor formation was assessed in offspring at the age of 84 weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

Accumulation of 8-hydroxydeoxyguanosine, L-arginine and Glucose Metabolites by Liver Tumor Cells Are the Important Characteristic Features of Metabolic Syndrome and Non-Alcoholic Steatohepatitis-Associated Hepatocarcinogenesis.

To uncover mechanisms and explore novel biomarkers of obesity, type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH)-associated hepatocarcinogenesis, cellular and molecular alterations in the liver, and hepatocellular carcinomas (HCCs) were investigated in NASH model 60-week-old Tsumura, Suzuki, Obese Diabetic (TSOD) mice and NASH HCC patients. Markedly elevated lipid deposition, inflammation, fibrosis, and peroxisome proliferation in the liver, preneoplastic lesions, and HCCs of TSOD mice were accompanied by accumulation of polysaccharides in the cellular cytoplasm and nuclei and increase of oxidative DNA damage marker, 8-hydroxydeoxyguanosine (8-OHdG) formation in the liver and altered foci. Metabolomics of TSOD mice HCCs demonstrated significant elevation of the concentration of amino acid L-arginine, phosphocreatine, S-adenosylmethionine/S-adenosylhomocysteine ratio, adenylate, and guanylate energy charges in coordination with tremendous rise of glucose metabolites, mostly fructose 1,6-diphosphate. L-arginine accumulation in HCCs was associated with significant under-expression of arginase 1 (ARG1), suppression of the urea cycle, methionine and putrescine degradation pathways, activation of Ser and Thr kinase Akt AKT, phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, ß-catenin, mammalian target of rapamycin (mTOR), and cell proliferation. Furthermore, clinicopathological analysis in 20 metabolic syndrome/NASH and 80 HCV-positive HCC patients demonstrated significant correlation of negative ARG1 expression with poor tumor differentiation, higher pathological stage, and significant decrease of survival in metabolic syndrome/NASH-associated HCC patients, thus indicating that ARG1 could become a potential marker for NASH HCC. From these results, formation of oxidative stress and 8-OHdG in the DNA and elevation of glucose metabolites and L-arginine due to ARG1 suppression in mice liver cells are the important characteristics of T2DM/NASH-associated hepatocarcinogenesis, which may take part in activating oxidative stress resistance, synthesis of phosphocreatine, cell signaling, methylation, and proliferation.

Role of γ-H2AX as a biomarker for detection of bladder carcinogens in F344 rats.

Phosphorylation of histone H2AX at serine 139 (γ-H2AX) is known to be induced by direct DNA damage or cellular metabolic imbalances and malfunctions. Previous studies have reported that γ-H2AX is a useful biomarker for early detection of genotoxic bladder carcinogens in rats. The purpose of the present study was to determine the role of γ-H2AX as a biomarker for detection of non-genotoxic bladder carcinogens in rats. Six-week-old male F344 rats were treated with 15 different chemicals for 4 weeks. Immunohistochemical analyses revealed that all three genotoxic bladder carcinogens and six out of seven non-genotoxic bladder carcinogens significantly increased γ-H2AX formation in the bladder urothelium of rats. In addition, four out of five rat bladder noncarcinogens did not increase γ-H2AX formation in the bladder urothelium regardless of genotoxicity. These results suggest that γ-H2AX is a useful biomarker for detection of both genotoxic and non-genotoxic bladder carcinogens in rats.

Quantitative analysis of mutagenicity and carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in F344 gpt delta transgenic rats.

Quantitative analysis of the mutagenicity and carcinogenicity of the low doses of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the mutagenicity and carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 gpt delta transgenic rats were fed diets supplemented with 0, 0.1, 1, 10 or 100 ppm IQ for 4 weeks. The frequencies of gpt transgene mutations in the liver were significantly increased in the 10 and 100 ppm groups. In addition, the mutation spectra was altered in the 1, 10 and 100 ppm groups: frequencies of G:C to T:A transversion were significantly increased in groups administered 1, 10 and 100 ppm IQ in a dose-dependent manner, and the frequencies of G:C to A:T transitions, A:T to T:A transversions and A:T to C:G transversions were significantly increased in the 100 ppm group. Increased frequencies of single base pair deletions and Spi- mutants in the liver, and an increase in glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, was also observed in the 100 ppm group. In contrast, neither mutations nor mutation spectra or GST-P-positive foci were statistically altered by administration of IQ at 0.1 ppm. We estimated the point of departure for the mutagenicity and carcinogenicity of IQ using the no-observed-effect level approach and the Benchmark dose approach to characterise the dose-response relationship of low doses of IQ. Our findings demonstrate the existence of no effect levels of IQ for both in vivo mutagenicity and hepatocarcinogenicity. The findings of the present study will facilitate an understanding of the carcinogenic effects of low doses of IQ and help to determine a margin of exposure that may be useful for practical human risk assessment.

Promotion effects of acetoaceto-o-toluidide on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder carcinogenesis in rats.

Recent epidemiological studies have indicated that occupational exposure to the aromatic amine acetoaceto-o-toluidide (AAOT) was associated with a marked increase in urinary bladder cancers in Japan. However, little is known about the carcinogenicity of AAOT. To evaluate the urinary bladder carcinogenicity of AAOT, male and female F344 rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks followed by dietary administration of 0, 0.167, 0.5, or 1.5% AAOT for 31 weeks. The incidences and multiplicities of bladder tumors were significantly increased in the 0.5 and 1.5% groups of male and female rats in a dose-response manner. AAOT and seven downstream metabolites were detected in the urine of the male and female rats administered AAOT with levels increasing in a dose-dependent manner. The most abundant urinary metabolite of AAOT was the human bladder carcinogen o-toluidine (OTD), which was at least one order of magnitude higher than AAOT and the other AAOT metabolites. In a second experiment, male F344 rats were administered 0, 0.167, or 1.5% AAOT for 4 weeks. Gene expression analyses revealed that the expression of JUN and its downstream target genes was increased in the urothelium of male rats treated with 1.5% AAOT. These results demonstrate that AAOT promotes BBN-induced urinary bladder carcinogenesis in rats and suggest that overexpressed of JUN and its downstream target genes may be involved the bladder carcinogenicity of AAOT. In conclusion, AAOT, like other carcinogenic aromatic amines, is likely to be a carcinogen to the urinary bladder, and OTD metabolized from AAOT is the ultimate carcinogen.

A chronic toxicity study of diphenylarsinic acid in the drinking water of C57BL/6J mice for 52 weeks.

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to mice in their drinking water for 52 weeks. DPAA was administered to mice at concentrations of 0, 6.25, 12.5, and 25 ppm in their drinking water for 52 weeks. There were no significant differences in final body weights between the control groups and the DPAA treatment groups in male or female mice. Relative liver weights were significantly increased in males treated with 25 ppm DPAA, and absolute liver weights were significantly decreased in female mice treated with 25 ppm DPAA. In female mice, cholangitis and simple bile duct hyperplasia were observed in the 12.5 and 25 ppm DPAA groups, and focal necrosis of hepatocytes was observed in the 25 ppm DPAA group. Proteomic analysis and Ingenuity Pathway Analysis identified 18 proteins related to hepatotoxicity that were overexpressed in the female 25 ppm group. The phase I metabolic enzyme CYP2E1 was one of these overexpressed proteins. Immunostaining confirmed high expression of CYP2E1 in the livers of females in the 25 ppm group. These results suggest that DPAA is toxic to the intrahepatic bile duct epithelium and hepatocytes in female mice and that CYP2E1 might be involved in DPAA-associated toxicity. The no-observed-adverse-effect levels of DPAA were 12.5 ppm (1.6 mg/kg bw/day) for males and 6.25 ppm (1.1 mg/kg bw/day) for females under the conditions of this study.

In vivo positive mutagenicity of 1,4-dioxane and quantitative analysis of its mutagenicity and carcinogenicity in rats.

1,4-Dioxane is a widely used synthetic industrial chemical and its contamination of drinking water and food is a potential health concern. It induces liver tumors when administered in the drinking water to rats and mice. However, the mode of action (MOA) of the hepatocarcinogenicity of 1,4-dioxane remains unclear. Importantly, it is unknown if 1,4-dioxane is genotoxic, a key consideration for risk assessment. To determine the in vivo mutagenicity of 1,4-dioxane, gpt delta transgenic F344 rats were administered 1,4-dioxane at various doses in the drinking water for 16 weeks. The overall mutation frequency (MF) and A:T- to -G:C transitions and A:T- to -T:A transversions in the gpt transgene were significantly increased by administration of 5000 ppm 1,4-dioxane. A:T- to -T:A transversions were also significantly increased by administration of 1000 ppm 1,4-dioxane. Furthermore, the DNA repair enzyme MGMT was significantly induced at 5000 ppm 1,4-dioxane, implying that extensive genetic damage exceeded the repair capacity of the cells in the liver and consequently led to liver carcinogenesis. No evidence supporting other MOAs, including induction of oxidative stress, cytotoxicity, or nuclear receptor activation, that could contribute to the carcinogenic effects of 1,4-dioxane were found. These findings demonstrate that 1,4-dioxane is a genotoxic hepatocarcinogen and induces hepatocarcinogenesis through a mutagenic MOA in rats. Because our data indicate that 1,4-dioxane is a genotoxic carcinogen, we estimated the point of departure of the mutagenicity and carcinogenicity of 1,4-dioxane using the no-observed effect-level approach and the Benchmark dose approach to characterize its dose-response relationship at low doses.

Chronic dietary toxicity and carcinogenicity studies of dammar resin in F344 rats.

Dammar resin is a natural food additive and flavoring substance present in many foods and drinks. The present study evaluates the chronic toxicity and carcinogenicity of dietary dammar resin in F344 rats. Dietary concentrations in the 52-week chronic toxicity study were 0, 0.03, 0.125, 0.5, or 2%. The major treatment-related deleterious effects were body weight suppression, increased relative liver weight, and low hemoglobin levels in males and females. Foci of cellular alteration in the liver were observed in the male 2% group, but not in any other group. The no-observed-adverse-effect level for chronic toxicity was 0.125% for males (200.4 mg/kg b.w./day) and females (241.9 mg/kg b.w./day). Dietary concentrations in the 104-week carcinogenicity study were 0, 0.03, 0.5, or 2%. Dammar resin induced hemorrhagic diathesis in males and females, possibly via the inhibition of extrinsic and intrinsic coagulation pathways. Incidences of hepatocellular adenomas and carcinomas were significantly increased in the male 2% group, but not in any other group. In the 4-week subacute toxicity study, the livers of male rat-fed diet-containing 2% dammar resin had increased levels of protein oxidation and increased the expression of two anti-apoptotic and seven cytochrome P450 (CYP) genes. There was also an increased tendency of oxidative DNA damage. These findings demonstrate that dammar resin is hepatocarcinogenic in male F344 rats and underlines the roles of inhibition of apoptosis, induction of CYP enzymes, and oxidative stress in dammar resin-induced hepatocarcinogenesis.

Carbonic anhydrase 2 is a novel invasion-associated factor in urinary bladder cancers.

Rat bladder cancer is nearly always papillary non-invasive urothelial carcinoma (UC). To establish an animal model mimicking invasive UC that arises from papillary non-invasive UC in the bladder, male human c-Ha-ras proto-oncogene transgenic rats (Hras128) were treated with 0.05% N-butyl-N-(hydroxybutyl)nitrosameine (BBN) in their drinking water and/or 0.1% phenylethyl isothiocyanate (PEITC) in their diet as follows: BBN (8 weeks)→PEITC (8 weeks); PEITC (8 weeks)→BBN (8 weeks); BBN alone (16 weeks); PEITC alone (16 weeks); and no treatment. At the end of week 16, the highest incidence of invasive UC was observed in the BBN→PEITC group. Therefore, we used Hras128 rats treated with BBN followed by PEITC as a model of invasive bladder cancer to identify invasion-associated proteins. Proteome analysis was performed to compare the protein profiles of invasive and non-invasive UC in Hras128 rats. We identified 49 proteins that were either overexpressed or underexpressed in invasive UC but not in non-invasive UC. Immunohistochemical analysis of carbonic anhydrase 2 (CA2), an overexpressed protein, showed that the relative number of CA2-positive UC was significantly higher for invasive UC compared to non-invasive UC in rats. Moreover, the incidence of CA2-positive cancers was also significantly higher for human muscle-invasive bladder cancer (MIBC) compared to non-MIBC (NMIBC) and was positively associated with the progression of NMIBC. Our findings indicate that CA2 is an invasion-associated factor and suggest that it could serve as a potential therapeutic molecular target for bladder cancers.

Enhanced Susceptibility of Ogg1 Mutant Mice to Multiorgan Carcinogenesis.

The role of deficiency of oxoguanine glycosylase 1 (Ogg1) Mmh homolog, a repair enzyme of the 8-hydroxy-2'-deoxyguanosine (8-OHdG) residue in DNA, was investigated using the multiorgan carcinogenesis bioassay in mice. A total of 80 male and female six-week-old mice of C57BL/6J background carrying a mutant Mmh allele of the Mmh/Ogg1 gene (Ogg1-/-) and wild type (Ogg1+/+) mice were administered N-diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), N-bis (2-hydroxypropyl) nitrosamine (DHPN) and 1,2-dimethylhydrazine dihydrochloride (DMH) (DMBDD) to induce carcinogenesis in multiple organs, and observed up to 34 weeks. Significant increase of lung adenocarcinomas incidence was observed in DMBDD-treated Ogg1-/- male mice, but not in DMBDD-administered Ogg1+/+ animals. Furthermore, incidences of lung adenomas were significantly elevated in both Ogg1-/- males and females as compared with respective Ogg1-/- control and DMBDD-treated Ogg1+/+ groups. Incidence of total liver tumors (hepatocellular adenomas, hemangiomas and hemangiosarcomas) was significantly higher in the DMBDD-administered Ogg1-/- males and females. In addition, in DMBDD-treated male Ogg1-/- mice, incidences of colon adenomas and total colon tumors showed a trend and a significant increase, respectively, along with significant rise in incidence of simple hyperplasia of the urinary bladder, and a trend to increase for renal tubules hyperplasia in the kidney. Furthermore, incidence of squamous cell hyperplasia in the forestomach of DMBDD-treated Ogg1-/- male mice was significantly higher than that of Ogg1+/+ males. Incidence of small intestine adenomas in DMBDD Ogg1-/- groups showed a trend for increase, as compared to the wild type mice. The current results demonstrated increased susceptibility of Ogg1 mutant mice to the multiorgan carcinogenesis induced by DMBDD. The present bioassay could become a useful tool to examine the influence of various targets on mouse carcinogenesis.

Proteome Characteristics of Non-Alcoholic Steatohepatitis Liver Tissue and Associated Hepatocellular Carcinomas.

To uncover mechanisms of nonalcoholic steatohepatitis (NASH) associated hepatocarcinogenesis, we compared the proteomes of human NASH-associated liver biopsies, resected hepatocellular carcinomas (HCCs) and HCCs of HCV⁺ patients with normal liver tissue of patients with gastrointestinal tumor metastasis, in formalin-fixed paraffin-embedded samples obtained after surgery in our hospital during the period from 2006 to 2011. In addition, proteome analysis of liver tumors in male STAM NASH-model mice was performed. Similar changes in the proteome spectrum such as overexpression of enzymes involved in lipid, cholesterol and bile acid biosynthesis and examples associated with suppression of fatty acid oxidation and catabolism, alcohol metabolism, mitochondrial function as well as low expression levels of cytokeratins 8 and 18 were observed in both human NASH biopsies and NASH HCCs, but not HCV⁺ HCCs. Alterations in downstream protein expression pointed to significant activation of transforming growth factor ß, SMAD family member 3, ß-catenin, Nrf2, SREBP-LXRα and nuclear receptor-interacting protein 1 (NRIP1), and inhibition of PPARs and p53 in human NASH biopsies and/or HCCs, suggesting their involvement in accumulation of lipids, development of fibrosis, oxidative stress, cell proliferation and suppression of apoptosis in NASH hepatocarcinogenesis. In STAM mice, PPARs inhibition was not obvious, while expression of cytokeratins 8 and 18 was elevated, indicative of essential differences between human and mouse NASH pathogenesis.

Diphenylarsinic acid exerts promotion effects on hepatobiliary carcinogenesis in a rat medium-term multiorgan carcinogenicity bioassay.

We have previously demonstrated that diphenylarsinic acid (DPAA) promotes liver carcinogenesis in rats in a medium-term liver carcinogenicity bioassay. However, the effects of DPAA on other organs have not been determined. In the present study, the effects of DPAA on carcinogenesis were investigated using a rat multiorgan carcinogenicity bioassay. A total of 60 six-week-old male F344 rats were treated with the carcinogens diethylnitrosamine, N-butyl-N-(4-hydroxybutyl) nitrosamine, N-methyl-N-nitrosourea, N-bis (2-hydroxypropyl) nitrosamine, and 1,2-dimethylhydrazine dihydrochloride to initiate carcinogenesis in multiple organs. After initiation, DPAA was given at a dose of 0, 5, or 20 ppm in drinking water for 27 weeks. The incidences of moderate and severe bile duct hyperplasia were significantly increased in the 20 ppm DPAA group (29.4%, 70.6%, respectively) compared with the 0 ppm DPAA group (0%, 0%, respectively), and the incidence and multiplicity of cholangioma were significantly increased in the 20 ppm DPAA group (29.4%, 0.4 ± 0.8/rat) compared with the 0 ppm DPAA group (0%, 0/rat). The total number and average area of glutathione S-transferase placenta form-positive foci, preneoplastic lesions in rat livers, were significantly increased in the 20 ppm DPAA group (10.5 ± 2.2/cm2, 5.3 ± 1.7 mm2/cm2) compared with the 0 ppm DPAA group (6.2 ± 2.9/cm2, 2.4 ± 1.4 mm2/cm2). In conclusion, our results demonstrate that DPAA promotes hepatobiliary carcinogenesis in a rat medium-term multiorgan carcinogenicity bioassay; no promotion effects were observed in other organs.

CD44 variant 9 is a potential biomarker of tumor initiating cells predicting survival outcome in hepatitis C virus-positive patients with resected hepatocellular carcinoma.

This study investigated whether the expression of CD44 variant 9 (CD44v9) might be a functional marker of tumor-initiating stem-like cells in primary hepatocellular carcinomas (HCCs) of hepatitis C virus (HCV)(+) patients and provide an indicator of patient survival, as well as associated mechanisms. A total of 90 HCV(+) HCC patients who underwent surgery from 2006 to 2011 were enrolled and monitored for 2-8 years. Expression of CD44v9 was validated immunohistochemically in all HCCs, followed by comparative proteome, survival, and clinicopathological analyses. CD44 variant 8--10 was further evaluated in diethylnitrosamine-induced HCCs of C57Bl/6J mice. Focally localized CD44v(+) cells with a membranous staining pattern were detected in human HCV(+) and mouse HCCs. CD44v9(+) cells of HCCs were predominantly negative for Ki67 and P-p38, indicating decrease of cell proliferation in the CD44v9(+) tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2-mediated signaling, DNA repair, and inhibition of xenobiotic metabolism. CD44v9 IHC evaluation in 90 HCV(+) HCC cases revealed that positive expression was significantly associated with poor overall and recurrence-free survival, a younger age, poor histological differentiation of HCCs, and high alkaline phosphatase levels compared with patients with negative expression. CD44v9 is concluded to be a potential biomarker of tumor-initiating stem-like cells and a prognostic marker in HCV(+) HCC patients associated with Nrf2-mediated resistance to oxidative stress.

Qualitative and quantitative approaches in the dose-response assessment of genotoxic carcinogens.

Qualitative and quantitative approaches are important issues in field of carcinogenic risk assessment of the genotoxic carcinogens. Herein, we provide quantitative data on low-dose hepatocarcinogenicity studies for three genotoxic hepatocarcinogens: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodiethylamine (DEN). Hepatocarcinogenicity was examined by quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci, which are the preneoplastic lesions in rat hepatocarcinogenesis and the endpoint carcinogenic marker in the rat liver medium-term carcinogenicity bioassay. We also examined DNA damage and gene mutations which occurred through the initiation stage of carcinogenesis. For the establishment of points of departure (PoD) from which the cancer-related risk can be estimated, we analyzed the above events by quantitative no-observed-effect level and benchmark dose approaches. MeIQx at low doses induced formation of DNA-MeIQx adducts; somewhat higher doses caused elevation of 8-hydroxy-2'-deoxyquanosine levels; at still higher doses gene mutations occurred; and the highest dose induced formation of GST-P positive foci. These data indicate that early genotoxic events in the pathway to carcinogenesis showed the expected trend of lower PoDs for earlier events in the carcinogenic process. Similarly, only the highest dose of IQ caused an increase in the number of GST-P positive foci in the liver, while IQ-DNA adduct formation was observed with low doses. Moreover, treatment with DEN at low doses had no effect on development of GST-P positive foci in the liver. These data on PoDs for the markers contribute to understand whether genotoxic carcinogens have a threshold for their carcinogenicity. The most appropriate approach to use in low dose-response assessment must be approved on the basis of scientific judgment.

Examination of in vivo mutagenicity of sodium arsenite and dimethylarsinic acid in gpt delta rats.

Arsenic is a well-known human bladder and liver carcinogen, but its exact mechanism of carcinogenicity is not fully understood. Dimethylarsinic acid (DMAV) is a major urinary metabolite of sodium arsenite (iAsIII) and induces urinary bladder cancers in rats. DMAV and iAsIII are negative in in vitro mutagenicity tests. However, their in vivo mutagenicities have not been determined. The purpose of present study is to evaluate the in vivo mutagenicities of DMAV and iAsIII in rat urinary bladder epithelium and liver using gpt delta F344 rats. Ten-week old male gpt delta F344 rats were randomized into 3 groups and administered 0, 92mg/L DMAV, or 87mg/L iAsIII (each 50mg/L As) for 13weeks in the drinking water. In the mutation assay, point mutations are detected in the gpt gene by 6-thioguanine selection (gpt assay) and deletion mutations are identified in the red/gam genes by Spi- selection (Spi- assay). Results of the gpt and Spi- assays showed that DMAV and iAsIII had no effects on the mutant frequencies or mutation spectrum in urinary bladder epithelium or liver. These findings indicate that DMAV and iAsIII are not mutagenic in urinary bladder epithelium or liver in rats.

Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model.

To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective of initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis.