kc-hits: a tool to aid in the evaluation and classification of chemical carcinogens.

MOTIVATION: The evaluation of chemicals for their carcinogenic hazard requires the analysis of a wide range of data and the characterization of these results relative to the key characteristics of carcinogens. The workflow used historically requires many manual steps that are labor-intensive and can introduce errors, bias and inconsistencies. RESULTS: The automation of parts of the evaluation workflow using the kc-hits software has led to significant improvements in process efficiency, as well as more consistent and comprehensive results. AVAILABILITY AND IMPLEMENTATION: https://gitlab.com/i1650/kc-hits.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Butyrate-containing structured lipids act on HDAC4, HDAC6, DNA damage and telomerase activity during promotion of experimental hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) presents with a high treatment resistance and poor prognosis. Early diagnosis and preventive approaches such as chemoprevention are essential for the HCC control. Therefore, we evaluated the chemopreventive effects of butyrate-containing structured lipids (STLs) administered during the promotion stage of hepatocarcinogenesis in rats submitted to the 'resistant hepatocyte' (RH) model. Administration of butyrate-containing STLs inhibited the incidence and mean number of visible hepatic nodules per rat and reduced the number and area of glutathione S-transferase placental form-positive (GST-P+) preneoplastic focal lesions in the livers. This was accompanied by the induction of apoptosis and an increased level of hepatic butyric acid. Treatment with butyrate-containing STLs resulted in increased histone H3 lysine 9 (H3K9) acetylation, reduction of total histone deacetylase (HDAC) activity, and lower levels of HDAC4 and HDAC6 proteins. The chemopreventive effect of butyrate-containing STLs was also associated with the increased nuclear compartmentalization of p53 protein and reduced expression of the Bcl-2 protein. In addition, rats treated with butyrate-containing STLs showed decreased DNA damage and telomerase activity in the livers. These results demonstrate that the suppressive activity of butyrate-containing STLs is associated with inhibition of elevated during hepatocarcinogenesis chromatin-modifying proteins HDAC4 and HDAC6, subcellular redistribution of the p53 protein, and decreased DNA damage and telomerase activity.

Characterization of the variability in the extent of nonalcoholic fatty liver induced by a high-fat diet in the genetically diverse Collaborative Cross mouse model.

Interindividual variability and sexual dimorphisms in the development of nonalcoholic fatty liver disease (NAFLD) are still poorly understood. In the present study, male and female strains of Collaborative Cross (CC) mice were fed a high-fat and high-sucrose (HF/HS) diet or a control diet for 12 weeks to investigate interindividual- and sex-specific variations in the development of NAFLD. The severity of liver steatosis varied between sexes and individual strains and was accompanied by an elevation of serum markers of insulin resistance, including increases in total cholesterol, low-density lipoproteins, high-density lipoproteins, phospholipids, and glucose. The development of NAFLD was associated with overexpression of the critical fatty acid uptake and de novo lipogenesis genes Pparg, Mogat1, Cd36, Acaab1, Fabp2, and Gdf15 in male and female mice. The expression of Pparg, Mogat1, and Cd36 was positively correlated with liver triglycerides in male mice, and Mogat1 and Cd36 expression were positively correlated with liver triglycerides in female mice. Our results indicate the value of CC mice in combination with HF/HS diet-induced alterations as an approach to study the susceptibility and interindividual variabilities in the pathogenesis of nonalcoholic fatty liver and early nonalcoholic steatohepatitis at the population level, uncovering of susceptible and resistant cohorts, and identifying sex-specific molecular determinants of disease susceptibility.

Genotoxic and Epigenotoxic Alterations in the Lung and Liver of Mice Induced by Acrylamide: A 28 Day Drinking Water Study.

Acrylamide has been classified as a "Group 2A carcinogen" (probably carcinogenic to humans) by the International Agency for Research on Cancer. The carcinogenicity of acrylamide is attributed to its well-recognized genotoxicity. In the present study, we investigated the effect of acrylamide on epigenetic alterations in mice. Female B6C3F1 mice received acrylamide in drinking water for 28 days, at doses previously used in a 2 year cancer bioassay (0, 0.0875, 0.175, 0.35, and 0.70 mM), and the genotoxic and epigenetic effects were investigated in lungs, a target organ for acrylamide carcinogenicity, and livers, a nontarget organ. Acrylamide exposure resulted in a dose-dependent formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine and N3-(2-carbamoyl-2-hydroxyethyl)adenine in liver and lung DNA. In contrast, the profiles of global epigenetic alterations differed between the two tissues. In the lungs, acrylamide exposure resulted in a decrease of histone H4 lysine 20 trimethylation (H4K20me3), a common epigenetic feature of human cancer, while in the livers, there was increased acetylation of histone H3 lysine 27 (H3K27ac), a gene transcription activating mark. Treatment with 0.70 mM acrylamide also resulted in substantial alterations in the DNA methylation and whole transcriptome in the lungs and livers; however, there were substantial differences in the trends of DNA methylation and gene expression changes between the two tissues. Analysis of differentially expressed genes showed a marked up-regulation of genes and activation of the gene transcription regulation pathway in livers, but not lungs. This corresponded to increased histone H3K27ac and DNA hypomethylation in livers, in contrast to hypermethylation and transcription silencing in lungs. Our results demonstrate that acrylamide induced global epigenetic alterations independent of its genotoxic effects, suggesting that epigenetic events may determine the organ-specific carcinogenicity of acrylamide. Additionally this study provides strong support for the importance of epigenetic alterations, in addition to genotoxic events, in the mechanism of carcinogenesis induced by genotoxic chemical carcinogens.

Population-Based Analysis of DNA Damage and Epigenetic Effects of 1,3-Butadiene in the Mouse.

Metabolism of 1,3-butadiene, a known human and rodent carcinogen, results in formation of reactive epoxides, a key event in its carcinogenicity. Although mice exposed to 1,3-butadiene present DNA adducts in all tested tissues, carcinogenicity is limited to liver, lung, and lymphoid tissues. Previous studies demonstrated that strain- and tissue-specific epigenetic effects in response to 1,3-butadiene exposure may influence susceptibly to DNA damage and serve as a potential mechanism of tissue-specific carcinogenicity. This study aimed to investigate interindividual variability in the effects of 1,3-butadiene using a population-based mouse model. Male mice from 20 Collaborative Cross strains were exposed to 0 or 635 ppm 1,3-butadiene by inhalation (6 h/day, 5 days/week) for 2 weeks. We evaluated DNA damage and epigenetic effects in target (lung and liver) and nontarget (kidney) tissues of 1,3-butadiene-induced carcinogenesis. DNA damage was assessed by measuring N-7-(2,3,4-trihydroxybut-1-yl)-guanine (THB-Gua) adducts. To investigate global histone modification alterations, we evaluated the trimethylation and acetylation of histones H3 and H4 across tissues. Changes in global cytosine DNA methylation were evaluated from the levels of methylation of LINE-1 and SINE B1 retrotransposons. We quantified the degree of variation across strains, deriving a chemical-specific human variability factor to address population variability in carcinogenic risk, which is largely ignored in current cancer risk assessment practice. Quantitative trait locus mapping identified four candidate genes related to chromatin remodeling whose variation was associated with interstrain susceptibility. Overall, this study uses 1,3-butadiene to demonstrate how the Collaborative Cross mouse population can be used to identify the mechanisms for and quantify the degree of interindividual variability in tissue-specific effects that are relevant to chemically induced carcinogenesis.

Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.

The substantial rise in the prevalence of nonalcoholic steatohepatitis (NASH), an advanced form of nonalcoholic fatty liver disease, and the strong association between NASH and the development of hepatocellular carcinoma indicate the urgent need for a better understanding of the underlying mechanisms. In the present study, by using the Stelic animal model of NASH and NASH-derived liver carcinogenesis, we investigated the role of the folate-dependent 1-carbon metabolism in the pathogenesis of NASH. We demonstrated that advanced NASH and NASH-related liver carcinogenesis are characterized by a significant dysregulation of 1-carbon homeostasis, with diminished expression of key 1-carbon metabolism genes, especially a marked inhibition of the S-adenosylhomocysteine hydrolase ( Ahcy) gene and an increased level of S-adenosyl-l-homocysteine (SAH). The reduction in Ahcy expression was associated with gene-specific cytosine DNA hypermethylation and enrichment of the gene promoter by trimethylated histone H3 lysine 27 and deacetylated histone H4 lysine 16, 2 main transcription-inhibiting markers. These results indicate that epigenetically mediated inhibition of Ahcy expression may be a driving force in causing SAH elevation and subsequent downstream disturbances in transsulfuration and transmethylation pathways during the development and progression of NASH.-Pogribny, I. P., Dreval, K., Kindrat, I., Melnyk, S., Jimenez, L., de Conti, A., Tryndyak, V., Pogribna, M., Ortega, J. F., James, S. J., Rusyn, I., Beland, F. A. Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.

Identification of chromatin-accessible domains in non-alcoholic steatohepatitis-derived hepatocellular carcinoma.

Non-alcoholic steatohepatitis (NASH) is becoming one of the major causes of hepatocellular carcinoma (HCC) in the United States and Western countries; however, the molecular mechanisms associated with NASH-related liver carcinogenesis are not well understood. In the present study, we investigated cancer-associated chromatin alterations using a model that resembles the development of NASH-related HCC in humans. An assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) identified 1677 tumor-specific chromatin-accessible regions in NASH-derived HCC tissue samples. Using a combined analysis of ATAC-seq and global gene expression data, we identified 199 differentially expressed genes, 139 up-regulated and 60 down-regulated. Interestingly, 15 of the 139 up-regulated genes had accessible chromatin sites within 5 Kb of the transcription start site (TSS), including Apoa4, Anxa2, Serpine1, Igfbp1, and Tubb2a, genes critically involved in the development of NASH and HCC. We demonstrate that the mechanism for the up-regulation of these genes is associated with the enrichment of chromatin-accessible regions by transcription factors, especially NFATC2, and histone H3K4me1 and H3K27ac gene transcription-activating marks. These data underline the important role of chromatin accessibility perturbations in reshaping of the chromatin landscape in NASH-related HCC.

ß-ionone modulates the expression of miRNAs and genes involved in the metastatic phenotype of microdissected persistent preneoplastic lesions in rats submitted to hepatocarcinogenesis.

MicroRNAs (miRNAs) are post-transcriptional gene expression regulators which expression is frequently altered in hepatocellular carcinoma (HCC). ß-ionone (ßI) is noted for its ability to inhibit persistent preneoplastic lesions (pPNLs) in liver rats. We evaluated the expression of miRNAs involved in carcinogenesis and possible targets modulated by ßI, in pPNLs and surrounding of microdissected tissues. Rats subjected to resistant hepatocyte model were treated during promotion stage with ßI (16 mg/100 g body weight) or corn oil (CO; 0.25 mL/100 g body weight; controls). Five animals receive no treatment (NT). In CO group, 38 and 29 miRNAs showed reduced expression relative to NT (P < 0.05) in pPNLs and surrounding, respectively. No miRNAs showed increased expression in surrounding of the CO compared to NT group; however, 30 miRNAs showed increased expression (P ≤ 0.05) in pPNLs of the CO group. There was no difference between ßI and CO groups (P > 0.05) in the expression of miRNAs in surrounding. In pPNLs ßI increased expression of miR-122 and miR-34a (P ≤ 0.05) and reduced of Igf2 (P ≤ 0.05), target of the latter, compared to CO. Additionally, ßI decreased the expression of miR-181c and its target Gdf2 (P ≤ 0.05). ßI reduced the expression of miR-181b and miR-708 (P ≤ 0.05) and increased the expression of their respective target mRNAs Timp3 and Mtss1 (P ≤ 0.05), relative to CO group. Modulation of miRNAs target genes by ßI was confirmed in vitro. ßI is a promising chemopreventive agent in the initial stages of hepatocarcinogenesis, as it modulates the expression of the miRNAs and target genes that can alter the metastatic phenotype of HCC. © 2016 Wiley Periodicals, Inc.

Furan-induced transcriptomic and gene-specific DNA methylation changes in the livers of Fischer 344 rats in a 2-year carcinogenicity study.

Furan is a significant food contaminant and a potent hepatotoxicant and rodent liver carcinogen. The carcinogenic effect of furan has been attributed to genotoxic and non-genotoxic, including epigenetic, changes in the liver; however, the mechanisms of the furan-induced liver tumorigenicity are still unclear. The goal of the present study was to investigate the role of transcriptomic and epigenetic events in the development of hepatic lesions in Fischer (F344) rats induced by furan treatment in a classic 2-year rodent tumorigenicity bioassay. High-throughput whole-genome transcriptomic analysis demonstrated distinct alterations in gene expression in liver lesions induced in male F344 rats treated with 0.92 or 2.0 mg furan/kg body weight (bw)/day for 104 weeks. Compared to normal liver tissue, 1336 and 1541 genes were found to be differentially expressed in liver lesions in rats treated with 0.92 and 2.0 mg furan/kg bw/day, respectively, among which 1001 transcripts were differentially expressed at both doses. Pairing transcriptomic and next-generation bisulfite sequencing analyses of the common differentially expressed genes identified 42 CpG island-containing genes in which the methylation level was correlated inversely with gene expression. Forty-eight percent of these genes (20 genes, including Areg, Jag1, and Foxe1) that exhibited the most significant methylation and gene expression changes were involved in key pathways associated with different aspects of liver pathology. Our findings illustrate that gene-specific DNA methylation changes have functional consequences and may be an important component of furan hepatotoxicity and hepatocarcinogenicity.

Transcriptomic responses provide a new mechanistic basis for the chemopreventive effects of folic acid and tributyrin in rat liver carcinogenesis.

The steady increase in the incidence and mortality of hepatocellular carcinoma (HCC) signifies a crucial need to understand better its pathogenesis to improve clinical management and prevention of the disease. The aim of this study was to investigate molecular mechanisms for the chemopreventive effects of folic acid and tributyrin alone or in combination on rat hepatocarcinogenesis. Male Wistar rats were subjected to a classic "resistant hepatocyte" model of liver carcinogenesis and treated with folic acid and tributyrin alone or in combination for 5 weeks during promotion stage. Treatment with folic acid and tributyrin alone or in combination strongly inhibited the development of glutathione-S-transferase placental form (GSTP)-positive foci. Microarray analysis showed significant changes in gene expression. A total of 498, 655 and 940 of differentially expressed genes, involved in cell cycle, p53-signaling, angiogenesis and Wnt pathways, was identified in the livers of rats treated with folic acid, tributyrin or folic acid and tributyrin. A detailed analysis of these differentially expressed genes revealed that treatments inhibited angiogenesis in the preneoplastic livers. This was evidenced by the fact that 30 out of 77 differentially expressed genes common to all three treatments are involved in the regulation of the angiogenesis pathway. The inhibition of angiogenesis was confirmed by reduced levels of CD34 protein. In conclusion, the tumor-suppressing activity of folic acid and tributyrin is associated with inhibition of angiogenesis at early stages of rat liver carcinogenesis. Importantly, the combination of folic acid and tributyrin has stronger chemopreventive effect than each of the compounds alone.

Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury.

Dysregulation of one-carbon metabolism-related metabolic processes is a major contributor to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). It is well established that genetic and gender-specific variations in one-carbon metabolism contribute to the vulnerability to NAFLD in humans. To examine the role of one-carbon metabolism dysregulation in the pathogenesis and individual susceptibility to NAFLD, we used a "population-based" mouse model where male mice from 7 inbred were fed a choline- and folate-deficient (CFD) diet for 12 wk. Strain-dependent down-regulation of several key one-carbon metabolism genes, including methionine adenosyltransferase 1α (Mat1a), cystathionine-ß-synthase (Cbs), methylenetetrahydrofolate reductase (Mthfr), adenosyl-homocysteinase (Ahcy), and methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), was observed. These changes were strongly associated with interstrain variability in liver injury (steatosis, necrosis, inflammation, and activation of fibrogenesis) and hyperhomocysteinemia. Mechanistically, the decreased expression of Mat1a, Ahcy, and Mthfd1 was linked to a reduced level and promoter binding of transcription factor CCAAT/enhancer binding protein ß (CEBPß), which directly regulates their transcription. The strain specificity of diet-induced dysregulation of one-carbon metabolism suggests that interstrain variation in the regulation of one-carbon metabolism may contribute to the differential vulnerability to NFLD and that correcting the imbalance may be considered as preventive and treatment strategies for NAFLD.

ß-ionone inhibits persistent preneoplastic lesions during the early promotion phase of rat hepatocarcinogenesis: TGF-α, NF-κB, and p53 as cellular targets.

Dietary isoprenic derivatives such as ß-ionone (ßI) are a promising class of chemopreventive agents. In this study, cellular aspects of ßI protective activities during early hepatocarcinogenesis were evaluated. Male Wistar rats were submitted to "resistant hepatocyte" model and then received daily 16 mg/100 g body weight (b.w.) of ßI (ßI group) or only 0.25 mL/100 g b.w. of corn oil (vehicle, control group [CO]) during 4 wk, specifically during early promotion phase. Compared to controls, ßI inhibited (P < 0.05) the development of persistent preneoplastic lesions (pPNL), considered to be potential hepatocellular carcinoma (HCC) progression sites, and increased remodeling PNL (rPNL) (P < 0.05) that tend to regress to a normal phenotype. Increased ßI hepatic levels (P < 0.05), in the ßI group, were associated with its chemopreventive actions. Compared to control rats, ßI reduced the frequency of both pPNL and rPNL positive for tumor growth factor (TGF)-α (P < 0.05), reduced the frequency of pPNL stained for p65 (nuclear factor-kappaB; NF-κB) (P < 0.05), and reduced the frequency of pPNL positive for cytoplasmic p53 (P < 0.05). Our data demonstrated that ßI targets TGF-α, NF-κB, and p53 in initial phases of hepatocarcinogenesis and specifically inhibits PNL with increased probability to progress to HCC. This isoprenoid may represent a chemopreventive agent of choice for HCC control.

The chemopreventive activity of the butyric acid prodrug tributyrin in experimental rat hepatocarcinogenesis is associated with p53 acetylation and activation of the p53 apoptotic signaling pathway.

The reversibility of non-genotoxic phenotypic alterations has been explored in order to develop novel preventive and therapeutic approaches for cancer control. Previously, it has been demonstrated that histone deacetylase (HDAC) inhibitor tributyrin, a butyric acid prodrug, to have chemopreventive effects on rat hepatocarcinogenesis. The goal of this study was to determine molecular mechanisms associated with the chemopreventive activity of tributyrin. Male Wistar rats were allocated randomly to untreated control group and two experimental groups. Rats in the experimental group 1 were treated with maltodextrin (3g/kg body wt), and rats in experimental group 2 were treated with tributyrin (2g/kg body wt) daily for 8 weeks. Two weeks after treatment initiation, rats from experimental groups were subjected to a 'resistant hepatocyte' model of hepatocarcinogenesis. Treatment with tributyrin resulted in lower HDAC activity and Hdac3 and Hdac4 gene expression, and an increase of histone H3 lysine 9 and 18 and histone H4 lysine 16 acetylation as compared with the experimental group 1. In addition to the increase in histone acetylation, tributyrin caused an increase in the acetylation of the nuclear p53 protein. These changes were accompanied by a normalization of the p53-signaling network, particularly by the upregulation of pro-apoptotic genes, and a consequent increase of apoptosis and autophagy in the livers of tributyrin-treated rats. These results indicate that the chemopreventive activity of tributyrin may be related to an increase of histone and p53 acetylation, which could lead to the induction of the p53 apoptotic pathway.