A preclinical model of severe NASH-like liver injury by chronic administration of a high-fat and high-sucrose diet in mice.

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease, affecting 38% of adults globally. If left untreated, NAFLD may progress to more advanced forms of the disease, including non-alcoholic steatohepatitis (NASH), liver cirrhosis, and fibrosis. Early NAFLD detection is critical to prevent disease progression. Using an obesogenic high-fat and high-sucrose (HF/HS) diet, we characterized the progression of NAFLD in male and female Collaborative Cross CC042 mice after 20-, 40-, and 60-week intervals of chronic HF/HS diet feeding. The incidence and severity of liver steatosis, inflammation, and fibrosis increased in both sexes over time, with male mice progressing to a NASH-like disease state faster than female mice, as indicated by earlier and more pronounced changes in liver steatosis. Histopathological indication of macrovesicular steatosis and gene expression changes of key lipid metabolism genes were found to be elevated in both sexes after 20 weeks of HF/HS diet. Measurement of circulating markers of inflammation (CXCL10 and TNF-α), histopathological analysis of immune cell infiltrates, and gene expression changes in inflammation-related genes indicated significant liver inflammation after 40 and 60 weeks of HF/HS diet exposure in both sexes. Liver fibrosis, as assessed by Picosirius red and Masson's trichrome staining and changes in expression of key fibrosis related genes indicated significant changes after 40 and 60 weeks of HF/HS diet exposure. In conclusion, we present a preclinical animal model of dietary NAFLD progression, which recapitulates human pathophysiological and pathomorphological changes, that could be used to better understand the progression of NAFLD and support development of new therapeutics.

Effect of an obesogenic high-fat and high-sucrose diet on hepatic gene expression signatures in male Collaborative Cross mice.

Nonalcoholic fatty liver disease (NAFLD), the most prevalent chronic liver disease, is characterized by substantial variations in case-level severity. In this study, we used a genetically diverse Collaborative Cross (CC) mouse population model to analyze the global transcriptome and clarify the molecular mechanisms involved in hepatic fat accumulation that determine the level and severity of NAFLD. Twenty-four strains of male CC mice were maintained on a high-fat/high-sucrose (HF/HS) diet for 12 wk, and their hepatic gene expression profiles were determined by next-generation RNA sequencing. We found that the development of the nonalcoholic fatty liver (NAFL) phenotype in CC mice coincided with significant changes in the expression of hepatic genes at the population level, evidenced by the presence of 724 differentially expressed genes involved in lipid and carbohydrate metabolism, cell morphology, vitamin and mineral metabolism, energy production, and DNA replication, recombination, and repair. Importantly, expression of 68 of these genes strongly correlated with the extent of hepatic lipid accumulation in the overall population of HF/HS diet-fed male CC mice. Results of partial least squares (PLS) modeling showed that these derived hepatic gene expression signatures help to identify the individual mouse strains that are highly susceptible to the development of NAFLD induced by an HF/HS diet. These findings imply that gene expression profiling, combined with a PLS modeling approach, may be a useful tool to predict NAFLD severity in genetically diverse patient populations.NEW & NOTEWORTHY Feeding male Collaborative Cross mice an obesogenic diet allows modeling NAFLD at the population level. The development of NAFLD coincided with significant hepatic transcriptomic changes in this model. Genes (724) were differentially expressed and expression of 68 genes strongly correlated with the extent of hepatic lipid accumulation. Partial least squares modeling showed that derived hepatic gene expression signatures may help to identify individual mouse strains that are highly susceptible to the development of NAFLD.

Gene expression analyses reveal potential mechanism of inorganic arsenic-induced apoptosis in zebrafish.

Our previous study showed that sodium arsenite (200 mg/L) affected the nervous system and induced motor neuron development via the Sonic hedgehog pathway in zebrafish larvae. To gain more insight into the effects of arsenite on other signaling pathways, including apoptosis, we have performed quantitative polymerase chain reaction array-based gene expression analyses. The 96-well array plates contained primers for 84 genes representing 10 signaling pathways that regulate several biological functions, including apoptosis. We exposed eggs at 5 h postfertilization until the 72 h postfertilization larval stage to 200 mg/L sodium arsenite. In the Janus kinase/signal transducers and activators of transcription, nuclear factor κ-light-chain-enhancer of activated B cells, and Wingless/Int-1 signaling pathways, the expression of only one gene in each pathway was significantly altered. The expression of multiple genes was altered in the p53 and oxidative stress pathways. Sodium arsenite induced excessive apoptosis in the larvae. This compelled us to analyze specific genes in the p53 pathway, including cdkn1a, gadd45aa, and gadd45ba. Our data suggest that the p53 pathway is likely responsible for sodium arsenite-induced apoptosis. In addition, sodium arsenite significantly reduced global DNA methylation in the zebrafish larvae, which may indicate that epigenetic factors could be dysregulated after arsenic exposure. Together, these data elucidate potential mechanisms of arsenic toxicity that could improve understanding of arsenic's effects on human health.

Epigenetic effects of low-level sodium arsenite exposure on human liver HepaRG cells.

Chronic exposure to inorganic arsenic is associated with a variety of adverse health effects, including lung, bladder, kidney, and liver cancer. Several mechanisms have been proposed for arsenic-induced tumorigenesis; however, insufficient knowledge and many unanswered questions remain to explain the integrated molecular pathogenesis of arsenic carcinogenicity. In the present study, using non-tumorigenic human liver HepaRG cells, we investigated epigenetic alterations upon prolonged exposure to a noncytotoxic concentration of sodium arsenite (NaAsO2). We demonstrate that continuous exposure of HepaRG cells to 1 µM sodium arsenite (NaAsO2) for 14 days resulted in substantial cytosine DNA demethylation and hypermethylation across the genome, among which the claudin 14 (CLDN14) gene was hypermethylated and the most down-regulated gene. Another important finding was a profound loss of histone H3 lysine 36 (H3K36) trimethylation, which was accompanied by increased damage to genomic DNA and an elevated de novo mutation frequency. These results demonstrate that continuous exposure of HepaRG cells to a noncytotoxic concentration of NaAsO2 results in substantial epigenetic abnormalities accompanied by several carcinogenesis-related events, including induction of epithelial-to-mesenchymal transition, damage to DNA, inhibition of DNA repair genes, and induction of de novo mutations. Importantly, this study highlights the intimate mechanistic link and interplay between two fundamental cancer-associated events, epigenetic and genetic alterations, in arsenic-associated carcinogenesis.

Status of hepatic DNA methylome predetermines and modulates the severity of non-alcoholic fatty liver injury in mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a major health problem and a leading cause of chronic liver disease in the United States and Western countries. In humans, genetic factors greatly influence individual susceptibility to NAFLD; nonetheless, the effect of inter-individual differences in the normal liver epigenome with regard to the susceptibility to NAFLD has not been determined. RESULTS: In the present study, we investigated the association between the DNA methylation status in the livers of A/J and WSB/EiJ mice and the severity of NAFLD-associated liver injury. We demonstrate that A/J and WSB/EiJ mice, which are characterized by significant differences in the severity of liver injury induced by a choline- and folate-deficient (CFD) diet exhibit substantial differences in cytosine DNA methylation in their normal livers. Furthermore, feeding A/J and WSB/EiJ mice a CFD diet for 12 weeks resulted in different trends and changes in hepatic cytosine DNA methylation. CONCLUSION: Our findings indicate a primary role of hepatic DNA methylation in the pathogenesis of NAFLD and suggest that individual variations in DNA methylation across the genome may be a factor determining and influencing the vulnerability to NAFLD.

Cellular and molecular alterations in a human hepatocellular in vitro model of nonalcoholic fatty liver disease development and stratification.

The rapidly increasing incidence of nonalcoholic fatty liver disease (NAFLD) is a growing health crisis worldwide. If not detected early, NAFLD progression can lead to irreversible pathological states, including liver fibrosis and cirrhosis. Using in vitro models to understand the molecular pathogenesis has been extremely beneficial; however, most studies have utilized only short-term exposures, highlighting a limitation in current research to model extended fat-induced liver injury. We treated Hep3B cells continuously with a low dose of oleic and palmitic free fatty acids (FFAs) for 7 or 28 days. Transcriptomic analysis identified dysregulated molecular pathways and differential expression of 984 and 917 genes after FFA treatment for 7 and 28 days respectively. DNA methylation analysis of altered DNA methylated regions (DMRs) found 7 DMRs in common. Pathway analysis of differentially expressed genes (DEGs) revealed transcriptomic changes primarily involved in lipid metabolism, small molecule biochemistry, and molecular transport. Western blot analysis revealed changes in PDK4 and CPT1A protein levels, indicative of mitochondrial stress. In line with this, there was mitochondrial morphological change demonstrating breakdown of the mitochondrial network. This in vitro model of human NAFL mimics results observed in human patients and may be used as a pre-clinical model for drug intervention.

Evaluating the toxicokinetics of some metabolites of a C6 polyfluorinated compound, 6:2 fluorotelomer alcohol in pregnant and nonpregnant rats after oral exposure to the parent compound.

The 6:2 fluorotelomer alcohol (6:2 FTOH) is a common impurity in per- and polyfluoroalkyl substances (PFASs) used in many applications. Our previous toxicokinetic (TK) evaluation of 6:2 FTOH calculated times to steady state (tss) of one of its metabolites, 5:3 fluorotelomer carboxylic acid (5:3A), in the plasma and tissues of up to a year after oral exposure to rats. Our current work further elucidated the TK of 5:3A and other metabolites of 6:2 FTOH in pregnant and nonpregnant rats after repeated oral exposure and examined the role of renal transporters in the biopersistence of 5:3A. The tss values for 5:3A in serum and tissues of adult nonpregnant animals ranged from 150 days to over a year. 4:3 fluorotelomer carboxylic acid (4:3A) was an additional potentially-biopersistent metabolite. 5:3A was the major metabolite of 6:2 FTOH in serum of pregnant dams and fetuses at each time interval. 5:3A was not a substrate for renal transporters in a human kidney cell line in vitro, indicating that renal reuptake of 5:3A is unlikely contribute to its biopersistence. Further research is needed to identify the underlying processes and evaluate the impact of these 6:2 FTOH metabolites on human health.

Acetaminophen-induced acute liver injury in HCV transgenic mice.

The exact etiology of clinical cases of acute liver failure is difficult to ascertain and it is likely that various co-morbidity factors play a role. For example, epidemiological evidence suggests that coexistent hepatitis C virus (HCV) infection increased the risk of acetaminophen-induced acute liver injury, and was associated with an increased risk of progression to acute liver failure. However, little is known about possible mechanisms of enhanced acetaminophen hepatotoxicity in HCV-infected subjects. In this study, we tested a hypothesis that HCV-Tg mice may be more susceptible to acetaminophen hepatotoxicity, and also evaluated the mechanisms of acetaminophen-induced liver damage in wild type and HCV-Tg mice expressing core, E1 and E2 proteins. Male mice were treated with a single dose of acetaminophen (300 or 500 mg/kg in fed animals; or 200 mg/kg in fasted animals; i.g.) and liver and serum endpoints were evaluated at 4 and 24h after dosing. Our results suggest that in fed mice, liver toxicity in HCV-Tg mice is not markedly exaggerated as compared to the wild-type mice. In fasted mice, greater liver injury was observed in HCV-Tg mice. In fed mice dosed with 300 mg/kg acetaminophen, we observed that liver mitochondria in HCV-Tg mice exhibited signs of dysfunction showing the potential mechanism for increased susceptibility.

An in vitro investigation of metabolically sensitive biomarkers in breast cancer progression.

Epigenetic biomarkers are emerging as determinants of breast cancer prognosis. Breast cancer cells display unique alterations in major cellular metabolic pathways and it is becoming widely recognized that enzymes that regulate epigenetic alterations are metabolically sensitive. In this study, we used microarray data from the GEO database to compare gene expression for regulators of metabolism and epigenetic alterations among non-invasive epithelial (MCF-7, MDA-MB-361, and T-47D) and invasive mesenchymal (MDA-MB-231, Hs-578T, and BT-549) breast cancer cell lines. The expression of genes, including GLS1, GFPT2, LDHA, HDAC9, MYST2, and SUV420H2, was assessed using RT-PCR. There was differential expression between epithelial and mesenchymal cell lines. MYST2 and SUV420H2 regulate the levels of the epigenetic biomarkers histone H4 lysine 16 acetylation (H4K16ac) and histone H4 lysine 20 trimethylation (H4K20me3), respectively. Reduced amounts of H4K16ac and H4K20me3 correlated with lower levels of MYST2 and SUV420H2 in mesenchymal cells and, along with reduced amounts of histone H3 lysine 9 acetylation (H3K9ac), were found to distinguish epithelial from mesenchymal cells. In addition, both GLS1 and GFPT2 play roles in glutamine metabolism and were observed to be more highly expressed in mesenchymal cell lines, and when glutamine and glutamate levels reported in the NCI-60 metabolomics dataset were compared, the ratio of glutamate/glutamine was found to be higher in mesenchymal cells. Blocking the conversion of glutamine to glutamate using an allosteric inhibitor, Compound 968, against GLS1, increased H4K16ac in T-47D and MDA-MB-231 cells, linking glutamine metabolism to a particular histone modification in breast cancer. These findings support the concept that metabolically sensitive histone modifications and corresponding histone modifying enzymes can be used as diagnostic and prognostic biomarkers for breast cancer. It also further emphasizes the importance of glutamine metabolism in tumor progression and that inhibitors of cellular metabolic pathways may join histone deacetylase inhibitors as a form of epigenetic therapy.

Plasma microRNAs are sensitive indicators of inter-strain differences in the severity of liver injury induced in mice by a choline- and folate-deficient diet.

MicroRNAs (miRNAs) are a class of small, conserved, tissue-specific regulatory non-coding RNAs that modulate a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, including nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the present study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used as potential biomarkers for the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically diverse strains of inbred male mice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were fed a choline- and folate-deficient (CFD) diet for 12weeks. This diet induced liver injury in all mouse strains; however, the extent of NAFLD-associated pathomorphological changes in the livers was strain-specific, with A/J, C57BL/6J, and C3H/HeJ mice being the least sensitive and WSB/EiJ mice being the most sensitive. The morphological changes in the livers were accompanied by differences in the levels of hepatic and plasma miRNAs. The levels of circulating miR-34a, miR-122, miR-181a, miR-192, and miR-200b miRNAs were significantly correlated with a severity of NAFLD-specific liver pathomorphological features, with the strongest correlation occurring with miR-34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD.

Non-alcoholic fatty liver disease-associated DNA methylation and gene expression alterations in the livers of Collaborative Cross mice fed an obesogenic high-fat and high-sucrose diet.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disease, and patient susceptibility to its onset and progression is influenced by several factors. In this study, we investigated whether altered hepatic DNA methylation in liver tissue correlates with the degree of severity of NAFLD-like liver injury induced by a high-fat and high-sucrose (HF/HS) diet in Collaborative Cross (CC) mice. Using genome-wide targeted bisulphite DNA methylation next-generation sequencing, we found that mice with different non-alcoholic fatty liver (NAFL) phenotypes could be distinguished by changes in hepatic DNA methylation profiles. Specifically, NAFL-prone male CC042 mice exhibited more prominent DNA methylation changes compared with male CC011 mice and female CC011 and CC042 mice that developed only a mild NAFL phenotype. Moreover, these mouse strains demonstrated different patterns of DNA methylation. While the HF/HS diet induced both DNA hypomethylation and DNA hypermethylation changes in all the mouse strains, the NAFL-prone male CC042 mice demonstrated a global predominance of DNA hypermethylation, whereas a more pronounced DNA hypomethylation pattern developed in the mild-NAFL phenotypic mice. In a targeted analysis of selected genes that contain differentially methylated regions (DMRs), we identified NAFL phenotype-associated differences in DNA methylation and gene expression of the Apoa4, Gls2, and Apom genes in severe NAFL-prone mice but not in mice with mild NAFL phenotypes. These changes in the expression of Apoa4 and Gls2 coincided with similar findings in a human in vitro cell model of diet-induced steatosis and in patients with NAFL. These results suggest that changes in the expression and DNA methylation status of these three genes may serve as a set of predictive markers for the development of NAFLD.

Temporal dynamics of SARS-CoV-2 genome and detection of variants of concern in wastewater influent from two metropolitan areas in Arkansas.

Although SARS-CoV-2 can cause severe illness and death, a percentage of the infected population is asymptomatic. This, along with other factors, such as insufficient diagnostic testing and underreporting due to self-testing, contributes to the silent transmission of SARS-CoV-2 and highlights the importance of implementing additional surveillance tools. The fecal shedding of the virus from infected individuals enables its detection in community wastewater, and this has become a valuable public health tool worldwide as it allows the monitoring of the disease on a populational scale. Here, we monitored the presence of SARS-CoV-2 and its dynamic genomic changes in wastewater sampled from two metropolitan areas in Arkansas during major surges of COVID-19 cases and assessed how the viral titers in these samples related to the clinical case counts between late April 2020 and January 2022. The levels of SARS-CoV-2 RNA were quantified by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) using a set of TaqMan assays targeting three different viral genes (encoding ORF1ab polyprotein, surface glycoprotein, and nucleocapsid phosphoprotein). An allele-specific RT-qPCR approach was used to screen the samples for SARS-CoV-2 mutations. The identity and genetic diversity of the virus were further investigated through amplicon-based RNA sequencing, and SARS-CoV-2 variants of concern were detected in wastewater samples throughout the duration of this study. Our data show how changes in the virus genome can affect the sensitivity of specific RT-qPCR assays used in COVID-19 testing with the surge of new variants. A significant association was observed between viral titers in wastewater and recorded number of COVID-19 cases in the areas studied, except when assays failed to detect targets due to the presence of particular variants. These findings support the use of wastewater surveillance as a reliable complementary tool for monitoring SARS-CoV-2 and its genetic variants at the community level.

Lipidomic profiling of the hepatic esterified fatty acid composition in diet-induced nonalcoholic fatty liver disease in genetically diverse Collaborative Cross mice.

Non-alcoholic fatty liver disease (NAFLD), one of the most common forms of chronic liver disease, is characterized by the excessive accumulation of lipid species in hepatocytes. Recent studies have indicated that in addition to the total lipid quantities, changes in lipid composition are a determining factor in hepatic lipotoxicity. Using ultra-high performance liquid chromatography coupled with electrospray tandem mass spectrometry, we analyzed the esterified fatty acid composition in 24 strains of male and female Collaborative Cross (CC) mice fed a high fat/high sucrose (HF/HS) diet for 12 weeks. Changes in lipid composition were found in all strains after the HF/HS diet, most notably characterized by increases in monounsaturated fatty acids (MUFA) and decreases in polyunsaturated fatty acids (PUFA). Similar changes in MUFA and PUFA were observed in a choline- and folate-deficient (CFD) mouse model of NAFLD, as well as in hepatocytes treated in vitro with free fatty acids. Analysis of fatty acid composition revealed that alterations were accompanied by an increase in the estimated activity of MUFA generating SCD1 enzyme and an estimated decrease in the activity of PUFA generating FADS1 and FADS2 enzymes. PUFA/MUFA ratios were inversely correlated with lipid accumulation in male and female CC mice fed the HF/HS diet and with morphological markers of hepatic injury in CFD diet-fed mouse model of NAFLD. These results demonstrate that different models of NAFLD are characterized by similar changes in the esterified fatty acid composition and that alterations in PUFA/MUFA ratios may serve as a diagnostic marker for NAFLD severity.

Epigenetic changes induced in mice liver by methionine-supplemented and methionine-deficient diets.

A diet deficient in donors of methyl group, such as methionine, affects DNA methylation and hepatic lipid metabolism. Methionine also affects other epigenetic mechanisms, such as microRNAs. We investigated the effects of methionine-supplemented or methionine-deficient diets on the expression of chromatin-modifying genes, global DNA methylation, the expression and methylation of genes related to lipid metabolism, and the expression of microRNAs in mouse liver. Female Swiss albino mice were fed a control diet (0.3% methionine), a methionine-supplemented diet (2% methionine), and a methionine-deficient diet (0% methionine) for 10 weeks. The genes most affected by the methionine-supplemented diet were associated with histone and DNA methyltransferases activity, while the methionine-deficient diet mostly altered the expression of histone methyltransferases genes. Both diets altered the global DNA methylation and the expression and gene-specific methylation of the lipid metabolism gene Apoa5. Both diets altered the expression of several liver homeostasis-related microRNAs, including miR-190b-5p, miR-130b-3p, miR-376c-3p, miR-411-5p, miR-29c-3p, miR-295-3p, and miR-467d-5p, with the methionine-deficient diet causing a more substantial effect. The effects of improper amounts of methionine in the diet on liver pathologies may involve a cooperative action of chromatin-modifying genes, which results in an aberrant pattern of global and gene-specific methylation, and microRNAs responsible for liver homeostasis.

Role of ferritin alterations in human breast cancer cells.

Breast cancer is the most common malignancy in women. Successful treatment of breast cancer relies on a better understanding of the molecular mechanisms involved in breast cancer initiation and progression. Recent studies have suggested a crucial role of perturbations in ferritin levels and tightly associated with this, the deregulation of intracellular iron homeostasis; however, the underlying molecular mechanisms for the cancer-linked ferritin alterations remain largely unknown and often with conflicting conclusions. Therefore, this study was undertaken to define the role of ferritin in breast cancer. We determined that human breast cancer cells with an epithelial phenotype, such as MCF-7, MDA-MB-361, T-47D, HCC70 and cells, expressed low levels of ferritin light chain, ferritin heavy chain, transferrin, transferring receptor, and iron-regulatory proteins 1 and 2. In contrast, expression of these proteins was substantially elevated in breast cancer cells with an aggressive mesenchymal phenotype, such as Hs-578T, BT-549, and especially MDA-MB-231 cells. The up-regulation of ferritin light chain and ferritin heavy chain in MDA-MB-231 cells was accompanied by alterations in the subcellular distribution of these proteins as characterized by an increased level of nuclear ferritin and a lower level of the cellular labile iron pool as compared to MCF-7 cells. We established that ferritin heavy chain is a target of miRNA miR-200b, suggesting that its up-regulation in MDA-MB-231 cells may be triggered by the low expression of miR-200b. Ectopic up-regulation of miR-200b by transfection of MDA-MB-231 cells with miR-200b substantially decreased the level of ferritin heavy chain. More importantly, miR-200b-induced down-regulation of ferritin was associated with an increased sensitivity of the MDA-MB-231 cells to the chemotherapeutic agent doxorubicin. These results suggest that perturbations in ferritin levels are associated with the progression of breast cancer toward a more advanced malignant phenotype.

The role of epigenetic events in genotoxic hepatocarcinogenesis induced by 2-acetylaminofluorene.

It is well established that genotoxic reactivity of chemical carcinogens or their metabolites is a critical event in the initiation of tumorigenesis. However, the underlying mechanisms of events following initiation are less well understood, and with respect to genotoxic liver carcinogenesis, it is largely unknown how the initiated cells progress to form preneoplastic hepatic foci. In the present study, we investigated the underlying events associated with tumor-promoting activity of 2-acetylaminofluorene (2-AAF), a powerful complete genotoxic rat liver carcinogen. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 24 weeks, and the status of cytosine DNA methylation, histone methylation, and microRNA expression was determined in the livers of control and 2-AAF-fed rats. The results demonstrate that stages of multistage carcinogenesis following the initiation are driven primarily by carcinogen-induced epigenetic alterations. This was evidenced by altered global histone lysine methylation patterns, increased histone H3 lysine 9 and histone H3 lysine 27 trimethylation in the promoter regions of Rassf1a, p16(INK4a), Socs1, Cdh1, and Cx26 tumor suppressor genes, early Rassf1a and p16(INK4a) promoter CpG island hypermethylation, and altered microRNA expression in preneoplastic livers of rats exposed to 2-AAF. These changes were accompanied by dysregulation of the balance between cell proliferation and apoptosis, a fundamental pro-tumorigenic event in hepatocarcinogenesis. These results signify the fundamental role of epigenetic alterations in genotoxic liver carcinogenesis.

Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats.

Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.

Difference in expression of hepatic microRNAs miR-29c, miR-34a, miR-155, and miR-200b is associated with strain-specific susceptibility to dietary nonalcoholic steatohepatitis in mice.

The importance of dysregulation of microRNA (miRNA) expression in nonalcoholic steatohepatitis (NASH) has been increasingly recognized; however, the association between altered expression of miRNAs and pathophysiological features of NASH and whether there is a connection between susceptibility to NASH and altered expression of miRNAs are largely unknown. In this study, male inbred C57BL/6J and DBA/2J mice were fed a lipogenic methyl-deficient diet that causes liver injury similar to human NASH, and the expression of miRNAs and the level of proteins targeted by these miRNAs in the livers were determined. Administration of the methyl-deficient diet triggered NASH-specific changes in the livers of C57BL/6J and DBA/2J mice, with the magnitude being more severe in DBA/2J mice. This was evidenced by a greater extent of expression of fibrosis-related genes in the livers of methyl-deficient DBA/2J mice. The development of NASH was accompanied by prominent changes in the expression of miRNAs, including miR-29c, miR-34a, miR-155, and miR-200b. Interestingly, changes in the expression of these miRNAs and protein levels of their targets, including Cebp-ß, Socs 1, Zeb-1, and E-cadherin, in the livers of DBA/2J mice fed a methyl-deficient diet were more pronounced as compared with those in C57BL/6J mice. These results show that alterations in the expression of miRNAs are a prominent event during development of NASH induced by methyl deficiency and strongly suggest that severity of NASH and susceptibility to NASH may be determined by variations in miRNA expression response. More important, our data provide a mechanistic link between alterations in miRNA expression and pathophysiological and pathomorphological features of NASH.

E-cadherin transcriptional down-regulation by epigenetic and microRNA-200 family alterations is related to mesenchymal and drug-resistant phenotypes in human breast cancer cells.

The conversion of early stage tumors into invasive malignancies with an aggressive phenotype has been associated with the irreversible loss of E-cadherin expression. The loss of E-cadherin expression in human tumors, including breast cancer, has been attributed to promoter CpG island hypermethylation and direct inhibition by transcriptional repressors. Recent evidence demonstrates that up-regulation of E-cadherin by microRNA-200b (miR-200b) and miR-200c through direct targeting of transcriptional repressors of E-cadherin, ZEB1, and ZEB2, inhibits epithelial-to-mesenchymal transition (EMT), a crucial process in the tumor progression. We demonstrate that microRNA miR-200 family-mediated transcriptional up-regulation of E-cadherin in mesenchymal MDA-MB-231 and BT-549 cells is associated directly with translational repression of ZEB1 and indirectly with increased acetylation of histone H3 at the E-cadherin promoter. The increase in histone H3 acetylation may be attributed to the disruption of repressive complexes between ZEB1 and histone deacetylases and to the inhibition of SIRT1, a class III histone deacetylase. These events inhibit EMT and reactivate a less aggressive epithelial phenotype in cancer cells. Additionally, disruption of ZEB1-histone deacetylase repressor complexes and down-regulation of SIRT1 histone deacetylase up-regulate proapoptotic genes in the p53 apoptotic pathway resulting in the increased sensitivity of cancer cells to the chemotherapeutic agent doxorubicin.

Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin.

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer. Acquired cancer chemoresistance is a multifactorial phenomenon, involving various mechanisms and processes. Recent studies suggest that chemoresistance may be linked to drug-induced dysregulation of microRNA function. Furthermore, mounting evidence indicates the existence of similarities between drug-resistant and metastatic cancer cells in terms of resistance to apoptosis and enhanced invasiveness. We studied the role of miRNA alterations in the acquisition of cisplatin-resistant phenotype in MCF-7 human breast adenocarcinoma cells. We identified a total of 103 miRNAs that were overexpressed or underexpressed (46 upregulated and 57 downregulated) in MCF-7 cells resistant to cisplatin. These differentially expressed miRNAs are involved in the control of cell signaling, cell survival, DNA methylation and invasiveness. The most significantly dysregulated miRNAs were miR-146a, miR-10a, miR-221/222, miR-345, miR-200b and miR-200c. Furthermore, we demonstrated that miR-345 and miR-7 target the human multidrug resistance-associated protein 1. These results suggest that dysregulated miRNA expression may underlie the abnormal functioning of critical cellular processes associated with the cisplatin-resistant phenotype.