An entropy weight method to integrate big omics and mechanistically evaluate DILI.

BACKGROUND AND AIMS: DILI accounts for more than half of acute liver failure cases in the United States and is a major health care issue for the public worldwide. As investigative toxicology is playing an evolving role in the pharmaceutical industry, mechanistic insights into drug hepatotoxicity can facilitate drug development and clinical medication. METHODS: By integrating multisource datasets including gene expression profiles of rat livers from open TG-GATE database and DrugMatrix, drug labels from FDA Liver Toxicity Knowledge Base, and clinical reports from LiverTox, and with the employment of bioinformatic and computational tools, this study developed an approach to characterize and predict DILI based on the molecular understanding of the processes (toxicity pathways). RESULTS: A panel of 11 pathways widely covering biological processes and stress responses was established using a training set of six positive and one negative DILI drugs from open TG-GATEs. An entropy weight method-based model was developed to weight responsive genes within a pathway, and an interpretable machine-learning (ML) model XGBoot-SHAP was trained to rank the importance of pathways to the panel activity. The panel activity was proven to differentiate between injured and noninjured sample points and characterize DILI manifestation using six training drugs. Next, the model was tested using an additional 89 drugs (61 positives + 28 negatives), and a precision of 86% and higher can be achieved. CONCLUSIONS: This study provides a novel approach to mechanisms-driven prediction modeling, as well as big data integration for insights into pharmacology and other human biology areas.

Identifying piRNAs that regulate BaP-induced lung injuries: A bottom-up approach from toxicity pathway investigation to animal validation.

PIWI-interacting RNAs (piRNAs) is an emerging class of small non-coding RNAs that has been recently reported to have functions in infertility, tumorigenesis, and multiple diseases in humans. Previously, 5 toxicity pathways were proposed from hundreds of toxicological studies that underlie BaP-induced lung injuries, and a "Bottom-up" approach was established to identify small non-coding RNAs that drive BaP-induced pulmonary effects by investigating the activation of these pathways in vitro, and the expression of the candidate microRNAs were validated in tissues of patients with lung diseases from publications. Here in this study, we employed the "Bottom-up" approach to identifying the roles of piRNAs and further validated the mechanisms in vivo using mouse acute lung injury model. Specifically, by non-coding RNA profiling in in vitro BaP exposure, a total of 3 suppressed piRNAs that regulate 5 toxicity pathways were proposed, including piR-004153 targeting CYP1A1, FGFR1, ITGA5, IL6R, NGRF, and SDHA, piR-020326 targeting CDK6, and piR-020388 targeting RASD1. Animal experiments demonstrated that tail vein injection of respective formulated agomir-piRNAs prior to BaP exposure could all alleviate acute lung injury that was shown by histopathological and biochemical evidences. Immunohistochemical evaluation focusing on NF-kB and Bcl-2 levels showed that exogenous piRNAs protect against BaP-induced inflammation and apoptosis, which further support that the inhibition of the 3 piRNAs had an important impact on BaP-induced lung injuries. This mechanism-driven, endpoint-supported result once again confirmed the plausibility and efficiency of the approach integrating in silico, in vitro, and in vivo evidences for the purpose of identifying key molecules.

miR-145a-5p/SIK1/cAMP-dependent alteration of synaptic structural plasticity drives cognitive impairment induced by coke oven emissions.

Exposure to fine particulate matter (PM) is associated with the neurodegenerative diseases. Coke oven emissions (COEs) in occupational environment are important sources of PM. However, its neurotoxicity is still unclear. Therefore, evaluating the toxicological effects of COE on the nervous system is necessary. In the present study, we constructed mouse models of COE exposure by tracheal instillation. Mice exposed to COE showed signs of cognitive impairment. This was accompanied by a decrease in miR-145a-5p and an increase in SIK1 expression in the hippocampus, along with synaptic structural damage. Our results demonstrated that COE-induced miR-145a-5p downregulation could increase the expression of SIK1 and phosphorylated SIK1, inhibiting the cAMP/PKA/CREB pathway by activating PDE4D, which was associated with reduced synaptic structural plasticity. Furthermore, restoring of miR-145a-5p expression based on COE exposure in HT22 cells could partially reversed the negative effects of COE exposure through the SIK1/PDE4D/cAMP axis. Collectively, our findings link epigenetic regulation with COE-induced neurotoxicity and imply that miR-145a-5p could be an early diagnostic marker for neurological diseases in patients with COE occupational exposure.

Genomic Characterization Revealed PM2.5-Associated Mutational Signatures in Lung Cancer Including Activation of APOBEC3B.

Fine particulate matter (PM2.5) exposure causes DNA mutations and abnormal gene expression leading to lung cancer, but the detailed mechanisms remain unknown. Here, analysis of genomic and transcriptomic changes upon a PM2.5 exposure-induced human bronchial epithelial cell-based malignant transformed cell model in vitro showed that PM2.5 exposure led to APOBEC mutational signatures and transcriptional activation of APOBEC3B along with other potential oncogenes. Moreover, by analyzing mutational profiles of 1117 non-small cell lung cancers (NSCLCs) from patients across four different geographic regions, we observed a significantly higher prevalence of APOBEC mutational signatures in non-smoking NSCLCs than smoking in the Chinese cohorts, but this difference was not observed in TCGA or Singapore cohorts. We further validated this association by showing that the PM2.5 exposure-induced transcriptional pattern was significantly enriched in Chinese NSCLC patients compared with other geographic regions. Finally, our results showed that PM2.5 exposure activated the DNA damage repair pathway. Overall, here we report a previously uncharacterized association between PM2.5 and APOBEC activation, revealing a potential molecular mechanism of PM2.5 exposure and lung cancer.

Carbon dots induce pathological damage to the intestine via causing intestinal flora dysbiosis and intestinal inflammation.

BACKGROUND: Carbon dots (CDs), as excellent antibacterial nanomaterials, have gained great attention in treating infection-induced diseases such as periodontitis and stomatitis. Given the eventual exposure of CDs to the intestine, elucidating the effect of CDs on intestinal health is required for the safety evaluation of CDs. RESULTS: Herein, CDs extracted from ε-poly-L-lysine (PL) were chosen to explore the modulation effect of CDs on probiotic behavior in vitro and intestinal remodeling in vivo. Results verify that PL-CDs negatively regulate Lactobacillus rhamnosus (L. rhamnosus) growth via increasing reactive oxygen species (ROS) production and reducing the antioxidant activity, which subsequently destroys membrane permeability and integrity. PL-CDs are also inclined to inhibit cell viability and accelerate cell apoptosis. In vivo, the gavage of PL-CDs is verified to induce inflammatory infiltration and barrier damage in mice. Moreover, PL-CDs are found to increase the Firmicutes to Bacteroidota (F/B) ratio and the relative abundance of Lachnospiraceae while decreasing that of Muribaculaceae. CONCLUSION: Overall, these evidences indicate that PL-CDs may inevitably result in intestinal flora dysbiosis via inhibiting probiotic growth and simultaneously activating intestinal inflammation, thus causing pathological damage to the intestine, which provides an effective and insightful reference for the potential risk of CDs from the perspective of intestinal remodeling.

Integration of metabolomics and proteomics reveals the underlying hepatotoxic mechanism of perfluorooctane sulfonate (PFOS) and 6:2 chlorinated polyfluoroalkyl ether sulfonic acid (6:2 Cl-PFESA) in primary human hepatocytes.

Perfluorooctane sulfonate (PFOS) and its alternative 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) are ubiquitous in various environmental and human samples. They have been reported to have hepatotoxicity effects, but the potential mechanisms remain unclear. Herein, we integrated metabolomics and proteomics analysis to investigate the altered profiles in metabolite and protein levels in primary human hepatocytes (PHH) exposed to 6:2 Cl-PFESA and PFOS at human exposure relevant concentrations. Our results showed that 6:2 Cl-PFESA exhibited higher perturbation effects on cell viability, metabolome and proteome than PFOS. Integration of metabolomics and proteomics revealed that the alteration of glycerophospholipid metabolism was the critical pathway of 6:2 Cl-PFESA and PFOS-induced lipid metabolism disorder in primary human hepatocytes. Interestingly, 6:2 Cl-PFESA-induced cellular metabolic process disorder was associated with the cellular membrane-bounded signaling pathway, while PFOS was associated with the intracellular transport process. Moreover, the disruption effects of 6:2 Cl-PFESA were also involved in inositol phosphate metabolism and phosphatidylinositol signaling system. Overall, this study provided comprehensive insights into the hepatic lipid toxicity mechanisms of 6:2 Cl-PFESA and PFOS in human primary hepatocytes.

MiR-766-3p and miR-671-5p attenuate aristolochic acid-induced hepatotoxicity by directly targeting the key bioactivating enzyme NQO1.

Aristolochic acid (AA) as an emerging contaminant in herbal medicines or crops has been well-recognized for causing nephropathy since 1990s. Over the last decade, mounting evidence has linked AA to liver injury; however, the underlying mechanism is poorly elucidated. MicroRNAs respond to environmental stress and mediate multiple biological processes, thus showing biomarker potentials prognostically or diagnostically. In the present study, we investigated the role of miRNAs in AA-induced hepatotoxicity, specifically in regulating NQO1, the key enzyme responsible for AA bioactivation. In silico analysis showed that hsa-miR-766-3p and hsa-miR-671-5p were significantly associated with AAI exposure as well as NQO1 induction. A 28-day rat experiment of 20 mg/kg AA exposure demonstrated a 3-fold increase of NQO1 and an almost 50 % decrease of the homologous miR-671 that were accompanied with liver injury, which was consistent with in silico prediction. Further mechanistic investigation using Huh7 cells with IC50 of AAI at 146.5 µM showed both hsa-miR-766-3p and hsa-miR-671-5p were able to directly bind to and down-regulate NQO1 basal expression. In addition, both miRNAs were shown to suppress AAI-induced NQO1 upregulation in Huh7 cells at a cytotoxic concentration of 70 µM, and consequently alleviate AAI-induced cellular effects, including cytotoxicity and oxidative stress. Together, these data illustrate that miR-766-3p and miR-671-5p attenuate AAI-induced hepatotoxicity, and thus have monitoring and diagnostic potentials.

Perturbation of Specific Signaling Pathways Is Involved in Initiation of Mouse Liver Fibrosis.

BACKGROUND AND AIMS: To identify the regulatory role of protein phosphatase 2A (PP2A) in the development of liver disease, we generated a mouse model with hepatocyte-specific deletion of Ppp2r1a gene (encoding PP2A Aα subunit). APPROACH AND RESULTS: Homozygote (HO) mice and matched wild-type littermates were investigated at 3, 6, 9, 12, 15, and 18 months of age. Pathological examination showed that PP2A Aα deficiency in hepatocytes resulted in progressive liver fibrosis phenotype from 9 months of age. No hepatocyte death was observed in HO mice. However, perturbation of pathways including epidermal growth factor receptor 1 (EGFR1), amino acid metabolism, and translation factors as well as leptin and adiponectin led to pronounced hepatic fibrosis. In vitro studies demonstrated the involvement of specific B subunit complexes in the regulation of EGFR1 signaling pathway and cross talk between defected hepatocytes and stimulation of interstitial hyperplasia. It is noteworthy that HO mice failed to develop hepatocellular carcinoma for as long as 22 months of age. We further demonstrate that PP2A Aß-containing holoenzymes played a critical role in preventing hepatocyte apoptosis and antagonizing tumorigenesis through specific pathways on Aα loss. Furthermore, PP2A Aα and Aß were functionally distinct, and the Aß isoform failed to substitute for Aα in the development of inflammation and liver fibrosis. CONCLUSIONS: These observations identify pathways that contribute to the pathogenesis of liver fibrosis and provide putative therapeutic targets for its treatment.

The path via pathway-based approaches towards safety assessment: A concise review.

For decades, chemical safety assessment has been proposed to shift from animal testing to in vitro testing systems in response to the call for the 3R. In Europe, the answer was to combine various information sources in integrated testing strategies (ITS); In the US, it was in 2007 when the landmark report by the National Research Council put forward a vision of in vitro toxicity testing paradigm. Since then, efforts to develop pathway-based assessment framework have been on the track. In 2010, systems biology brought out a conceptual framework called adverse outcome pathway (AOP), which took one step further from toxicity pathway to regulatory toxicology. Computational modeling, high-throughput screening, high-content omics have all been approached to facilitate this progress. This paper briefly reviewed the achievement of pathway-based chemical assessment since 2007, discussed potential pitfalls and challenges that mechanism-driven chemical assessment may undergo, and presented future perspectives of safety assessment that is to be based on computational system biology.

Single-cell transcriptomics reveals immune dysregulation mediated by IL-17A in initiation of chronic lung injuries upon real-ambient particulate matter exposure.

BACKGROUND: Long-term exposure to fine particulate matter (PM2.5) increases susceptibility to chronic respiratory diseases, including inflammation and interstitial fibrosis. However, the regulatory mechanisms by which the immune response mediates the initiation of pulmonary fibrosis has yet to be fully characterized. This study aimed to illustrate the interplay between different cell clusters and key pathways in triggering chronic lung injuries in mice following PM exposure. RESULTS: Six-week-old C57BL/6J male mice were exposed to PM or filtered air for 16 weeks in a real-ambient PM exposure system in Shijiazhuang, China. The transcriptional profiles of whole lung cells following sub-chronic PM exposure were characterized by analysis of single-cell transcriptomics. The IL-17A knockout (IL-17A-/-) mouse model was utilized to determine whether the IL-17 signaling pathway mediated immune dysregulation in PM-induced chronic lung injuries. After 16-week PM exposure, chronic lung injuries with excessive collagen deposition and increased fibroblasts, neutrophils, and monocytes were noted concurrent with a decreased number of major classes of immune cells. Single-cell analysis showed that activation of the IL-17 signaling pathway was involved in the progression of pulmonary fibrosis upon sub-chronic PM exposure. Depletion of IL-17A led to significant decline in chronic lung injuries, which was mainly triggered by reduced recruitment of myeloid-derived suppressor cells (MDSCs) and downregulation of TGF-ß. CONCLUSION: These novel findings demonstrate that immunosuppression via the IL-17A pathway plays a critical role in the initiation of chronic lung injuries upon sub-chronic PM exposure.

GLIS3 mediated by the Rap1/PI3K/AKT signal pathway facilitates real-ambient PM2.5 exposure disturbed thyroid hormone homeostasis regulation.

Exposure to fine particulate matter (PM2.5) could damage multiple organs and systems. Recent epidemiological studies have shown that PM2.5 can disrupt dynamic balance of thyroid hormone (TH). However, the underlying mechanism by which PM2.5 interferes with TH remains unclear. This study evaluated the role of Gli-similar3 (GLIS3) in the effect of PM2.5 on TH synthesis in mice using a real-ambient exposure system, in Shijiazhuang City, Hebei Province. The PM2.5exposure group (PM) and filtered air group (FA) were placed in the exposure device for four and eight weeks. The results showed that the PM2.5 exposure altered the structure of the thyroid gland. Moreover, after PM2.5 exposure for eight weeks, the exposure level of free thyroxine (FT4) increased and the expression level of thyroid stimulating hormone (TSH) decreased in serum of mice. In addition, PM2.5 exposure significantly increased the expression of proteins related to thyroid hormone synthesis, such as sodium iodide transporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG). Next, we found that GLIS3 and thyroid transcription factor Paired box 8 (PAX8) also increased after PM2.5 exposure. In order to further explore the potential molecular mechanism, we carried out transcriptome sequencing. KEGG analysis of the top 10 pathways revealed that the Ras-associated protein 1 (Rap1) signaling pathway could activate transcription factors and is related to thyroid cell survival. Additionally, PM2.5 exposure significantly increased the protein levels of Rap1 and its active form (Rap1 +GTP). We speculate that the active state of Rap1 is believed to be involved in activating the expression of transcription factor GLIS3. In conclusion, PM2.5 exposure induces histological changes in the thyroid gland and thyroid dysfunction in mice. The exposure activates GLIS3 through the Rap1/PI3K/AKT pathway to promote the expression of proteins related to thyroid hormone synthesis, leading to increased dysregulating TH homeostasis.

A toxicity pathway-based approach for modeling the mode of action framework of lead-induced neurotoxicity.

BACKGROUND: The underlying mechanisms of lead (Pb) toxicity are not fully understood, which makes challenges to the traditional risk assessment. There is growing use of the mode of action (MOA) for risk assessment by integration of experimental data and system biology. The current study aims to develop a new pathway-based MOA for assessing Pb-induced neurotoxicity. METHODS: The available Comparative Toxicogenomic Database (CTD) was used to search genes associated with Pb-induced neurotoxicity followed by developing toxicity pathways using Ingenuity Pathway Analysis (IPA). The spatiotemporal sequence of disturbing toxicity pathways and key events (KEs) were identified by upstream regulator analysis. The MOA framework was constructed by KEs in biological and chronological order. RESULTS: There were a total of 71 references showing the relationship between lead exposure and neurotoxicity, which contained 2331 genes. IPA analysis showed that the neuroinflammation signaling pathway was the core toxicity pathway in the enriched pathways relevant to Pb-induced neurotoxicity. The upstream regulator analysis demonstrated that the aryl hydrocarbon receptor (AHR) signaling pathway was the upstream regulator of the neuroinflammation signaling pathway (11.76% overlap with upstream regulators, |Z-score|=1.451). Therefore, AHR activation was recognized as the first key event (KE1) in the MOA framework. The following downstream molecular and cellular key events were also identified. The pathway-based MOA framework of Pb-induced neurotoxicity was built starting with AHR activation, followed by an inflammatory response and neuron apoptosis. CONCLUSION: Our toxicity pathway-based approach not only advances the development of risk assessment for Pb-induced neurotoxicity but also brings new insights into constructing MOA frameworks of risk assessment for new chemicals.

Lipid metabolism disorders effects of 6:2 chlorinated polyfluorinated ether sulfonate through Hsa-miRNA-532-3p/Acyl-CoA oxidase 1(ACOX1) pathway.

6:2 Chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), an alternative product of perfluorooctane sulfonate (PFOS), has been frequently detected in various environmental, wildlife, and human samples. A few studies revealed the hepatotoxicity of 6:2 Cl-PFESA in animals, but the underlying toxicity mechanisms remain largely unknown. In this study, we investigated the lipid metabolism disorders of 6:2 Cl-PFESA through miRNA-gene interaction mode in Huh-7 cells. Our results showed that 6:2 Cl-PFESA significantly promoted cellular lipid accumulation and increased the expression of Acyl-CoA oxidase 1 (ACOX1), with the lowest effective concentrations (LOECs) of 3 µM. In silico analysis showed that hsa-miR-532-3p is a potential miRNA molecule targeting ACOX1. Fluorescent-based RNA electrophoretic mobility shift assay (FREMSA) and ACOX1-mediated luciferase reporter gene assays showed that hsa-miR-532-3p could directly bind to ACOX1 and inhibit its transcription activity. Besides, 6:2 Cl-PFESA decreased the expression of hsa-miR-532-3p in the PPARα-independent manner. Overexpression of hsa-miR-532-3p promoted 6:2 Cl-PFESA-induced cellular lipid accumulation and decreased the ACOX1 production in Huh-7 cells. Taken together, at human exposure relevant concentrations, 6:2 Cl-PFESA might upregulate the expression levels of ACOX1 through downregulating hsa-miR-532-3p, and disturbed lipid homeostasis in Huh-7 cells, which revealed a novel epigenetic mechanism of 6:2 Cl-PFESA-induced hepatic lipid toxic effects.

MicroRNA hsa-miR-1301-3p Regulates Human ADH6, ALDH5A1 and ALDH8A1 in the Ethanol-Acetaldehyde-Acetate Metabolic Pathway.

Alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) are vital enzymes involved in the metabolism of a variety of alcohols. Differences in the expression and enzymatic activity of human ADHs and ALDHs correlate with individual variability in metabolizing alcohols and drugs and in the susceptibility to alcoholic liver disease. MicroRNAs (miRNAs) function as epigenetic modulators to regulate the expression of drug-metabolizing enzymes. To characterize miRNAs that target ADHs and ALDHs in human liver cells, we carried out a systematic bioinformatics analysis to analyze free energies of the interaction between miRNAs and their cognate sequences in ADH and ALDH transcripts and then calculated expression correlations between miRNAs and their targeting ADH and ALDH genes using a public data base. Candidate miRNAs were selected to evaluate bioinformatic predictions using a series of biochemical assays. Our results showed that 11 miRNAs have the potential to modulate the expression of two ADH and seven ALDH genes in the human liver. We found that hsa-miR-1301-3p suppressed the expression of ADH6, ALDH5A1, and ALDH8A1 in liver cells and blocked their induction by ethanol. In summary, our results revealed that hsa-miR-1301-3p plays an important role in ethanol metabolism by regulating ADH and ALDH gene expression. SIGNIFICANCE STATEMENT: Systematic bioinformatics analysis showed that 11 microRNAs might play regulatory roles in the expression of two alcohol dehydrogenase (ADH) and seven aldehyde dehydrogenase (ALDH) genes in the human liver. Experimental evidences proved that hsa-miR-1301-3p suppressed the expression of ADH6, ALDH5A1, and ALDH8A1 in liver cells and decreased their inducibility by ethanol.

Coordinated Regulation of UGT2B15 Expression by Long Noncoding RNA LINC00574 and hsa-miR-129-5p in HepaRG Cells.

Recent studies have shown that microRNAs and long noncoding RNAs (lncRNAs) regulate the expression of drug metabolizing enzymes (DMEs) in human hepatic cells and that a set of DMEs, including UDP glucuronosyltransferase (UGT) 2B15, is down-regulated dramatically in liver cells by toxic acetaminophen (APAP) concentrations. In this study we analyzed mRNA, microRNA, and lncRNA expression profiles in APAP-treated HepaRG cells to explore noncoding RNA-dependent regulation of DME expression. The expression of UGT2B15 and lncRNA LINC00574 was decreased in APAP-treated HepaRG cells. UGT2B15 levels were diminished by LINC00574 suppression using antisense oligonucleotides or small interfering RNA. Furthermore, we found that hsa-miR-129-5p suppressed LINC00574 and decreased UGT2B15 expression via LINC00574 in HepaRG cells. In conclusion, our results indicate that LINC00574 acts as an important regulator of UGT2B15 expression in human hepatic cells, providing experimental evidence and new clues to understand the role of cross-talk between noncoding RNAs. SIGNIFICANCE STATEMENT: We showed a molecular network that displays the cross-talk and consequences among mRNA, micro RNA, long noncoding RNA, and proteins in acetaminophen (APAP)-treated HepaRG cells. APAP treatment increased the level of hsa-miR-129-5p and decreased that of LINC00574, ultimately decreasing the production of UDP glucuronosyltransferase (UGT) 2B15. The proposed regulatory network suppresses UGT2B15 expression through interaction of hsa-miR-129-5p and LINC00574, which may be achieved potentially by recruiting RNA binding proteins.

Caloric restriction attenuates C57BL/6 J mouse lung injury and extra-pulmonary toxicity induced by real ambient particulate matter exposure.

BACKGROUND: Caloric restriction (CR) is known to improve health and extend lifespan in human beings. The effects of CR on adverse health outcomes in response to particulate matter (PM) exposure and the underlying mechanisms have yet to be defined. RESULTS: Male C57BL/6 J mice were fed with a CR diet or ad libitum (AL) and exposed to PM for 4 weeks in a real-ambient PM exposure system located at Shijiazhuang, China, with a daily mean concentration (95.77 µg/m3) of PM2.5. Compared to AL-fed mice, CR-fed mice showed attenuated PM-induced pulmonary injury and extra-pulmonary toxicity characterized by reduction in oxidative stress, DNA damage and inflammation. RNA sequence analysis revealed that several pulmonary pathways that were involved in production of reactive oxygen species (ROS), cytokine production, and inflammatory cell activation were inactivated, while those mediating antioxidant generation and DNA repair were activated in CR-fed mice upon PM exposure. In addition, transcriptome analysis of murine livers revealed that CR led to induction of xenobiotic metabolism and detoxification pathways, corroborated by increased levels of urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) and decreased cytotoxicity measured in an ex vivo assay. CONCLUSION: These novel results demonstrate, for the first time, that CR in mice confers resistance against pulmonary injuries and extra-pulmonary toxicity induced by PM exposure. CR led to activation of xenobiotic metabolism and enhanced detoxification of PM-bound chemicals. These findings provide evidence that dietary intervention may afford therapeutic means to reduce the health risk associated with PM exposure.

Long noncoding RNA LINC00844-mediated molecular network regulates expression of drug metabolizing enzymes and nuclear receptors in human liver cells.

Noncoding RNAs, such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), regulate gene expression in many physiological and pathological processes, including drug metabolism. Drug metabolizing enzymes (DMEs) are critical components in drug-induced liver toxicity. In this study, we used human hepatic HepaRG cells treated with 5 or 10 mM acetaminophen (APAP) as a model system and identified LINC00844 as a toxicity-responsive lncRNA. We analyzed the expression profiles of LINC00844 in different human tissues. In addition, we examined the correlations between the levels of LINC00844 and those of key DMEs and nuclear receptors (NRs) for APAP metabolism in humans. Our results showed that lncRNA LINC00844 is enriched in the liver and its expression correlates positively with mRNA levels of CYP3A4, CYP2E1, SULT2A1, pregnane X receptor (PXR), and hepatocyte nuclear factor (HNF) 4α. We demonstrated that LINC00844 regulates the expression of these five genes in HepaRG cells using gain- and loss-of-function assays. Further, we discovered that LINC00844 is localized predominantly in the cytoplasm and acts as an hsa-miR-486-5p sponge, via direct binding, to protect SULT2A1 from miRNA-mediated gene silencing. Our data also demonstrated a functional interaction between LINC00844 and hsa-miR-486-5p in regulating DME and NR expression in HepaRG cells and primary human hepatocytes. We depicted a LINC00844-mediated regulatory network that involves miRNA and NRs and influences DME expression in response to APAP toxicity.

Integration of proteomics, lipidomics, and metabolomics reveals novel metabolic mechanisms underlying N, N-dimethylformamide induced hepatotoxicity.

N, N-Dimethylformamide (DMF) is a universal organic solvent which widely used in various industries, and a considerable amount of DMF is detected in industrial effluents. Accumulating animal and epidemiological studies have identified liver injury as an early toxic effect of DMF exposure; however, the detailed mechanisms remain poorly understood. In this study, we systematically integrated the quantitative proteomics, lipidomics, and metabolomics data obtained from the primary human hepatocytes exposed to DMF, to depict the complicated biochemical reactions correlated to liver damage. Eventually, we identified 284 deregulated proteins (221 downregulated and 63 upregulated) and 149 deregulated lipids or metabolites (99 downregulated and 50 upregulated) induced by DMF exposure. Further, the integration of the protein-metabolite (lipid) interactions revealed that N-glycan biosynthesis (involved in the endoplasmic reticulum stress and the unfolded protein response), bile acid metabolism (involved in the lipid metabolism and the inflammatory process), and mitochondrial dysfunction and glutathione depletion (both contributed to reactive oxygen species) were the typical biochemical reactions disturbed by DMF exposure. In summary, our study identified the versatile protein, lipid, and metabolite molecules in multiple signaling and metabolic pathways involved in DMF induced liver injury, and provided new insights to elucidate the toxic mechanisms of DMF.

Integrative Functional Genomics Implicates EPB41 Dysregulation in Hepatocellular Carcinoma Risk.

Genome-wide association studies (GWASs) have provided many insights into cancer genetics. However, the molecular mechanisms of many susceptibility SNPs defined by GWASs in cancer heritability and in promoting cancer risk remain elusive. New research strategies, including functional evaluations, are warranted to systematically explore truly causal genetic variants. In this study, we developed an integrative functional genomics methodology to identify cancer susceptibility SNPs in transcription factor-binding sites across the whole genome. Employing integration of functional genomic data from c-Myc cistromics, 1000 Genomes, and the TRANSFAC matrix, we successfully annotated 12 SNPs present in the c-Myc cistrome with properties consistent with modulating c-Myc binding affinity in hepatocellular carcinoma (HCC). After genotyping these 12 SNPs in 1,806 HBV-related HCC case subjects and 1,708 control subjects, we identified a HCC susceptibility SNP, rs157224G>T, in Chinese populations (T allele: odds ratio = 1.64, 95% confidence interval = 1.32-2.02; p = 5.2 × 10(-6)). This polymorphism leads to HCC predisposition through modifying c-Myc-mediated transcriptional regulation of EPB41, with the risk rs157224T allele showing significantly decreased gene expression. Based on cell proliferation, wound healing, and transwell assays as well as the mouse xenograft model, we identify EPB41 as a HCC susceptibility gene in vitro and in vivo. Consistent with this notion, we note that EPB41 expression is significantly decreased in HCC tissue specimens, especially in portal vein metastasis or intrahepatic metastasis, compared to normal tissues. Our results highlight the involvement of regulatory genetic variants in HCC and provide pathogenic insights of this malignancy via a genome-wide approach.

Regulation of cytochrome P450 expression by microRNAs and long noncoding RNAs: Epigenetic mechanisms in environmental toxicology and carcinogenesis.

Environmental exposures to hazardous chemicals are associated with a variety of human diseases and disorders, including cancers. Phase I metabolic activation and detoxification reactions catalyzed by cytochrome P450 enzymes (CYPs) affect the toxicities of many xenobiotic compounds. Proper regulation of CYP expression influences their biological effects. Noncoding RNAs (ncRNAs) are involved in regulating CYP expression, and ncRNA expression is regulated in response to environmental chemicals. The mechanistic interactions between ncRNAs and CYPs associated with the toxicity and carcinogenicity of environmental chemicals are described in this review, focusing on microRNA-dependent CYP regulation. The role of long noncoding RNAs in regulating CYP expression is also presented and new avenues of research concerning this regulatory mechanism are described.