Single-cell RNA sequencing (scRNA-seq) technologies allow the dissection of gene expression at single-cell resolution, which greatly revolutionizes transcriptomic studies. A number of scRNA-seq ...protocols have been developed, and these methods possess their unique features with distinct advantages and disadvantages. Due to technical limitations and biological factors, scRNA-seq data are noisier and more complex than bulk RNA-seq data. The high variability of scRNA-seq data raises computational challenges in data analysis. Although an increasing number of bioinformatics methods are proposed for analyzing and interpreting scRNA-seq data, novel algorithms are required to ensure the accuracy and reproducibility of results. In this review, we provide an overview of currently available single-cell isolation protocols and scRNA-seq technologies, and discuss the methods for diverse scRNA-seq data analyses including quality control, read mapping, gene expression quantification, batch effect correction, normalization, imputation, dimensionality reduction, feature selection, cell clustering, trajectory inference, differential expression calling, alternative splicing, allelic expression, and gene regulatory network reconstruction. Further, we outline the prospective development and applications of scRNA-seq technologies.
In addition to primary human hepatocytes, hepatoma cell lines, and transfected nonhepatoma, hepatic cell lines have been used for pharmacological and toxicological studies. However, a systematic ...evaluation and a general report of the gene expression spectra of drug-metabolizing enzymes and transporters (DMETs) in these in vitro systems are not currently available. To fill this information gap and to provide references for future studies, we systematically characterized the basal gene expression profiles of 251 drug-metabolizing enzymes in untreated primary human hepatocytes from six donors, four commonly used hepatoma cell lines (HepG2, Huh7, SK-Hep-1, and Hep3B), and one transfected human liver epithelial cell line. A large variation in DMET expression spectra was observed between hepatic cell lines and primary hepatocytes, with the complete absence or much lower abundance of certain DMETs in hepatic cell lines. Furthermore, the basal DMET expression spectra of five hepatic cell lines are summarized, providing references for researchers to choose carefully appropriate in vitro models for their studies of drug metabolism and toxicity, especially for studies with drugs in which toxicities are mediated through the formation of reactive metabolites.
Leflunomide, used for the treatment of rheumatoid arthritis, has been reported to cause severe liver problems and liver failure; however, the underlying mechanisms are not clear. In this study, we ...used multiple approaches including genomic analysis to investigate and characterize the possible molecular mechanisms of the cytotoxicity of leflunomide in hepatic cells. We found that leflunomide caused endoplasmic reticulum (ER) stress and activated an unfolded protein response, as evidenced by increased expression of related genes including CHOP and GADD34; and elevated protein levels of typical ER stress markers including CHOP, ATF-4, p-eIF2α, and spliced XBP1. The secretion of Gaussia luciferase was suppressed in cells treated with leflunomide in an ER stress reporter assay. Inhibition of ER stress with an ER stress inhibitor 4-phenylbutyrate, and knockdown of ATF-4 and CHOP genes partially protected cells upon leflunomide exposure. In addition, both genomic and biochemical analyses revealed that JNK and ERK1/2 of MAPK signaling pathways were activated, and both contributed to the leflunomide-induced cytotoxicity. Inhibiting JNK activation using a JNK inhibitor attenuated the ER stress and cytotoxicity of leflunomide, whereas inhibiting ERK1/2 using an ERK1/2 inhibitor or ERK1/2 siRNA increased the adverse effect caused by leflunomide, suggesting opposite roles for the two pathways. In summary, our data indicate that both ER stress and the activation of JNK and ERK1/2 contribute to leflunomide-induced cytotoxicity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The rat has been used extensively as a model for evaluating chemical toxicities and for understanding drug mechanisms. However, its transcriptome across multiple organs, or developmental stages, has ...not yet been reported. Here we show, as part of the SEQC consortium efforts, a comprehensive rat transcriptomic BodyMap created by performing RNA-Seq on 320 samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats. We catalogue the expression profiles of 40,064 genes, 65,167 transcripts, 31,909 alternatively spliced transcript variants and 2,367 non-coding genes/non-coding RNAs (ncRNAs) annotated in AceView. We find that organ-enriched, differentially expressed genes reflect the known organ-specific biological activities. A large number of transcripts show organ-specific, age-dependent or sex-specific differential expression patterns. We create a web-based, open-access rat BodyMap database of expression profiles with crosslinks to other widely used databases, anticipating that it will serve as a primary resource for biomedical research using the rat model.
Dronedarone is used to treat patients with cardiac arrhythmias and has been reported to be associated with liver injury. Our previous mechanistic work demonstrated that DNA damage-induced apoptosis ...contributes to the cytotoxicity of dronedarone. In this study, we examined further the underlying mechanisms and found that after a 24-h treatment of HepG2 cells, dronedarone caused cytotoxicity, G1-phase cell cycle arrest, suppression of topoisomerase II, and DNA damage in a concentration-dependent manner. We also investigated the role of cytochrome P450s (CYPs)-mediated metabolism in the dronedarone-induced toxicity using our previously established HepG2 cell lines expressing individually 14 human CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). We demonstrated that CYP3A4, 3A5, and 2D6 were the major enzymes that metabolize dronedarone, and that CYP3A7, 2E1, 2C19, 2C18, 1A1, and 2B6 also metabolize dronedarone, but to a lesser extent. Our data showed that the cytotoxicity of dronedarone was decreased in CYP3A4-, 3A5-, or 2D6-overexpressing cells compared to the control HepG2 cells, indicating that the parent dronedarone has higher potency than the metabolites to induce cytotoxicity in these cells. In contrast, cytotoxicity was increased in CYP1A1-overexpressing cells, demonstrating that CYP1A1 exerts an opposite effect in dronedarone’s toxicity, comparing to CYP3A4, 3A5, or 2D6. We also studied the involvement of topoisomerase II in dronedarone-induced toxicity, and demonstrated that the overexpression of topoisomerase II caused an increase in cell viability and a decrease in γ-H2A.X induction, suggesting that suppression of topoisomerase II may be one of the mechanisms involved in dronedarone-induced liver toxicity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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Cytochrome P450 1A2 (CYP1A2) is one of the most abundant and important drug metabolizing enzymes in human liver. However, little is known about the post-transcriptional regulation of ...CYP1A2, especially the mechanisms involving microRNAs (miRNAs). This study applied a systematic approach to investigate the post-transcriptional regulation of CYP1A2 by miRNAs. Candidate miRNAs targeting the 3′-untranslated region (3′-UTR) of CYP1A2 were screened in silico, resulting in the selection of sixty-two potential miRNAs for further analysis. The levels of two miRNAs, hsa-miR-132-5p and hsa-miR-221-5p, were inversely correlated with the expression of CYP1A2 mRNA transcripts in normal human liver tissue samples represented in The Cancer Genome Atlas (TCGA) dataset. The interactions between these miRNAs and cognate CYP1A2 mRNA sequences were evaluated using luciferase reporter gene studies and electrophoretic mobility shift assays, by which a direct interaction was confirmed involving hsa-miR-132-5p and a cognate binding site present in the CYP1A2 3′-UTR. Experiments by which hsa-miR-132-5p or random miRNA controls were introduced into HepG2, Huh-7 and HepaRG hepatic cell lines showed that only hsa-miR-132-5p suppressed the endogenous and lansoprazole-induced expression of CYP1A2, at biological activity, protein production, and mRNA transcript levels. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays showed that hsa-miR-132-5p attenuates CYP1A2-mediated, lansoprazole-enhanced, flutamide-induced hepatic cell toxicity. Results from multilayer experiments demonstrate that hsa-miR-132-5p suppresses the expression of CYP1A2 and that this suppression is able to decrease the extent of an adverse drug-drug interaction involving lansoprazole and flutamide.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Sertraline, an antidepressant, is commonly used to manage mental health symptoms related to depression, anxiety disorders, and obsessive–compulsive disorder. The use of sertraline has been associated ...with rare but severe hepatotoxicity. Previous research demonstrated that mitochondrial dysfunction, apoptosis, and endoplasmic reticulum stress were involved in sertraline-associated cytotoxicity. In this study, we reported that after a 24-h treatment in HepG2 cells, sertraline caused cytotoxicity, suppressed topoisomerase I and IIα, and damaged DNA in a concentration-dependent manner. We also investigated the role of cytochrome P450 (CYP)-mediated metabolism in sertraline-induced toxicity using our previously established HepG2 cell lines individually expressing 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). We demonstrated that CYP2D6, 2C19, 2B6, and 2C9 metabolize sertraline, and sertraline-induced cytotoxicity was significantly decreased in the cells expressing these CYPs. Western blot analysis demonstrated that the induction of ɣH2A.X (a hallmark of DNA damage) and topoisomerase inhibition were partially reversed in CYP2D6-, 2C19-, 2B6-, and 2C9-overexpressing HepG2 cells. These data indicate that DNA damage and topoisomerase inhibition are involved in sertraline-induced cytotoxicity and that CYPs-mediated metabolism plays a role in decreasing the toxicity of sertraline.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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