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Interaction network between the genes of 4 different lists by using ToppCluster (Kaimal et al., 2010). Cluster 1 (red): 7 genes of the mtDNA coding for mitochondrial proteins which have shown concordance between methylation patterns and gene expression; Cluster 2 (blue): 5 genes which have shown increased numbers of neighbors in the networks; Cluster 3 (purple): 38 genes that showed concordance with protein expression and are known to encode for mitochondrial proteins by using MitoCarta2.0 (Calvo et al., 2016); and Cluster 4 (green): 16 transcription factors of the mitochondria-nucleus signaling pathway which have shown modified mRNA expression values after 2 and/or 3 days of VPA-treatment. The concordant gene/protein CPT1A has been marked yellow, since it is the direct target of VPA.
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... interestingly, in contrast to the earlier hypothesis that it is toxicant-related oxidative stress which activates the mitochondria-nu- cleus signaling pathway (Jazwinski, 2013;Kotiadis et al., 2014;Ryan and Hoogenraad, 2007) we now propose that mitochondria-nucleus communications result from direct gene-gene interactions induced by VPA (Fig. 8). In more detail, the mitochondrial gene MT-CO2 interacts with 1 of the 5 network genes, namely FN1 (directly, as well as in- directly via prohibitin (PHB)). This FN1 seems to play a pivotal role in this interaction network, since it showed also connections with 2 other network genes (namely CAV1 and FLNA) and with 2 TFs involved in the mitochondria-nucleus signaling (namely PRKCB and MYC). Notably, the importance of FN1 in the mitochondrial nuclear crosstalk has been noted in a previous study where FN1 was upregulated in a breast epi- thelial cell line devoid of mtDNA (ρ 00 cells) compared to a breast car- cinoma cell line ( Kulawiec et al., 2008). Furthermore, MYC interacts with 3 other TFs relevant for toxicant-induced mitochondria-nucleus signaling, with 4 of the 5 network genes, and most importantly, with 4 genes concordantly regulating mitochondrial ...
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... of the 6 genes (except of GNAS), which developed an increasing number of neighbors during VPA-treatment which dropped again after washout (Fig. 7E), play a role in mitochondria formation and func- tioning ( Lehwald et al., 2012;Mukherjee et al., 2014;Schmidt et al., 2008;Seibenhener et al., 2013;Volonte et al., 2016;Wu et al., 2005;Yu et al., 2017). FN1 knock-down has been shown to induce mitochon- drial-dependent apoptosis by decreasing CA 2+ ( Wu et al., 2005). Fur- thermore, IQGAP1 is known to play a role in β-catenin expression ( Schmidt et al., 2008), which has an important role in mitochondrial homeostasis ( Lehwald et al., 2012). In addition, FLNA and CAV1 are required for the transport of ribonucleoprotein to the mitochondria ( Mukherjee et al., 2014). CAV1 has also been shown to reduce the ATP- production via mitochondrial dysfunction ( Volonte et al., 2016;Yu et al., 2017). SQSTM1 deficiency causes defects in mitochondrial membrane potential which can lead to mitochondrial dysfunction ( Seibenhener et al., 2013). Interestingly, the mitochondria-nucleus signaling TF MYC proto-oncogene bHLH transcription factor (MYC) interconnected the network building genes FN1, SQSTM1, CAV1, and FLNA (Fig. 8). Hereby, the downregulated expressions of FN1, CAV1, and FLNA during VPA-treatment correspond with the downregulation of MYC on day 1 and day 2. MYC expression had normalized after the WO period, possibly through hypermethylation (Supplementary Table ...
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... summary, our cross-omics analysis of dynamic perturbations caused by the prototypical liver toxicant VPA in human hepatocytes has revealed new insights in the mitochondrial-nuclear crosstalk endorsing VPA-induced mitochondrial toxicity as apparent from reduced ATP production which was maintained even after terminating VPA treat- ment, as well as reducing oxidative VPA metabolism as an adaptive response to the continuing VPA challenge. We propose that in the early phase of treatment VPA-generated oxidative stress downregulates the electron transport chain and the expression of a range of ATP-synthases which leads to an initial decrease in ATP production as a first mani- festation of mitochondrial toxicity. Subsequently, through interference with the HDAC/DNMT gene families, VPA induces a transient hy- permethylation which further downregulates mitochondrial genes thus increasingly impairing mitochondrial function and ATP production. One of these mitochondrial genes, MT-CO2, thereupon activates the mitochondria-nucleus signaling pathway mediated by FN1 which over time establishes an interaction with the downregulated nuclear TF MYC. Thereafter, MYC decreases CPS1 expression which then down- regulates CPT1A, proteins of CPS1 and CPT1A being downregulated even after washout. This leads to the downregulation of a small reg- ulatory network consisting of six concordant mitochondrial genes (Fig. 8). This gene-gene interaction network is confirmed by the fact that proteins of CPS1, CPT1A, COQ9, AGK, and CPT2 were down- regulated even after terminating VPA treatment which matches with the observed repressed ATP-production after washout (Fig. 2). This impairment of the mitochondrial function has been considered as one of the reasons for steatosis/hepatotoxicity ( Begriche et al., 2006;Pessayre et al., 1999;Silva et al., ...
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... et al., 2016;Yu et al., 2017). SQSTM1 deficiency causes defects in mitochondrial membrane potential which can lead to mitochondrial dysfunction ( Seibenhener et al., 2013). Interestingly, the mitochondria-nucleus signaling TF MYC proto-oncogene bHLH transcription factor (MYC) interconnected the network building genes FN1, SQSTM1, CAV1, and FLNA (Fig. 8). Hereby, the downregulated expressions of FN1, CAV1, and FLNA during VPA-treatment correspond with the downregulation of MYC on day 1 and day 2. MYC expression had normalized after the WO period, possibly through hypermethylation (Supplementary Table ...
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... interestingly, in contrast to the earlier hypothesis that it is toxicant-related oxidative stress which activates the mitochondria-nucleus signaling pathway (Jazwinski, 2013;Kotiadis et al., 2014;Ryan and Hoogenraad, 2007) we now propose that mitochondria-nucleus communications result from direct gene-gene interactions induced by VPA (Fig. 8). In more detail, the mitochondrial gene MT-CO2 interacts with 1 of the 5 network genes, namely FN1 (directly, as well as indirectly via prohibitin (PHB)). This FN1 seems to play a pivotal role in this interaction network, since it showed also connections with 2 other network genes (namely CAV1 and FLNA) and with 2 TFs involved in the ...
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... by FN1 which over time establishes an interaction with the downregulated nuclear TF MYC. Thereafter, MYC decreases CPS1 expression which then downregulates CPT1A, proteins of CPS1 and CPT1A being downregulated even after washout. This leads to the downregulation of a small regulatory network consisting of six concordant mitochondrial genes (Fig. 8). This gene-gene interaction network is confirmed by the fact that proteins of CPS1, CPT1A, COQ9, AGK, and CPT2 were downregulated even after terminating VPA treatment which matches with the observed repressed ATP-production after washout (Fig. 2). This impairment of the mitochondrial function has been considered as one of the reasons ...
Citations
... HepG2 cells were cultured on 2D/3D growth substrates for 8 days prior to harvesting for RNAseq ( Figure 4A). The expression of mechanotransduction-related genes ( Figure 4B) and liver-related genes ( Figure 4C) were compared among 2D/3D-primed populations, human liver and the published transcriptome of 1-day cultured 2D primary hepatocytes [36]. ...
It is widely recognised that cells respond to their microenvironment, which has implications for cell culture practices. Growth cues provided by 2D cell culture substrates are far removed from native 3D tissue structure in vivo. Geometry is one of many factors that differs between in vitro culture and in vivo cellular environments. Cultured cells are far removed from their native counterparts and lose some of their predictive capability and reliability. In this study, we examine the cellular processes that occur when a cell is cultured on 2D or 3D surfaces for a short period of 8 days prior to its use in functional assays, which we term: “priming”. We follow the process of mechanotransduction from cytoskeletal alterations, to changes to nuclear structure, leading to alterations in gene expression, protein expression and improved functional capabilities. In this study, we utilise HepG2 cells as a hepatocyte model cell line, due to their robustness for drug toxicity screening. Here, we demonstrate enhanced functionality and improved drug toxicity profiles that better reflect the in vivo clinical response. However, findings more broadly reflect in vitro cell culture practises across many areas of cell biology, demonstrating the fundamental impact of mechanotransduction in bioengineering and cell biology.
... The preparation of the samples processed here is described in Wolters et al. (2018). In brief, human hepatocytes were exposed to 15 mM VPA or 1% EtOH (CTL) and sampled after one, two, and three days of treatment. ...
Motivation:
Long-read transcriptome sequencing (LRTS) has the potential to enhance our understanding of alternative splicing, and the complexity of this process requires the use of versatile computational tools, with the ability to accommodate various stages of the workflow with maximum flexibility.
Results:
We introduce IsoTools, a Python-based LRTS analysis framework that offers a wide range of functionality for transcriptome reconstruction and quantification of transcripts. Furthermore, we integrate a graph-based method for identifying alternative splicing events and a statistical approach based on the beta-binomial distribution for detecting differential events. To demonstrate the effectiveness of our methods, we applied IsoTools to PacBio LRTS data of human hepatocytes treated with the HDAC inhibitor valproic acid. Our results indicate that LRTS can provide valuable insights into alternative splicing, particularly in terms of complex and differential splicing patterns, in comparison to short-read RNA-seq.
Availability and implementation:
IsoTools is available on GitHub and PyPI, and its documentation, including tutorials, CLI and API references, can be found at https://isotools.readthedocs.io/.
Supplementary information:
Supplementary data are available at Bioinformatics online.
... Spatial-omics technologies recognize subpopulations with the specificity of current -omic findings while also providing notable spatial context by overlaying -omics level data into tissue images, and it has the ability to perform indepth bimolecular profiling while preserving the native morphology of the cell for the investigation of cell-cell and cell-the extracellular matrix (ECM) interactions [164]. Omics analysis permitted discrimination between the toxicity of valproic acid (VPA) and adaptation, and omics researches revealed dynamic responses leading to constant mitochondrial dysfunction [165]. The plant omics analysis approach recognized grain samples of inferior quality and omics profiling can clarify the risk assessment procedure of new/GM plant varieties [166,167]. ...
Multi-omics approaches have developed as a profitable technique for plant systems, a popular method in medical and biological sciences underlining the necessity to outline new inte-grative technology and functions to facilitate the multi-scale depiction of biological systems. Understanding a biological system through various omics layers reveals supplementary sources of variability and probably inferring the sequence of cases leading to a definitive process. Manuscripts and reviews were searched on PubMed with the keywords of multi-omics, data analysis, omics, data analysis, data integration, deep learning multi-omics, and multi-omics integration. Articles that were published after 2010 were prioritized. The authors focused mainly on popular publications developing new approaches. Omics reveal interesting tools to produce behavioral and interactions data in microbial communities, and integrating omics details into microbial risk assessment will have an impact on food safety, and also on relevant spoilage control procedures. Omics datasets, comprehensively characterizing biological cases at a molecular level, are continually increasing in both dimensionality and complexity. Multi-omics data analysis is appropriate for treatment optimization, molecular testing and disease prognosis, and to achieve mechanistic understandings of diseases. New effective solutions for multi-omics data analysis together with well-designed components are recommended for many trials. The goal of this mini-review article is to introduce multi-omics technologies considering different multi-omics analyses.
... The preparation of the samples processed here are described in [29]. In brief, human hepatocytes were exposed to 15 mM valproic acid (VPA) or 1% EtOH (CTL) and sampled after one day, two days and three days of treatment. ...
Long-read transcriptome sequencing (LRTS) holds the promise to refine our understanding of alternative splicing, however, the nature of splicing is highly complex, and computational tools need to cope with this complexity by maximum flexibility at different steps of the work-flow. For this purpose we developed IsoTools, a Python-based LRTS analysis package. IsoTools provides broad functionality for transcriptome reconstruction and quantification of isoforms. Additionally, we implemented a graph-based method for the identification of alternative splicing events and a statistical approach based on the beta-binomial distribution for the detection of differential events. To demonstrate our methods, we generated PacBio LRTS data of human hepatocytes treated with the HDAC inhibitor valproic acid. Contrasted with short read RNA-seq, this analysis shows that LRTS provides valuable additional insights for a better understanding of alternative splicing, in particular with respect to complex novel and differential splicing patterns. IsoTools is available at https://github.com/MatthiasLienhard/isotools.
... The use of pooled human primary cells from multiple donors to create 3D cultures that are metabolically competent with normal karyotype and intact DNA repair system is gaining momentum to replace cell line analyses. For example, using such a hepatocyte 3D-culture system, 5-day exposure to valproic acid at a non-toxic but steatotic 15 mM concentration, followed by three-day withdrawal, led to persistent differentially methylated regions in 31 genes [649], with persistent disruption of energy metabolism [650]. The culture of pools from multiple donors of human primary cells might be a promising avenue for epigenetic testing, provided data reproducibility. ...
Epigenetics involves a series of mechanisms that entail histone and DNA covalent modifications and non-coding RNAs, and that collectively contribute to programing cell functions and differentiation. Epigenetic anomalies and DNA mutations are co-drivers of cellular dysfunctions, including carcinogenesis. Alterations of the epigenetic system occur in cancers whether the initial carcinogenic events are from genotoxic (GTxC) or non-genotoxic (NGTxC) carcinogens. NGTxC are not inherently DNA reactive, they do not have a unifying mode of action and as yet there are no regulatory test guidelines addressing mechanisms of NGTxC. To fil this gap, the Test Guideline Programme of the Organisation for Economic Cooperation and Development is developing a framework for an integrated approach for the testing and assessment (IATA) of NGTxC and is considering assays that address key events of cancer hallmarks. Here, with the intent of better understanding the applicability of epigenetic assays in chemical carcinogenicity assessment, we focus on DNA methylation and histone modifications and review: (1) epigenetic mechanisms contributing to carcinogenesis, (2) epigenetic mechanisms altered following exposure to arsenic, nickel, or phenobarbital in order to identify common carcinogen-specific mechanisms, (3) characteristics of a series of epigenetic assay types, and (4) epigenetic assay validation needs in the context of chemical hazard assessment. As a key component of numerous NGTxC mechanisms of action, epigenetic assays included in IATA assay combinations can contribute to improved chemical carcinogen identification for the better protection of public health.
... The preparation of the samples processed here are described in [41]. In brief, human hepatocytes were exposed to 15 mM valproic acid (VPA) or 1% EtOH (CTL) and sampled after one day, two days and three days of treatment. ...
Long-read transcriptome sequencing (LRTS) holds the promise to boost our understanding of alternative splicing. Recent advances in accuracy and throughput have diminished the major limitations and enabled the direct quantification of isoforms. Considering the complexity of the data and the broad range of potential applications, it is clear that highly flexible, accurate analysis tools are crucial. Here, we present IsoTools, a comprehensive Python-based analysis package, for the improvement of alternative and differential splicing analysis. IsoTools provides a comprehensive data structure that integrates genomic information from LRTS transcripts together with the reference annotation, and enables broad functionality to quality control, visualize and analyze the data. Additionally, we implemented a graph-based method for the identification of alternative splicing events and a statistical approach based on the beta binomial distribution for the detection of differential events. To demonstrate our methods, we generated PacBio Iso-Seq data of human hepatocytes treated with the HDAC inhibitor valproic acid, a compound known to induce widespread transcriptional changes. Contrasted with short read RNA-Seq of the same samples, this analysis shows that LRTS provides valuable additional insights for a better understanding of alternative splicing, in particular with respect to complex novel and differential splicing events. IsoTools is made available for the community along with extensive documentation at https://github.com/MatthiasLienhard/isotools.
... In another example, CysA downregulates OATP1B1, NTCP, Cytochrome P450 (CYP)3A4, and bile acid-CoA: amino acid N-acyltransferase (Wolters et al., 2016). Valproic acid suppresses the expression of several ATPsynthases, fatty acid transporters, and other mitochondrial genes leading to mitochondrial dysfunction, steatosis, and hepatotoxicity (Wolters et al., 2018). Transcriptomics can be applied to determine the hepatocellular responses that may protect from fatal hepatotoxicity and allow for recovery from DILI. ...
Drug-induced liver injury (DILI) is a significant clinical issue, affecting 1-1.5 million patients annually, and remains a major challenge during drug development-toxicity and safety concerns are the second highest reason for drug candidate failure. The future prevalence of DILI can be minimised by developing a greater understanding of the biological mechanisms behind DILI. Both qualitative and quantitative analytical techniques are vital to characterising and investigating DILI. A variety of assays are capable of characterising specific aspects of a drug's hepatotoxic nature, with multiplexed assays capable of characterising and scoring a drug's association with DILI. However, an even deeper insight into the perturbations to biological pathways involved in the mechanisms of DILI can be gained through the use of omics-based analytical techniques: genomics, transcriptomics, proteomics, and metabolomics. These omics analytical techniques can offer qualitative and quantitative insight into genetic susceptibilities to DILI, the impact of drug treatment on gene expression, and the effect on protein and metabolite abundance. This review will discuss the analytical techniques that can be applied to characterise and investigate the biological mechanisms of DILI and potential predictive biomarkers.
... One prominent example of the utility of this system is the identification of expression changes of genes involved in mitochondrial damage, RNA processing, transcription, and inflammation that clearly distinguish the idiosyncratic hepatotoxin trovafloxacin from structurally related nontoxic fluoroquinolones (Liguori et al. 2005;2008). Similarly, PHH monolayer cultures have provided insights into the toxicity mechanisms of cyclosporine A (Wolters et al. 2016), diclofenac (Sarkar et al. 2017), valproic acid (Wolters et al. 2018) and triazole antifungals (Goetz and Dix 2009). Furthermore, by leveraging transcriptomic data from the Open TG-GATES repository, a toxicogenomic database containing data from PHH exposed to 158 compounds, NRF2 activation was identified as a good indicator of the intrinsic biochemical reactivity of a compound which remains an important component for the comprehensive evaluation of compound systems toxicology (Copple et al. 2019). ...
Despite rigorous preclinical testing, clinical attrition rates in drug development remain high with drug-induced liver injury (DILI) remaining one of the most frequent causes of project failures. To understand DILI mechanisms, major efforts are put into the development of physiologically relevant cell models and culture paradigms with the aim to enhance preclinical to clinical result translation. While the majority of toxicogenomic studies have been based on cell lines, there are emerging trends toward the predominant use of stem cell-derived organoids and primary human hepatocytes in complex 3D cell models. Such studies have been successful in disentangling diverse toxicity mechanisms, including genotoxicity, mitochondrial injury, steatogenesis and cholestasis and can aid in distinguishing hepatotoxic from nontoxic structural analogs. Furthermore, by leveraging inter-individual differences of cells from different donors, these approaches can emulate the complexity of polygenic risk scores, which facilitates personalized drug-specific DILI risk analyses. In summary, toxicogenomic studies into drug-induced hepatotoxicity have majorly contributed to our mechanistic understanding of DILI and the incorporation of organotypic human 3D liver models into the preclinical testing arsenal promises to enhance biological insights during drug discovery, increase confidence in preclinical safety and minimize the translational gap.
... Yet, even though (cholelithiasis-induced) cholestasis is of high economic as well as clinical importance, its aetiology and pathogenesis are still not well-defined [683,688], which can partially be attributed to inadequate characterization of experimental cholestasis models. In this regard, transcriptomic analysis by means of RNA sequencing or microarray has become a standard research tool to compare and validate experimental model(s) in terms of relevance to human disease [422,577,590] and to elucidate the mechanistic basis of cholestasis at transcriptional level [8,422,608,650,689], but also to create a transcriptional signature specific and potentially predictive for a given disease [648,650,690]. With respect to the latter, we previously established a transcriptional blueprint based on the AOP of cholestasis [650]. ...
Cholestatic liver insults constitute a major manifestation of drug-induced liver injury. Current in vivo and in vitro approaches poorly detect drug-induced cholestatic liver injury, which is partly due to gaps in the mechanistic understanding of this type of hepatotoxicity. This doctoral project tackles this hurdle by providing a state-of-the-art scenario of the established as well as novel mechanisms that drive druginduced cholestatic liver injury. In a first study, a liver-based in vitro system was optimized to mechanistically study cholestasis, namely human hepatoma HepaRG cell cultures exposed to different cholestatic drugs and bile acids. This in vitro system has shed new light on the mechanisms underlying drug-induced cholestasis and has unveiled differences between different types of cholestasis as well as between the in vitro and in vivo situation. In a second study, the newly developed in vitro system in combination with other tools to predict cholestatic potential was evaluated for its application to chemicals outside the pharmaceutical area, in particular industrial chemicals, cosmetics ingredients, herbicides and food additives. It was found that further fit-for-purpose optimization is required for general use of the in vitro setting. A third study investigated liver samples of cholelithiasis-induced cholestasis patients and cholestatic mice by means of transcriptomic analysis in order to elucidate the mechanistic framework of different types of cholestasis and simultaneously characterize the human relevance of mouse models of cholestasis. Overall, this doctoral project has provided an important contribution to the elucidation of mechanisms underlying chemical-induced cholestasis, based on both in vitro as well as in vivo studies, and has yielded an in vitro setting fit for detecting drug-induced cholestasis.
... Exposure to VPA can, therefore, exert broad effects on gene expression by preventing global deacetylation of histones and promoting a permissive chromatin state. We have previously shown that prolonged exposure of primary human hepatocytes (PHHs) to VPA can induce gene expression changes as well as changes in 5-methylcytosine DNA methylation patterns Wolters et al. 2017Wolters et al. , 2018. Although these studies successfully identify cellular pathways related to mitochondrial functioning, TCA and β-oxidation as primary mechanisms affected by VPA exposure, their relative contribution to VPA-induced steatosis remains unclear. ...
... Primary human hepatocytes (PHHs) were cultured as described previously (Wolters et al. 2018). In brief, cryopreserved PHHs (Invitrogen) of three individuals (Hu8119, Hu1591 and Hu1540) were thawed and resuspended according to the supplier's instructions. ...
... In parallel PHHs were exposed to 1% milli-Q (vehicle control) in culture media for the same duration. We have previously found that 15 mM VPA was required to induce observable steatosis in PHHs Wolters et al. 2017Wolters et al. , 2018. Using the same PHH donor pool as used in the current study we previously determined 100%, 96% and 77% cell viability at days 1, 2, and 3, respectively, of daily exposure to 15 mM VPA and 97%, 82% and 44% cell viability at days 1, 2, and 3, respectively, of daily exposure to 30 mM VPA (Wolters et al. 2018). ...
Valproic acid (VPA) is a frequently prescribed anti-epileptic drug which is known to cause liver toxicity and steatosis through mitochondrial dysfunction. Nevertheless the mechanisms underlying these adverse effects are incompletely understood. In this study, we determined the effect of relatively short (3 h) or prolonged (72 h) exposure to VPA on mitochondrial function in primary human hepatocytes (PHHs). While 3 h VPA exposure did not affect oxygen consumption rates (OCRs) in PHHs, prolonged exposure (24–72 h) significantly reduced basal and maximal OCRs. Given that in particular prolonged VPA exposure is required to cause mitochondrial dysfunction, we investigated gene expression data after VPA exposure for 24, 48, 72 h and 72 h VPA followed by a 72 h washout period. We were able to reduce the comprehensive gene expression changes into a more comprehensible set of 18 TFs that were predicted to be persistently activated after 72 h of VPA exposure. Lentiviral knock-down of one of the candidate TFs, C/EBPα, partly rescued VPA-induced mitochondrial dysfunction. Furthermore, RNA-Seq analysis of shC/EBPα and shGFP control PHHs identified 24 genuine C/EBPα target genes that are regulated in response to prolonged VPA exposure in PHHs. Altogether this provides new insights on the involvement of C/EBPα in driving VPA-induced mitochondrial dysfunction in human liver cells. This hub gene, with its downstream regulators involved in this deregulation, thus represent potential new biomarkers for VPA-induced mitochondrial dysfunction.
