Effects of Different Combinations of Phytochemical-Rich Fruits and Vegetables on Chronic Disease Risk Markers and Gene Expression Changes: Insights from the MiBLEND Study, a Randomized Trial.

Adequate fruit and vegetable (F and V) intake, as recommended by the World Health Organization (over 400 g/day), is linked to reduced chronic disease risk. However, human intervention trials, especially with whole F and V and in complex combinations, are lacking. The MiBlend Study explored the effects of various phytochemical-rich F and V combinations on chronic disease risk markers, phytochemical absorption, and gene expression in blood. This randomized cross-over study involved participants consuming two of seven different F and V blends for 2 weeks (450 g/day), following a 2-week low F and V intake period (50 g/day). Each blend represented major phytochemical classes (flavonoids, anthocyanins, carotenoids, and glucosinolates) or combinations thereof. Markers of chronic disease risk, including DNA damage, oxidative stress, and retinal microvasculature, were measured. Increasing F and V intake significantly improved plasma antioxidant capacity, DNA damage protection, and retinal arteriolar dilation. Flavonoid-rich, carotenoid-rich, and complex blends notably reduced DNA damage susceptibility. Anthocyanin-rich and carotenoid-rich interventions were most effective in boosting antioxidant capacity, while blends high in flavonoids, especially combined with anthocyanins, significantly improved retinal microvasculature. Gene expression analysis revealed changes in DNA repair, signal transduction, and transcription processes, indicating mechanisms for these health benefits. The study suggests specific F and V blends can provide targeted health improvements, emphasizing the importance of both overall F and V intake and the specific phytochemical composition for personalized preventive strategies.

Beyond CSF and Neuroimaging Assessment: Evaluating Plasma miR-145-5p as a Potential Biomarker for Mild Cognitive Impairment and Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. New strategies for the early detection of MCI and sporadic AD are crucial for developing effective treatment options. Current techniques used for diagnosis of AD are invasive and/or expensive, so they are not suitable for population screening. Cerebrospinal fluid (CSF) biomarkers such as amyloid ß1-42 (Aß1-42), total tau (T-tau), and phosphorylated tau181 (P-tau181) levels are core biomarkers for early diagnosis of AD. Several studies have proposed the use of blood-circulating microRNAs (miRNAs) as potential novel early biomarkers for AD. We therefore applied a novel approach to identify blood-circulating miRNAs associated with CSF biomarkers and explored the potential of these miRNAs as biomarkers of AD. In total, 112 subjects consisting of 28 dementia due to AD cases, 63 MCI due to AD cases, and 21 cognitively healthy controls were included. We identified seven Aß1-42-associated plasma miRNAs, six P-tau181-associated plasma miRNAs, and nine Aß1-42-associated serum miRNAs. These miRNAs were involved in AD-relevant biological processes, such as PI3K/AKT signaling. Based on this signaling pathway, we constructed an miRNA-gene target network, wherein miR-145-5p has been identified as a hub. Furthermore, we showed that miR-145-5p performs best in the prediction of both AD and MCI. Moreover, miR-145-5p also improved the prediction performance of the mini-mental state examination (MMSE) score. The performance of this miRNA was validated using different datasets including an RT-qPCR dataset from plasma samples of 23 MCI cases and 30 age-matched controls. These findings indicate that blood-circulating miRNAs that are associated with CSF biomarkers levels and specifically plasma miR-145-5p alone or combined with the MMSE score can potentially be used as noninvasive biomarkers for AD or MCI screening in the general population, although studies in other AD cohorts are necessary for further validation.

Wnt7a Decreases Brain Endothelial Barrier Function Via ß-Catenin Activation.

The blood-brain barrier consists of tightly connected endothelial cells protecting the brain's microenvironment from the periphery. These endothelial cells are characterized by specific tight junction proteins such as Claudin-5 and Occludin, forming the endothelial barrier. Disrupting these cells might lead to blood-brain barrier dysfunction. The Wnt/ß-catenin signaling pathway can regulate the expression of these tight junction proteins and subsequent barrier permeability. The aim of this study was to investigate the in vitro effects of Wnt7a mediated ß-catenin signaling on endothelial barrier integrity. Mouse brain endothelial cells, bEnd.3, were treated with recombinant Wnt7a protein or XAV939, a selective inhibitor of Wnt/ß-catenin mediated transcription to modulate the Wnt signaling pathway. The involvement of Wnt/HIF1α signaling was investigated by inhibiting Hif1α signaling with Hif1α siRNA. Wnt7a stimulation led to activation and nuclear translocation of ß-catenin, which was inhibited by XAV939. Wnt7a stimulation decreased Claudin-5 expression mediated by ß-catenin and decreased endothelial barrier formation. Wnt7a increased Hif1α and Vegfa expression mediated by ß-catenin. However, Hif1α signaling pathway did not regulate tight junction proteins Claudin-5 and Occludin. Our data suggest that Wnt7a stimulation leads to a decrease in tight junction proteins mediated by the nuclear translocation of ß-catenin, which hampers proper endothelial barrier formation. This process might be crucial in initiating endothelial cell proliferation and angiogenesis. Although HIF1α did not modulate the expression of tight junction proteins, it might play a role in brain angiogenesis and underlie pathogenic mechanisms in Wnt/HIF1α signaling in diseases such as cerebral small vessel disease.

Hypoxic oligodendrocyte precursor cell-derived VEGFA is associated with blood-brain barrier impairment.

Cerebral small vessel disease is characterised by decreased cerebral blood flow and blood-brain barrier impairments which play a key role in the development of white matter lesions. We hypothesised that cerebral hypoperfusion causes local hypoxia, affecting oligodendrocyte precursor cell-endothelial cell signalling leading to blood-brain barrier dysfunction as an early mechanism for the development of white matter lesions. Bilateral carotid artery stenosis was used as a mouse model for cerebral hypoperfusion. Pimonidazole, a hypoxic cell marker, was injected prior to humane sacrifice at day 7. Myelin content, vascular density, blood-brain barrier leakages, and hypoxic cell density were quantified. Primary mouse oligodendrocyte precursor cells were exposed to hypoxia and RNA sequencing was performed. Vegfa gene expression and protein secretion was examined in an oligodendrocyte precursor cell line exposed to hypoxia. Additionally, human blood plasma VEGFA levels were measured and correlated to blood-brain barrier permeability in normal-appearing white matter and white matter lesions of cerebral small vessel disease patients and controls. Cerebral blood flow was reduced in the stenosis mice, with an increase in hypoxic cell number and blood-brain barrier leakages in the cortical areas but no changes in myelin content or vascular density. Vegfa upregulation was identified in hypoxic oligodendrocyte precursor cells, which was mediated via Hif1α and Epas1. In humans, VEGFA plasma levels were increased in patients versus controls. VEGFA plasma levels were associated with increased blood-brain barrier permeability in normal appearing white matter of patients. Cerebral hypoperfusion mediates hypoxia induced VEGFA expression in oligodendrocyte precursor cells through Hif1α/Epas1 signalling. VEGFA could in turn increase BBB permeability. In humans, increased VEGFA plasma levels in cerebral small vessel disease patients were associated with increased blood-brain barrier permeability in the normal appearing white matter. Our results support a role of VEGFA expression in cerebral hypoperfusion as seen in cerebral small vessel disease.

CODA: a combo-Seq data analysis workflow.

The analysis of the combined mRNA and miRNA content of a biological sample can be of interest for answering several research questions, like biomarkers discovery, or mRNA-miRNA interactions. However, the process is costly and time-consuming, separate libraries need to be prepared and sequenced on different flowcells. Combo-Seq is a library prep kit that allows us to prepare combined mRNA-miRNA libraries starting from very low total RNA. To date, no dedicated bioinformatics method exists for the processing of Combo-Seq data. In this paper, we describe CODA (Combo-seq Data Analysis), a workflow specifically developed for the processing of Combo-Seq data that employs existing free-to-use tools. We compare CODA with exceRpt, the pipeline suggested by the kit manufacturer for this purpose. We also evaluate how Combo-Seq libraries analysed with CODA perform compared with conventional poly(A) and small RNA libraries prepared from the same samples. We show that using CODA more successfully trimmed reads are recovered compared with exceRpt, and the difference is more dramatic with short sequencing reads. We demonstrate how Combo-Seq identifies as many genes and fewer miRNAs compared to the standard libraries, and how miRNA validation favours conventional small RNA libraries over Combo-Seq. The CODA code is available at https://github.com/marta-nazzari/CODA.

Transcriptomic Analysis in Human 3D Skin Model Injected with Resorbable Hyaluronic Acid Fillers Reveals Foreign Body Response.

Usage of injectable dermal fillers applied for aesthetic purposes has extensively increased over the years. As such, the number of related adverse reactions has increased, including patients showing severe complications such as product migration, topical swelling and inflammatory reactions of the skin. In order to understand the underlying molecular events of these adverse reactions we performed a genome-wide gene expression study on the multi-cell type human Phenion® Full-Thickness Skin Model exposed to five experimental hyaluronic acid (HA) preparations with increasing cross-linking degree, four commercial fillers from Perfectha®, and non-resorbable filler Bio-Alcamid®. In addition, we evaluated whether cross-linking degree or particle size of the HA-based fillers could be associated with the occurrence of adverse effects. In all cases, exposure to different HA fillers resulted in a clearly elevated gene expression of cytokines and chemokines related to acute inflammation as part of the foreign body response. Furthermore, for one experimental filler genes of OXPHOS complexes I-V were significantly down-regulated (adjusted p-value < 0.05), resulting in mitochondrial dysfunction which can be linked to over-expression of pro-inflammatory cytokines TNFα and IL-1ß and chemokine CCL2. Our hypothesis that cross-linking degree or particle size of the HA-based fillers is related to the biological responses induced by these fillers could only partially be confirmed for particle size. In conclusion, our innovative approach resulted in gene expression changes from a human 3D skin model exposed to dermal fillers that mechanistically substantiate aforementioned adverse reactions, and thereby adds to the weight of evidence that these fillers may induce inflammatory and fibrotic responses.

Lead-exposure associated miRNAs in humans and Alzheimer's disease as potential biomarkers of the disease and disease processes.

Alzheimer's disease (AD) is a neurodegenerative disease that eventually affects memory and behavior. The identification of biomarkers based on risk factors for AD provides insight into the disease since the exact cause of AD remains unknown. Several studies have proposed microRNAs (miRNAs) in blood as potential biomarkers for AD. Exposure to heavy metals is a potential risk factor for onset and development of AD. Blood cells of subjects that are exposed to lead detected in the circulatory system, potentially reflect molecular responses to this exposure that are similar to the response of neurons. In this study we analyzed blood cell-derived miRNAs derived from a general population as proxies of potentially AD-related mechanisms triggered by lead exposure. Subsequently, we analyzed these mechanisms in the brain tissue of AD subjects and controls. A total of four miRNAs were identified as lead exposure-associated with hsa-miR-3651, hsa-miR-150-5p and hsa-miR-664b-3p being negatively and hsa-miR-627 positively associated. In human brain derived from AD and AD control subjects all four miRNAs were detected. Moreover, two miRNAs (miR-3651, miR-664b-3p) showed significant differential expression in AD brains versus controls, in accordance with the change direction of lead exposure. The miRNAs' gene targets were validated for expression in the human brain and were found enriched in AD-relevant pathways such as axon guidance. Moreover, we identified several AD relevant transcription factors such as CREB1 associated with the identified miRNAs. These findings suggest that the identified miRNAs are involved in the development of AD and might be useful in the development of new, less invasive biomarkers for monitoring of novel therapies or of processes involved in AD development.

Evaluation of Adverse Effects of Resorbable Hyaluronic Acid Fillers: Determination of Macrophage Responses.

Resorbable tissue fillers for aesthetic purposes can induce severe complications including product migration, late swelling, and inflammatory reactions. The relation between product characteristics and adverse effects is not well understood. We hypothesized that the degree of cross-linking hyaluronic acid (HA) fillers was associated with the occurrence of adverse effects. Five experimental HA preparations similar to HA fillers were synthesized with an increasing degree of cross-linking. Furthermore, a series of commercial fillers (Perfectha®) was obtained that differ in degradation time based on the size of their particulate HA components. Cytotoxic responses and cytokine production by human THP-1-derived macrophages exposed to extracts of the evaluated resorbable HA fillers were absent to minimal. Gene expression analysis of the HA-exposed macrophages revealed the responses related to cell cycle control and immune reactivity. Our results could not confirm the hypothesis that the level of cross-linking in our experimental HA fillers or the particulate size of commercial HA fillers is related to the induced biological responses. However, the evaluation of cytokine induction and gene expression in macrophages after biomaterial exposure presents promising opportunities for the development of methods to identify cellular processes that may be predictive for biomaterial-induced responses in patients.

Integrating SNPs-based genetic risk factor with blood epigenomic response of differentially arsenic-exposed rural subjects reveals disease-associated signaling pathways.

Arsenic (As) contamination in groundwater is responsible for numerous adverse health outcomes among millions of people. Epigenetic alterations are among the most widely studied mechanisms of As toxicity. To understand how As exposure alters gene expression through epigenetic modifications, a systematic genome-wide study was designed to address the impact of multiple important single nucleotide polymorphisms (SNPs) related to As exposure on the methylome of drinking water As-exposed rural subjects from Pakistan. Urinary As levels were used to stratify subjects into low, medium and high exposure groups. Genome-wide DNA methylation was investigated using MeDIP in combination with NimbleGen 2.1 M Deluxe Promotor arrays. Transcriptome levels were measured using Agilent 8 × 60 K expression arrays. Genotyping of selected SNPs (As3MT, DNMT1a, ERCC2, EGFR and MTHFR) was measured and an integrated genetic risk factor for each respondent was calculated by assigning a specific value to the measured genotypes based on known risk allele numbers. To select a representative model related to As exposure we compared 9 linear mixed models comprising of model 1 (including the genetic risk factor), model 2 (without the genetic risk factor) and models with individual SNPs incorporated into the methylome data. Pathway analysis was performed using ConsensusPathDB. Model 1 comprising the integrated genetic risk factor disclosed biochemical pathways including muscle contraction, cardio-vascular diseases, ATR signaling, GPCR signaling, methionine metabolism and chromatin modification in association with hypo- and hyper-methylated gene targets. A unique pathway (direct P53 effector) was found associated with the individual DNMT1a polymorphism due to hyper-methylation of CSE1L and TRRAP. Most importantly, we provide here the first evidence of As-associated DNA methylation in relation with gene expression of ATR, ATF7IP, TPM3, UBE2J2. We report the first evidence that integrating SNPs data with methylome data generates a more representative epigenome profile and discloses a better insight in disease risks of As-exposed individuals.

Manganese-Induced Neurotoxicity through Impairment of Cross-Talk Pathways in Human Neuroblastoma Cell Line SH-SY5Y Differentiated with Retinoic Acid.

Manganese (Mn) is an important element; yet acute and/or chronic exposure to this metal has been linked to neurotoxicity and neurodegenerative illnesses such as Parkinson's disease and others via an unknown mechanism. To better understand it, we exposed a human neuroblastoma cell model (SH-SY5Y) to two Mn chemical species, MnCl2 and Citrate of Mn(II) (0-2000 µM), followed by a cell viability assay, transcriptomics, and bioinformatics. Even though these cells have been chemically and genetically modified, which may limit the significance of our findings, we discovered that by using RA-differentiated cells instead of undifferentiated SH-SY5Y cell line, both chemical species induce a similar toxicity, potentially governed by disruption of protein metabolism, with some differences. The MnCl2 altered amino acid metabolism, which affects RNA metabolism and protein synthesis. Citrate of Mn(II), however, inhibited the E3 ubiquitin ligases-target protein degradation pathway, which can lead to the buildup of damaged/unfolded proteins, consistent with histone modification. Finally, we discovered that Mn(II)-induced cytotoxicity in RA-SH-SY5Y cells shared 84 percent of the pathways involved in neurodegenerative diseases.

Comparing in vitro human liver models to in vivo human liver using RNA-Seq.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Transcriptome responses in blood reveal distinct biological pathways associated with arsenic exposure through drinking water in rural settings of Punjab, Pakistan.

BACKGROUND: Groundwater Arsenic (As) contamination is a global public health concern responsible for various health implications and a neglected area of environmental health research in Pakistan. Because of interindividual differences in genetic predisposition, As-related health issues may not be equally distributed among the As-exposed population. However, till date, no studies have been conducted including multiple SNPs involved in As metabolism and disease risk using a linear mixed effect model approach to analyze peripheral blood transcriptomics results. OBJECTIVES: In order to detect early responses on the gene expression level and to evaluate the impact of selected SNPs inferring disease risks associated with As exposure, we designed a systematic study to investigate blood transcriptomics profiles of 57 differentially exposed rural subjects living in drinking water As-contaminated settings of Lahore and Kasur districts in Punjab Province in southeast Pakistan. Exposure among the subjects was correlated with individual transcriptome responses applying urinary As profiles as the main biomarker for risk stratification. METHODS: We performed whole genome gene expression analysis in blood of subjects using microarrays. Linear effect mixed models were applied for evaluating the combined impact of SNPs hypothetically increasing the risk for As exposure-induced health effects (GSTM1, GSTT1, As3MT, DNMT1, MTHFR, ERCC2 and EGFR). RESULTS: Our findings confirmed important signaling, growth factor, cancer and other disease related pathways known to be associated with increased As exposure levels. In addition, upon implementing our integrative SNPs-based genetic risk factor, pathways associated with an increased risk of NAFLD and diabetes appeared significantly enhanced by down-regulation of genes NDUFV3, IKBKB, IL6R, ADIPOR1, PPARA, OGT and FOXO1. CONCLUSION: We report the first comprehensive study applying state-of-the-art bioinformatics approaches to address multiple SNP-based inter-individual variability in adverse molecular responses among subjects exposed to drinking water As contamination in Pakistan thereby providing strong evidence of various gene expression targets associated with development of known As-related diseases.

Short-term transcriptome and microRNAs responses to exposure to different air pollutants in two population studies.

Diesel vehicle emissions are the major source of genotoxic compounds in ambient air from urban areas. These pollutants are linked to risks of cardiovascular diseases, lung cancer, respiratory infections and adverse neurological effects. Biological events associated with exposure to some air pollutants are widely unknown but applying omics techniques may help to identify the molecular processes that link exposure to disease risk. Most data on health risks are related to long-term exposure, so the aim of this study is to investigate the impact of short-term exposure (two hours) to air pollutants on the blood transcriptome and microRNA expression levels. We analyzed transcriptomics and microRNA expression using microarray technology on blood samples from volunteers participating in studies in London, the Oxford Street cohort, and, in Barcelona, the TAPAS cohort. Personal exposure levels measurements of particulate matter (PM10, PM2.5), ultrafine particles (UFPC), nitrogen oxides (NO2, NO and NOx), black carbon (BC) and carbon oxides (CO and CO2) were registered for each volunteer. Associations between air pollutant levels and gene/microRNA expression were evaluated using multivariate normal models (MVN). MVN-models identified compound-specific expression of blood cell genes and microRNAs associated with air pollution despite the low exposure levels, the short exposure periods and the relatively small-sized cohorts. Hsa-miR-197-3p, hsa-miR-29a-3p, hsa-miR-15a-5p, hsa-miR-16-5p and hsa-miR-92a-3p are found significantly expressed in association with exposures. These microRNAs target also relevant transcripts, indicating their potential relevance in the research of omics-biomarkers responding to air pollution. Furthermore, these microRNAs are also known to be associated with diseases previously linked to air pollution exposure including several cancers such lung cancer and Alzheimer's disease. In conclusion, we identified in this study promising compound-specific mRNA and microRNA biomarkers after two hours of exposure to low levels of air pollutants during two hours that suggest increased cancer risks.

Integrative omics data analyses of repeated dose toxicity of valproic acid in vitro reveal new mechanisms of steatosis induction.

Valproic acid (VPA) is a very potent anti-cancer and neuro-protective drug probably by its HDAC inhibiting properties, which may cause steatosis in the liver. The present study investigates the effect of repetitive VPA treatment of primary human hepatocytes (PHH) on whole genome gene expression-, DNA methylation-, and miRNA changes, using microarrays and integrated data analyses. PHH were exposed to a non-cytotoxic dose of VPA for 5days daily which induced lipid accumulation. Part of the PHH was left untreated for 3days for studying the persistence of 'omics' changes. VPA treatment appeared to inhibit the expression of the transcription factors HNF1A and ONECUT1. HNF1A interacted with 41 differentially expressed genes of which 12 were also differentially methylated. None of the genes present in this network were regulated by a DE-miR. The subnetwork of ONECUT1 consisted of 44 differentially expressed genes of which 15 were differentially methylated, and 3 were regulated by a DE-miR. A number of genes in the networks are involved in fatty acid metabolism, and may contribute to the development of steatosis by increasing oxidative stress thereby causing mitochondrial dysfunction, and by shifting metabolism of VPA towards ß-oxidation due to reduced glucuronidation. Part of the changes remained persistent after washing out of VPA, like PMAIP1 which is associated with cellular stress in liver of patients with NASH. The MMP2 gene showed the highest number of interactions with other persistently expressed genes, among which LCN2 which is a key modulator of lipid homeostasis. Furthermore, VPA modulated the expression and DNA methylation level of nuclear receptors and their target genes involved in the adverse outcome pathway of steatosis, thereby expanding our current knowledge of the pathway. In particular, VPA modulated PPARγ, and PPARα, AHR and CD36 on both the gene expression and the DNA methylation level, thereby inhibiting ß-oxidation and increasing uptake of fatty acid into the hepatocytes, respectively. Overall, our integrative data analyses identified novel genes modulated by VPA, which provide more insight into the mechanisms of repeated dose toxicity of VPA, leading to steatosis.

Validation of gene expression profiles from cholestatic hepatotoxicants in vitro against human in vivo cholestasis.

Drug-induced liver injury remains the most common cause of acute liver failure and a frequently indicated reason for withdrawal of drugs. For the purpose of evaluating the relevance of liver cell models for assessing hepatotoxic risks in intact humans, we here aimed to benchmark 'omics-derived mechanistic data from three in vitro models for parenchymal liver function, intended for the investigation of drug-induced cholestasis, against 'omics data from cholestatic patients. Transcriptomic changes in HepG2 cells, primary mouse hepatocytes and primary human hepatocytes exposed to known cholestatic compounds were analyzed using microarrays. Some of the differentially expressed genes in HepG2 cells were also differentially expressed into the same direction in human cholestasis. The overlap between drug-induced transcriptomic responses in primary mouse hepatocytes and primary human hepatocytes appeared limited and no genes overlapping with in vivo cholestasis were found. Thereupon, a pathway for drug-induced cholestasis was used to map the drug-induced transcriptomic modifications involved in bile salt homeostasis. Indications of an adaptive response to prevent and reduce intracellular bile salt accumulation were observed in vivo as well as in the in vitro liver models. Furthermore, drug-specific changes were found, which may be indicative for their cholestatic properties. Furthermore, connectivity mapping was applied in order to investigate the predictive value of the in vitro models for in vivo cholestasis. This analysis resulted in a positive connection score for most compounds, which may indicate that for identifying cholestatic compounds the focus should be on gene expression signatures rather than on differentially expressed genes.

Integrative "-Omics" Analysis in Primary Human Hepatocytes Unravels Persistent Mechanisms of Cyclosporine A-Induced Cholestasis.

Cyclosporine A (CsA) is an undecapeptide with strong immunosuppressant activities and is used a lot after organ transplantation. Furthermore, it may induce cholestasis in the liver. In general, the drug-induced cholestasis (DIC) pathway includes genes involved in the uptake, synthesis, conjugation, and secretion of bile acids. However, whether CsA-induced changes in the cholestasis pathway in vitro are persistent for repeated dose toxicity has not yet been investigated. To explore this, primary human hepatocytes (PHH) were exposed to a subcytotoxic dose of 30 µM CsA daily for 3 and 5 days. To investigate the persistence of induced changes upon terminating CsA exposure after 5 days, a subset of PHH was subjected to a washout period (WO-period) of 3 days. Multiple -omics analyses, comprising whole genome analysis of DNA methylation, gene expression, and microRNA expression, were performed. The CsA-treatment resulted after 3 and 5 days, respectively, in 476 and 20 differentially methylated genes (DMGs), 1353 and 1481 differentially expressed genes (DEGs), and in 22 and 29 differentially expressed microRNAs (DE-miRs). Cholestasis-related pathways appeared induced during CsA-treatment. Interestingly, 828 persistent DEGs and 6 persistent DE-miRs but no persistent DMGs were found after the WO-period. These persistent DEGs and DE-miRs showed concordance for 22 genes. Furthermore, 29 persistent DEGs changed into the same direction as observed in livers from cholestasis patients. None of those 29 DEGs which among others relate to oxidative stress and lipid metabolism are yet present in the DIC pathway or cholestasis adverse outcome pathway (AOP) thus presenting novel findings. In summary, we have demonstrated for the first time a persistent impact of repeated dose administration of CsA on genes and microRNAs related to DIC in the gold standard human liver in vitro model with PHH.

Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma.

Chronic exposure to aflatoxin B1 (AFB1) has, in certain regions in the world, been strongly associated with hepatocellular carcinoma (HCC) development. AFB1 is a very potent hepatotoxic and carcinogenic mycotoxin which is frequently reported as a food contaminant. Epigenetic modifications provoked by environmental exposures, such as AFB1, may create a persistent epigenetic footprint. Deregulation of epigenetic mechanisms has actually been reported in HCC patients following AFB1 exposure; however, no attempts have yet been made to investigate early effects on the epigenome level which may be persistent on longer term, thereby possibly initiating carcinogenic events. In this study, we aim to identify methyl DNA-mRNA-interactions representative for a persistent epigenetic footprint associated with the early onset of AFB1-induced HCC. For this, primary human hepatocytes were exposed to 0.3µM of AFB1 for 5 days. Persistent epigenetic effects were measured 3 days after terminating the carcinogenic exposure. Whole genome DNA methylation changes and whole genome transcriptomic analysis were analyzed applying microarray technologies, and cross-omics interactions were evaluated. Upon combining transcriptomics data with results on DNA methylation, a range of persistent hyper- and hypo-methylated genes was identified which also appeared affected on the transcriptome level. For six of the hypo-methylated and up-regulated genes, namely TXNRD1, PCNA, CCNK, DIAPH3, RAB27A and HIST1H2BF, a clear role in carcinogenic events could be identified. This study is the first to report on a carcinogen-induced persistent impact on the epigenetic footprint in relation with the transcriptome which could be indicative for the early onset of AFB1-related development of HCC.

New insights into BaP-induced toxicity: role of major metabolites in transcriptomics and contribution to hepatocarcinogenesis.

Benzo(a)pyrene (BaP) is a ubiquitous carcinogen resulting from incomplete combustion of organic compounds and also present at high levels in cigarette smoke. A wide range of biological effects has been attributed to BaP and its genotoxic metabolite BPDE, but the contribution to BaP toxicity of intermediary metabolites generated along the detoxification path remains unknown. Here, we report for the first time how 3-OH-BaP, 9,10-diol and BPDE, three major BaP metabolites, temporally relate to BaP-induced transcriptomic alterations in HepG2 cells. Since BaP is also known to induce AhR activation, we additionally evaluated TCDD to source the expression of non-genotoxic AhR-mediated patterns. 9,10-Diol was shown to activate several transcription factor networks related to BaP metabolism (AhR), oxidative stress (Nrf2) and cell proliferation (HIF-1α, AP-1) in particular at early time points, while BPDE influenced expression of genes involved in cell energetics, DNA repair and apoptotic pathways. Also, in order to grasp the role of BaP and its metabolites in chemical hepatocarcinogenesis, we compared expression patterns from BaP(-metabolites) and TCDD to a signature set of approximately nine thousand gene expressions derived from hepatocellular carcinoma (HCC) patients. While transcriptome modulation by TCDD appeared not significantly related to HCC, BaP and BPDE were shown to deregulate metastatic markers via non-genotoxic and genotoxic mechanisms and activate inflammatory pathways (NF-κß signaling, cytokine-cytokine receptor interaction). BaP also showed strong repression of genes involved in cholesterol and fatty acid biosynthesis. Altogether, this study provides new insights into BaP-induced toxicity and sheds new light onto its mechanism of action as a hepatocarcinogen.

Interindividual variation in gene expression responses and metabolite formation in acetaminophen-exposed primary human hepatocytes.

Acetaminophen (APAP) is a readily available over-the-counter drug and is one of the most commonly used analgesics/antipyretics worldwide. Large interindividual variation in susceptibility toward APAP-induced liver failure has been reported. However, the exact underlying factors causing this variability in susceptibility are still largely unknown. The aim of this study was to better understand this variability in response to APAP by evaluating interindividual differences in gene expression changes and APAP metabolite formation in primary human hepatocytes (PHH) from several donors (n = 5) exposed in vitro to a non-toxic to toxic APAP dose range. To evaluate interindividual variation, gene expression data/levels of metabolites were plotted against APAP dose/donor. The correlation in APAP dose response between donors was calculated by comparing data points from one donor to the data points of all other donors using a Pearson-based correlation analysis. From that, a correlation score/donor for each gene/metabolite was defined, representing the similarity of the omics response to APAP in PHH of a particular donor to all other donors. The top 1 % highest variable genes were selected for further evaluation using gene set overrepresentation analysis. The biological processes in which the genes with high interindividual variation in expression were involved include liver regeneration, inflammatory responses, mitochondrial stress responses, hepatocarcinogenesis, cell cycle, and drug efficacy. Additionally, the interindividual variation in the expression of these genes could be associated with the variability in expression levels of hydroxyl/methoxy-APAP and C8H13O5N-APAP-glucuronide. The before-mentioned metabolites or their derivatives have also been reported in blood of humans exposed to therapeutic APAP doses. Possibly these findings can contribute to elucidating the causative factors of interindividual susceptibility toward APAP.

Extensive temporal transcriptome and microRNA analyses identify molecular mechanisms underlying mitochondrial dysfunction induced by multi-walled carbon nanotubes in human lung cells.

Understanding toxicity pathways of engineered nanomaterials (ENM) has recently been brought forward as a key step in twenty-first century ENM risk assessment. Molecular mechanisms linked to phenotypic end points is a step towards the development of toxicity tests based on key events, which may allow for grouping of ENM according to their modes of action. This study identified molecular mechanisms underlying mitochondrial dysfunction in human bronchial epithelial BEAS 2B cells following exposure to one of the most studied multi-walled carbon nanotubes (Mitsui MWCNT-7). Asbestos was used as a positive control and a non-carcinogenic glass wool material was included as a negative fibre control. Decreased mitochondrial membrane potential (MMP↓) was observed for MWCNTs at a biologically relevant dose (0.25 µg/cm(2)) and for asbestos at 2 µg/cm(2), but not for glass wool. Extensive temporal transcriptomic and microRNA expression analyses identified a 330-gene signature (including 26 genes with known mitochondrial function) related to MWCNT- and asbestos-induced MMP↓. Forty-nine of the MMP↓-associated genes showed highly similar expression patterns over time (six time points) and the majority was found to be regulated by two transcription factors strongly involved in mitochondrial homeostasis, APP and NRF1. In addition, four miRNAs were correlated with MMP↓ and one of them, miR-1275, was found to negatively correlate with a large part of the MMP↓-associated genes. Cellular processes such as gluconeogenesis, mitochondrial LC-fatty acid ß-oxidation and spindle microtubule function were enriched among the MMP↓-associated genes and miRNAs. These results are expected to be useful in the identification of key events in ENM-related toxicity pathways for the development of molecular screening techniques.