Publications by authors named "Jos C S Kleinjans"

129 Publications

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

Environ Pollut 2021 Oct 4;292(Pt A):118279. Epub 2021 Oct 4.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University, the Netherlands.

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.
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http://dx.doi.org/10.1016/j.envpol.2021.118279DOI Listing
October 2021

Small RNA Sequencing of Aqueous Humor and Plasma in Patients With Primary Open-Angle Glaucoma.

Invest Ophthalmol Vis Sci 2021 06;62(7):24

University Eye Clinic Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands.

Purpose: Identify differentially expressed microRNAs (miRNAs) in aqueous humor (AH) and blood of primary open-angle glaucoma (POAG) patients by using small RNA sequencing. These may provide insight into POAG pathophysiology or serve as diagnostic biomarker.

Methods: AH and plasma of nine POAG patients and 10 cataract control patients were small RNA sequenced on Illumina NovaSeq 6000. Identification of gene transcripts targeted by differentially expressed miRNAs was done with miRWalk and MirPath. These targets were used for pathway analysis and Gene Ontology enrichment. Diagnostic potential was evaluated by receiver operating characteristics analysis.

Results: We identified 715 miRNAs in plasma and 62 miRNAs in AH. Plasma miRNA profile did not differ between POAG and control. In contrast, in AH, seven miRNAs were differentially expressed. Hsa-miR-30a-3p, hsa-miR-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were upregulated, whereas hsa-miR-92a-3p, hsa-miR-451a, and hsa-miR-486-5p were downregulated in POAG. Compared to previous studies, hsa-mir-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were reported previously, strengthening their involvement in POAG whereas hsa-miR-30a-3p, hsa-miR-92a-3p, and hsa-miR-486-5p are implicated in POAG for the first time. Identified gene transcripts were involved in several pathways, some implicated in glaucoma before (e.g., TGF-β and neurotrophin signaling), whereas others are new (e.g., prolactin and apelin signaling). In respect to diagnostics, AH concentration of hsa-mir-143-3p had an area under the curve (AUC) of 0.889. Combined with hsa-miR-221-3p, AUC improved to 0.96.

Conclusions: Small RNA sequencing identified seven differentially expressed miRNAs in AH of POAG patients. The differentially expressed miRNAs may be useful as POAG biomarkers or could become targets for new therapeutic strategies.
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http://dx.doi.org/10.1167/iovs.62.7.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237107PMC
June 2021

New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids.

Arch Toxicol 2021 08 20;95(8):2691-2718. Epub 2021 Jun 20.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands.

5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.
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http://dx.doi.org/10.1007/s00204-021-03092-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298376PMC
August 2021

Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability.

Sci Rep 2021 06 2;11(1):11575. Epub 2021 Jun 2.

Division of Molecular and Computational Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081, HZ, Amsterdam, The Netherlands.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.
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http://dx.doi.org/10.1038/s41598-021-89550-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172841PMC
June 2021

microRNA expression profiles and personal monitoring of exposure to particulate matter.

Environ Pollut 2020 Aug 18;263(Pt B):114392. Epub 2020 Mar 18.

Italian Institute for Genomic Medicine (IIGM), c/o IRCCS Candiolo, 10060 Candiolo, Turin, Italy. Electronic address:

An increasing number of findings from epidemiological studies support associations between exposure to air pollution and the onset of several diseases, including pulmonary, cardiovascular and neurodegenerative diseases, and malignancies. However, intermediate, and potentially mediating, biological mechanisms associated with exposure to air pollutants are largely unknown. Previous studies on the human exposome have shown that the expression of certain circulating microRNAs (miRNAs), regulators of gene expression, are altered upon exposure to traffic-related air pollutants. In the present study, we investigated the relationship between particulate matter (PM) smaller than 2.5 μm (PM), PM absorbance (as a proxy of black carbon and soot), and ultrafine-particles (UFP, smaller than 0.1 μm), measured in healthy volunteers by 24 h personal monitoring (PEM) sessions and global expression levels of peripheral blood miRNAs. The PEM sessions were conducted in four European countries, namely Switzerland (Basel), United Kingdom (Norwich), Italy (Turin), and The Netherlands (Utrecht). miRNAs expression levels were analysed using microarray technology on blood samples from 143 participants. Seven miRNAs, hsa-miR-24-3p, hsa-miR-4454, hsa-miR-4763-3p, hsa-miR-425-5p, hsa-let-7d-5p, hsa-miR-502-5p, and hsa-miR-505-3p were significantly (FDR corrected) expressed in association with PM personal exposure, while no significant association was found between miRNA expression and the other pollutants. The results obtained from this investigation suggest that personal exposure to PM is associated with miRNA expression levels, showing the potential for these circulating miRNAs as novel biomarkers for air pollution health risk assessment.
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http://dx.doi.org/10.1016/j.envpol.2020.114392DOI Listing
August 2020

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

Environ Int 2020 02 18;135:105403. Epub 2019 Dec 18.

Grow School of Oncology and Developmental Biology, Department of Toxicogenomics, Maastricht University, the Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.envint.2019.105403DOI Listing
February 2020

Acetaminophen Overdose as a Potential Risk Factor for Parkinson's Disease.

Clin Transl Sci 2019 11 15;12(6):609-616. Epub 2019 Jul 15.

Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands.

Four complementary approaches were used to investigate acetaminophen overdose as a risk factor for Parkinson's disease (PD). Circulating microRNAs (miRNAs) serum profiles from acetaminophen-overdosed patients were compared with patients with terminal PD, revealing four shared miRNAs. Similarities were found among molecular structures of dopamine (DA), acetaminophen, and two known PD inducers indicating affinity for dopaminergic transport. Potential interactions between acetaminophen and the human DA transporter were confirmed by molecular docking modeling and binding free energy calculations. Thus, it is plausible that acetaminophen is taken up by the dopaminergic transport system into the substantia nigra (SN). A ChEMBL query identified proteins that are similarly targeted by DA and acetaminophen. Here, we highlight CYP3A4, present in the SN, a predominant metabolizer of acetaminophen into its toxic metabolite N-acetyl-p-benzoquinone imine and shown to be regulated in PD. Overall, based on our results, we hypothesize that overdosing of acetaminophen is a potential risk factor for parkinsonism.
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http://dx.doi.org/10.1111/cts.12663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853143PMC
November 2019

Drug-induced gene expression profile changes in relation to intestinal toxicity: State-of-the-art and new approaches.

Cancer Treat Rev 2019 Jul 27;77:57-66. Epub 2019 Jun 27.

Department of Toxicogenomics, GROW Institute and Developmental Biology, Maastricht University, Maastricht, the Netherlands.

One of the major complications that patients experience during pharmacological treatment is the occurrence of adverse drug reactions (ADRs). The most affected organs are the liver, kidney, heart and the gastrointestinal-immune system. In comparison to the other organs, less progress has been made on human-relevant prediction of drug-induced intestinal toxicity, evidencing current large data gaps. The most widely used drugs that are associated with intestinal damage include chemotherapeutics, such as 5-Fluorouracil or Tyrosine Kinase Inhibitors (TKIs), as well as non-steroidal anti-inflammatory drugs (NSAIDs). Chemotherapeutics are regarded as inducers of acute intestinal toxicity whereas NSAIDs are associated with chronic inflammation of the intestine. In view of the fact that only a few studies have been dedicated to studying cellular and genomic responses in relation to drug-induced intestinal ADRs, little is known about how intestinal toxicity develops after exposure to such drugs or which molecular mechanisms are involved. Therefore, new models and experiments are required to establish transcriptomic responses and alterations of molecular markers induced by different medicines. This review summarizes the available information about transcriptomic responses and biomarkers of toxicity induced by 5-FU, NSAIDS or TKIs in different experimental models. Future investigation should address the challenges in predicting intestinal toxicity induced by drugs and unveil specific gene expression profiles that can be applied in the development of safer drugs.
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http://dx.doi.org/10.1016/j.ctrv.2019.06.004DOI Listing
July 2019

DNA methylation profiling implicates exposure to PCBs in the pathogenesis of B-cell chronic lymphocytic leukemia.

Environ Int 2019 05 15;126:24-36. Epub 2019 Feb 15.

National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, 48 Vas. Constantinou Ave., Athens 11635, Greece. Electronic address:

Objectives: To characterize the impact of PCB exposure on DNA methylation in peripheral blood leucocytes and to evaluate the corresponding changes in relation to possible health effects, with a focus on B-cell lymphoma.

Methods: We conducted an epigenome-wide association study on 611 adults free of diagnosed disease, living in Italy and Sweden, in whom we also measured plasma concentrations of 6 PCB congeners, DDE and hexachlorobenzene.

Results: We identified 650 CpG sites whose methylation correlates strongly (FDR < 0.01) with plasma concentrations of at least one PCB congener. Stronger effects were observed in males and in Sweden. This epigenetic exposure profile shows extensive and highly statistically significant overlaps with published profiles associated with the risk of future B-cell chronic lymphocytic leukemia (CLL) as well as with clinical CLL (38 and 28 CpG sites, respectively). For all these sites, the methylation changes were in the same direction for increasing exposure and for higher disease risk or clinical disease status, suggesting an etiological link between exposure and CLL. Mediation analysis reinforced the suggestion of a causal link between exposure, changes in DNA methylation and disease. Disease connectivity analysis identified multiple additional diseases associated with differentially methylated genes, including melanoma for which an etiological link with PCB exposure is established, as well as developmental and neurological diseases for which there is corresponding epidemiological evidence. Differentially methylated genes include many homeobox genes, suggesting that PCBs target stem cells. Furthermore, numerous polycomb protein target genes were hypermethylated with increasing exposure, an effect known to constitute an early marker of carcinogenesis.

Conclusions: This study provides mechanistic evidence in support of a link between exposure to PCBs and the etiology of CLL and underlines the utility of omic profiling in the evaluation of the potential toxicity of environmental chemicals.
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http://dx.doi.org/10.1016/j.envint.2019.01.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063446PMC
May 2019

Identification of Sex-Specific Transcriptome Responses to Polychlorinated Biphenyls (PCBs).

Sci Rep 2019 01 24;9(1):746. Epub 2019 Jan 24.

Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands.

PCBs are classified as xenoestrogens and carcinogens and their health risks may be sex-specific. To identify potential sex-specific responses to PCB-exposure we established gene expression profiles in a population study subdivided into females and males. Gene expression profiles were determined in a study population consisting of 512 subjects from the EnviroGenomarkers project, 217 subjects who developed lymphoma and 295 controls were selected in later life. We ran linear mixed models in order to find associations between gene expression and exposure to PCBs, while correcting for confounders, in particular distribution of white blood cells (WBC), as well as random effects. The analysis was subdivided according to sex and development of lymphoma in later life. The changes in gene expression as a result of exposure to the six studied PCB congeners were sex- and WBC type specific. The relatively large number of genes that are significantly associated with PCB-exposure in the female subpopulation already indicates different biological response mechanisms to PCBs between the two sexes. The interaction analysis between different PCBs and WBCs provides only a small overlap between sexes. In males, cancer-related pathways and in females immune system-related pathways are identified in association with PCBs and WBCs. Future lymphoma cases and controls for both sexes show different responses to the interaction of PCBs with WBCs, suggesting a role of the immune system in PCB-related cancer development.
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http://dx.doi.org/10.1038/s41598-018-37449-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346099PMC
January 2019

Persistence of Epigenomic Effects After Recovery From Repeated Treatment With Two Nephrocarcinogens.

Front Genet 2018 3;9:558. Epub 2018 Dec 3.

Division of Molecular and Computational Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

The discovery of the epigenetic regulation of transcription has provided a new source of mechanistic understanding to long lasting effects of chemicals. However, this information is still seldom exploited in a toxicological context and studies of chemical effect after washout remain rare. Here we studied the effects of two nephrocarcinogens on the human proximal tubule cell line RPTEC/TERT1 using high-content mRNA microarrays coupled with miRNA, histone acetylation (HA) and DNA methylation (DM) arrays and metabolomics during a 5-day repeat-dose exposure and 3 days after washout. The mycotoxin ochratoxin A (OTA) was chosen as a model compound for its known impact on HA and DM. The foremost effect observed was the modulation of thousands of mRNAs and histones by OTA during and after exposure. In comparison, the oxidant potassium bromate (KBrO) had a milder impact on gene expression and epigenetics. However, there was no strong correlation between epigenetic modifications and mRNA changes with OTA while with KBrO the gene expression data correlated better with HA for both up- and down-regulated genes. Even when focusing on the genes with persistent epigenetic modifications after washout, only half were coupled to matching changes in gene expression induced by OTA, suggesting that while OTA causes a major effect on the two epigenetic mechanisms studied, these alone cannot explain its impact on gene expression. Mechanistic analysis confirmed the known activation of Nrf2 and p53 by KBrO, while OTA inhibited most of the same genes, and genes involved in the unfolded protein response. A few miRNAs could be linked to these effects of OTA, albeit without clear contribution of epigenetics to the modulation of the pathways at large. Metabolomics revealed disturbances in amino acid balance, energy catabolism, nucleotide metabolism and polyamine metabolism with both chemicals. In conclusion, the large impact of OTA on transcription was confirmed at the mRNA level but also with two high-content epigenomic methodologies. Transcriptomic data confirmed the previously reported activation (by KBrO) and inhibition (by OTA) of protective pathways. However, the integration of omic datasets suggested that HA and DM were not driving forces in the gene expression changes induced by either chemical.
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http://dx.doi.org/10.3389/fgene.2018.00558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286959PMC
December 2018

Phosphorylation of eIF2α promotes cell survival in response to benzo[a]pyrene exposure.

Toxicol In Vitro 2019 Feb 29;54:330-337. Epub 2018 Oct 29.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University, the Netherlands. Electronic address:

Cellular adaptation is important to cope with various stresses induced by altered environmental conditions. By controlling mRNA translation rates cells may adapt to stress to promote survival. Phosphorylation of eIF2α at serine 51 is one of the pathways controlling mRNA translation. Here we investigated the role of phosphorylated eIF2α during exposure to the environmental carcinogen benzo(a)pyrene (BaP). For our study we used mouse embryonic fibroblasts with a wild type eIF2α (MEF WT) and mouse embryonic fibroblasts with an eIF2α S51A knock-in mutation that cannot be phosphorylated. Here, we show that eIF2α phosphorylation occurs in MEF WT cells but not in MEF S51A cells. Survival of MEF S51A cells is profoundly reduced compared to MEF WT controls after BaP exposure. No differences in DNA damage or ROS production were observed between MEF WT and S51A cells. Disruption of eIF2α phosphorylation caused increased levels of apoptosis in response to BaP. This work demonstrates that eIF2α phosphorylation is important for reducing apoptosis and promoting cell survival in order to adapt to BaP exposure.
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http://dx.doi.org/10.1016/j.tiv.2018.10.015DOI Listing
February 2019

Comparison of statistical methods and the use of quality control samples for batch effect correction in human transcriptome data.

PLoS One 2018 30;13(8):e0202947. Epub 2018 Aug 30.

Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands.

Batch effects are technical sources of variation introduced by the necessity of conducting gene expression analyses on different dates due to the large number of biological samples in population-based studies. The aim of this study is to evaluate the performances of linear mixed models (LMM) and Combat in batch effect removal. We also assessed the utility of adding quality control samples in the study design as technical replicates. In order to do so, we simulated gene expression data by adding "treatment" and batch effects to a real gene expression dataset. The performances of LMM and Combat, with and without quality control samples, are assessed in terms of sensitivity and specificity while correcting for the batch effect using a wide range of effect sizes, statistical noise, sample sizes and level of balanced/unbalanced designs. The simulations showed small differences among LMM and Combat. LMM identifies stronger relationships between big effect sizes and gene expression than Combat, while Combat identifies in general more true and false positives than LMM. However, these small differences can still be relevant depending on the research goal. When any of these methods are applied, quality control samples did not reduce the batch effect, showing no added value for including them in the study design.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202947PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117018PMC
February 2019

Acute changes in DNA methylation in relation to 24 h personal air pollution exposure measurements: A panel study in four European countries.

Environ Int 2018 11 25;120:11-21. Epub 2018 Jul 25.

Division of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University, 3584 CM Utrecht, the Netherlands. Electronic address:

Background: One of the potential mechanisms linking air pollution to health effects is through changes in DNA-methylation, which so far has mainly been analyzed globally or at candidate sites.

Objective: We investigated the association of personal and ambient air pollution exposure measures with genome-wide DNA-methylation changes.

Methods: We collected repeated 24-hour personal and ambient exposure measurements of particulate matter (PM), PM absorbance, and ultrafine particles (UFP) and peripheral blood samples from a panel of 157 healthy non-smoking adults living in four European countries. We applied univariate mixed-effects models to investigate the association between air pollution and genome-wide DNA-methylation perturbations at single CpG (cytosine-guanine dinucleotide) sites and in Differentially Methylated Regions (DMRs). Subsequently, we explored the association of air pollution-induced methylation alterations with gene expression and serum immune marker levels measured in the same subjects.

Results: Personal exposure to PM was associated with methylation changes at 13 CpG sites and 69 DMRs. Two of the 13 identified CpG sites (mapped to genes KNDC1 and FAM50B) were located within these DMRs. In addition, 42 DMRs were associated with personal PM absorbance exposure, 16 DMRs with personal exposure to UFP, 4 DMRs with ambient exposure to PM, 16 DMRs with ambient PM absorbance exposure, and 15 DMRs with ambient UFP exposure. Correlation between methylation levels at identified CpG sites and gene expression and immune markers was generally moderate.

Conclusion: This study provides evidence for an association between 24-hour exposure to air pollution and DNA-methylation at single sites and regional clusters of CpGs. Analysis of differentially methylated regions provides a promising avenue to further explore the subtle impact of environmental exposures on DNA-methylation.
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http://dx.doi.org/10.1016/j.envint.2018.07.026DOI Listing
November 2018

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

Environ Pollut 2018 Nov 20;242(Pt A):182-190. Epub 2018 Jun 20.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, the Netherlands.

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 (PM, PM), ultrafine particles (UFPC), nitrogen oxides (NO, NO and NOx), black carbon (BC) and carbon oxides (CO and CO) 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.
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http://dx.doi.org/10.1016/j.envpol.2018.06.051DOI Listing
November 2018

Translational regulation is a key determinant of the cellular response to benzo[a]pyrene.

Toxicol Lett 2018 Oct 12;295:144-152. Epub 2018 Jun 12.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, 6200MD, The Netherlands. Electronic address:

Translational control is a cellular response mechanism which initiates adaptation during various stress situations. Here, we investigated the role of translational control after benzo[a]pyrene (BaP) exposure in primary mouse hepatocytes. Translated mRNAs were separated and captured based on the number of associated ribosomes using sucrose gradients and subjected to RNA sequencing (RNAseq) to investigate translational changes. Furthermore, unseparated RNA (total RNA) was used for RNAseq to determine the transcriptional alterations. We showed that, after 24 h of exposure to 10 μM BaP, the number of genes altered by changes in mRNA translation was substantially higher compared with the number of genes altered by transcription. Although part of the BaP regulated genes were regulated by both transcription and translation, we identified genes that were uniquely regulated by mRNA translation. These mRNA transcripts encode proteins that are involved in biological processes that are not affected by transcriptional regulation. Al together this work identified a new layer of gene expression regulation that might contribute to BaP-induced carcinogenesis.
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http://dx.doi.org/10.1016/j.toxlet.2018.06.009DOI Listing
October 2018

Epigenome-wide association study of adiposity and future risk of obesity-related diseases.

Int J Obes (Lond) 2018 12 1;42(12):2022-2035. Epub 2018 May 1.

Department of Medical Sciences, Unit of Cancer Epidemiology-CERMS, University of Turin, Turin, Italy.

Background: Obesity is an established risk factor for several common chronic diseases such as breast and colorectal cancer, metabolic and cardiovascular diseases; however, the biological basis for these relationships is not fully understood. To explore the association of obesity with these conditions, we investigated peripheral blood leucocyte (PBL) DNA methylation markers for adiposity and their contribution to risk of incident breast and colorectal cancer and myocardial infarction.

Methods: DNA methylation profiles (Illumina Infinium HumanMethylation450 BeadChip) from 1941 individuals from four population-based European cohorts were analysed in relation to body mass index, waist circumference, waist-hip and waist-height ratio within a meta-analytical framework. In a subset of these individuals, data on genome-wide gene expression level, biomarkers of glucose and lipid metabolism were also available. Validation of methylation markers associated with all adiposity measures was performed in 358 individuals. Finally, we investigated the association of obesity-related methylation marks with breast, colorectal cancer and myocardial infarction within relevant subsets of the discovery population.

Results: We identified 40 CpG loci with methylation levels associated with at least one adiposity measure. Of these, one CpG locus (cg06500161) in ABCG1 was associated with all four adiposity measures (P = 9.07×10 to 3.27×10) and lower transcriptional activity of the full-length isoform of ABCG1 (P = 6.00×10), higher triglyceride levels (P = 5.37×10) and higher triglycerides-to-HDL cholesterol ratio (P = 1.03×10). Of the 40 informative and obesity-related CpG loci, two (in IL2RB and FGF18) were significantly associated with colorectal cancer (inversely, P < 1.6×10) and one intergenic locus on chromosome 1 was inversely associated with myocardial infarction (P < 1.25×10), independently of obesity and established risk factors.

Conclusion: Our results suggest that epigenetic changes, in particular altered DNA methylation patterns, may be an intermediate biomarker at the intersection of obesity and obesity-related diseases, and could offer clues as to underlying biological mechanisms.
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http://dx.doi.org/10.1038/s41366-018-0064-7DOI Listing
December 2018

Rationale for Environmental Hygiene towards global protection of fetuses and young children from adverse lifestyle factors.

Environ Health 2018 04 23;17(1):42. Epub 2018 Apr 23.

Department of Radiotherapy and Experimental Cancerology, Ghent University, Ghent, Belgium.

Background: The regulatory management of chemicals and toxicants in the EU addresses hundreds of different chemicals and health hazards individually, one by one. An issue is that, so far, the possible interactions among chemicals or hazards are not considered as such. Another issue is the anticipated delay of several decades before effective protection of public health by regulatory decisions due to a time consuming process. Prenatal and early postnatal life is highly vulnerable to environmental health hazards with lifelong consequences, and a priority period for reduction of exposure. There are some initiatives regarding recommendations for pregnant women aiming at protection against one or another category of health hazard, however not validated by intervention studies.

Hypothesis: Here, we aim at strengthening the management of exposure to individual health hazards during pregnancy and lactation, with protective measures in a global strategy of Environmental Hygiene. We hypothesize that such a strategy could reduce both the individual effects of harmful agents in complex mixtures and the possible interactions among them. A panel of experts should develop and endorse implementable measures towards a protective behavior. Their application is meant to be preferably as a package of measures in order to maximize protection and minimize interactions in causing adverse effects. Testing our hypothesis requires biomonitoring studies and longitudinal evaluation of health endpoints in the offspring. Favorable effects would legitimate further action towards equal opportunity access to improved environmental health.

Conclusion: Environmental Hygiene is proposed as a global strategy aiming at effective protection of pregnant women, unborn children and infants against lifelong consequences of exposure to combinations of adverse lifestyle factors.
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http://dx.doi.org/10.1186/s12940-018-0385-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5914065PMC
April 2018

Pre-diagnostic blood immune markers, incidence and progression of B-cell lymphoma and multiple myeloma: Univariate and functionally informed multivariate analyses.

Int J Cancer 2018 09 26;143(6):1335-1347. Epub 2018 Apr 26.

Department of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden.

Recent prospective studies have shown that dysregulation of the immune system may precede the development of B-cell lymphomas (BCL) in immunocompetent individuals. However, to date, the studies were restricted to a few immune markers, which were considered separately. Using a nested case-control study within two European prospective cohorts, we measured plasma levels of 28 immune markers in samples collected a median of 6 years before diagnosis (range 2.01-15.97) in 268 incident cases of BCL (including multiple myeloma [MM]) and matched controls. Linear mixed models and partial least square analyses were used to analyze the association between levels of immune marker and the incidence of BCL and its main histological subtypes and to investigate potential biomarkers predictive of the time to diagnosis. Linear mixed model analyses identified associations linking lower levels of fibroblast growth factor-2 (FGF-2 p = 7.2 × 10 ) and transforming growth factor alpha (TGF-α, p = 6.5 × 10 ) and BCL incidence. Analyses stratified by histological subtypes identified inverse associations for MM subtype including FGF-2 (p = 7.8 × 10 ), TGF-α (p = 4.08 × 10 ), fractalkine (p = 1.12 × 10 ), monocyte chemotactic protein-3 (p = 1.36 × 10 ), macrophage inflammatory protein 1-alpha (p = 4.6 × 10 ) and vascular endothelial growth factor (p = 4.23 × 10 ). Our results also provided marginal support for already reported associations between chemokines and diffuse large BCL (DLBCL) and cytokines and chronic lymphocytic leukemia (CLL). Case-only analyses showed that Granulocyte-macrophage colony stimulating factor levels were consistently higher closer to diagnosis, which provides further evidence of its role in tumor progression. In conclusion, our study suggests a role of growth-factors in the incidence of MM and of chemokine and cytokine regulation in DLBCL and CLL.
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http://dx.doi.org/10.1002/ijc.31536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100111PMC
September 2018

Integrated 'omics analysis reveals new drug-induced mitochondrial perturbations in human hepatocytes.

Toxicol Lett 2018 Jun 6;289:1-13. Epub 2018 Mar 6.

Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Electronic address:

We performed a multiple 'omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III-V genes. Persistent transcripts and proteins were related to citric acid cycle and β-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2-FN1-MYC-CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation.
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http://dx.doi.org/10.1016/j.toxlet.2018.02.026DOI Listing
June 2018

A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases.

Toxicol In Vitro 2018 Apr 9;48:318-328. Epub 2018 Feb 9.

Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200, MD, Maastricht, The Netherlands.

Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.
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http://dx.doi.org/10.1016/j.tiv.2018.02.006DOI Listing
April 2018

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

Toxicology 2018 01 14;393:160-170. Epub 2017 Nov 14.

Department of Toxicogenomics, Grow School for Oncology and Developmental Biology, Maastricht University, P.o. Box 616, 6200 Md, Maastricht, The Netherlands.

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 β-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 β-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.
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http://dx.doi.org/10.1016/j.tox.2017.11.013DOI Listing
January 2018

Dose and Time Dependencies in Stress Pathway Responses during Chemical Exposure: Novel Insights from Gene Regulatory Networks.

Front Genet 2017 6;8:142. Epub 2017 Oct 6.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, Netherlands.

Perturbation of biological networks is often observed during exposure to xenobiotics, and the identification of disturbed processes, their dynamic traits, and dose-response relationships are some of the current challenges for elucidating the mechanisms determining adverse outcomes. In this scenario, reverse engineering of gene regulatory networks (GRNs) from expression data may provide a system-level snapshot embedded within accurate molecular events. Here, we investigate the composition of GRNs inferred from groups of chemicals with two distinct outcomes, namely carcinogenicity [azathioprine (AZA) and cyclophosphamide (CYC)] and drug-induced liver injury (DILI; diclofenac, nitrofurantoin, and propylthiouracil), and a non-carcinogenic/non-DILI group (aspirin, diazepam, and omeprazole). For this, we analyzed publicly available exposed human data, taking into account dose and time dependencies. Dose-Time Network Identification (DTNI) was applied to gene sets from exposed primary human hepatocytes using four stress pathways, namely endoplasmic reticulum (ER), NF-κB, NRF2, and TP53. Inferred GRNs suggested case specificity, varying in interactions, starting nodes, and target genes across groups. DILI and carcinogenic compounds were shown to directly affect all pathway-based GRNs, while non-DILI/non-carcinogenic chemicals only affected NF-κB. NF-κB-based GRNs clearly illustrated group-specific disturbances, with the cancer-related casein kinase being a target gene only in the carcinogenic group, and opposite regulation of NF-κB subunits being observed in DILI and non-DILI/non-carcinogenic groups. Target genes in NRF2-based GRNs shared by DILI and carcinogenic compounds suggested markers of hepatotoxicity. Finally, we indicate several of these group-specific interactions as potentially novel. In summary, our reversed-engineered GRNs are capable of revealing dose dependent, chemical-specific mechanisms of action in stress-related biological networks.
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http://dx.doi.org/10.3389/fgene.2017.00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649202PMC
October 2017

Evolving DNA methylation and gene expression markers of B-cell chronic lymphocytic leukemia are present in pre-diagnostic blood samples more than 10 years prior to diagnosis.

BMC Genomics 2017 Sep 13;18(1):728. Epub 2017 Sep 13.

Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48, Vassileos Constantinou Avenue, 11635, Athens, Greece.

Background: B-cell chronic lymphocytic leukemia (CLL) is a common type of adult leukemia. It often follows an indolent course and is preceded by monoclonal B-cell lymphocytosis, an asymptomatic condition, however it is not known what causes subjects with this condition to progress to CLL. Hence the discovery of prediagnostic markers has the potential to improve the identification of subjects likely to develop CLL and may also provide insights into the pathogenesis of the disease of potential clinical relevance.

Results: We employed peripheral blood buffy coats of 347 apparently healthy subjects, of whom 28 were diagnosed with CLL 2.0-15.7 years after enrollment, to derive for the first time genome-wide DNA methylation, as well as gene and miRNA expression, profiles associated with the risk of future disease. After adjustment for white blood cell composition, we identified 722 differentially methylated CpG sites and 15 differentially expressed genes (Bonferroni-corrected p < 0.05) as well as 2 miRNAs (FDR < 0.05) which were associated with the risk of future CLL. The majority of these signals have also been observed in clinical CLL, suggesting the presence in prediagnostic blood of CLL-like cells. Future CLL cases who, at enrollment, had a relatively low B-cell fraction (<10%), and were therefore less likely to have been suffering from undiagnosed CLL or a precursor condition, showed profiles involving smaller numbers of the same differential signals with intensities, after adjusting for B-cell content, generally smaller than those observed in the full set of cases. A similar picture was obtained when the differential profiles of cases with time-to-diagnosis above the overall median period of 7.4 years were compared with those with shorted time-to-disease. Differentially methylated genes of major functional significance include numerous genes that encode for transcription factors, especially members of the homeobox family, while differentially expressed genes include, among others, multiple genes related to WNT signaling as well as the miRNAs miR-150-5p and miR-155-5p.

Conclusions: Our findings demonstrate the presence in prediagnostic blood of future CLL patients, more than 10 years before diagnosis, of CLL-like cells which evolve as preclinical disease progresses, and point to early molecular alterations with a pathogenetic potential.
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http://dx.doi.org/10.1186/s12864-017-4117-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5598006PMC
September 2017

Identification of essential transcription factors for adequate DNA damage response after benzo(a)pyrene and aflatoxin B1 exposure by combining transcriptomics with functional genomics.

Toxicology 2017 09 4;390:74-82. Epub 2017 Sep 4.

Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200 MD, Maastricht, The Netherlands. Electronic address:

DNA damage mediates widespread changes in transcription through activation or repression of transcription factors (TFs). However, the consequences of regulating specific TFs for the outcome of the DNA repair process remain incompletely understood. Here, we combined transcriptomics and TF binding prediction with functional genomics to identify TFs essential for adequate DNA repair in HepG2 liver cells after a non-cytotoxic dose of carcinogens benzo(a)pyrene (BaP) (2μM) and aflatoxin B1 (AFB1) (5μM). BaP and AFB1 induced a largely common transcriptional response, mediated by similar TFs. A lentiviral shRNA screen knocking down the top31 identified TFs, was performed to determine their effect on DNA repair by assessing phosphorylation of H2AX (γ-H2AX). In addition to the top candidate p53, we identified several other interesting TFs that modulated γ-H2AX after BaP and AFB1 treatment. Validation studies confirmed the role of p53 in reducing γ-H2AX formation and DNA breaks measured by COMET assay after BaP and AFB1 exposure. Expression of the cell cycle inhibitor p21 was profoundly impaired upon p53 knock-down. In addition, the expression of 2 genes involved in nucleotide exchange repair, DDB2 and XPC was significantly reduced in p53 knock-down cells. Although p63 knock-down affected DNA damage upon BaP treatment this was not associated with altered expression of DDB2 or XPC. Finally, knock-down of ARNT reduced γ-H2AX in response to BaP, which was associated with reduced CYP1A1 expression. Importantly, our results suggest a new role for ARNT and its dimerization partner AHR in the occurrence of H2AX phosphorylation after AFB1 treatment. These data show that modulation of TF activity impacts on the repair of BaP- and AFB1-induced DNA damage. Our study also demonstrates the potential of combining functional genomics with genome-wide expression analysis to identify yet unknown causal relationships, thereby aiding in the interpretation of complex biological systems.
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http://dx.doi.org/10.1016/j.tox.2017.09.002DOI Listing
September 2017

Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes.

Chem Res Toxicol 2017 10 13;30(10):1847-1854. Epub 2017 Sep 13.

Department of Toxicogenomics, GROW School for Oncology and Developmental Biology, Maastricht University , P.O. Box 616, Maastricht 6200 MD, The Netherlands.

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation-sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645762PMC
October 2017

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

Toxicol In Vitro 2017 Oct 1;44:322-329. Epub 2017 Aug 1.

Department of Toxicogenomics, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands; Netherlands Toxicogenomics Centre, Maastricht, The Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tiv.2017.07.024DOI Listing
October 2017

Blood-based omic profiling supports female susceptibility to tobacco smoke-induced cardiovascular diseases.

Sci Rep 2017 02 22;7:42870. Epub 2017 Feb 22.

National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, 48 Vas. Constantinou Ave., Athens 11635, Greece.

We recently reported that differential gene expression and DNA methylation profiles in blood leukocytes of apparently healthy smokers predicts with remarkable efficiency diseases and conditions known to be causally associated with smoking, suggesting that blood-based omic profiling of human populations may be useful for linking environmental exposures to potential health effects. Here we report on the sex-specific effects of tobacco smoking on transcriptomic and epigenetic features derived from genome-wide profiling in white blood cells, identifying 26 expression probes and 92 CpG sites, almost all of which are affected only in female smokers. Strikingly, these features relate to numerous genes with a key role in the pathogenesis of cardiovascular disease, especially thrombin signaling, including the thrombin receptors on platelets F2R (coagulation factor II (thrombin) receptor; PAR1) and GP5 (glycoprotein 5), as well as HMOX1 (haem oxygenase 1) and BCL2L1 (BCL2-like 1) which are involved in protection against oxidative stress and apoptosis, respectively. These results are in concordance with epidemiological evidence of higher female susceptibility to tobacco-induced cardiovascular disease and underline the potential of blood-based omic profiling in hazard and risk assessment.
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http://dx.doi.org/10.1038/srep42870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320491PMC
February 2017

Associations Between Genome-wide Gene Expression and Ambient Nitrogen Oxides.

Epidemiology 2017 05;28(3):320-328

From the aDivision of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands; bMedical Research Council-Health Protection Agency Centre for Environment and Health, Department of Epidemiology and Biostatistics, Imperial College London, London, United Kingdom; cDepartment of Public Health and Clinical Medicine, Occupational and Environmental Medicine, Umeå University, Umeå, Sweden; dDepartment of Biobank Research, Umeå University, Umeå, Sweden; eMolecular and Nutritional Epidemiology Unit, Cancer Prevention and Research Institute (ISPO), Florence, Italy; fJulius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands; gHuGeF Foundation, Turin, Italy; hDepartment of Toxicogenomics, Maastricht University, Maastricht, The Netherlands; iEpidemiology Unit, Instituto Tumori, Milan, Italy; and jNational Hellenic Research Foundation, Institute of Biology, Pharmaceutical Chemistry and Biotechnology, Athens, Greece.

Background: We hypothesize that biological perturbations due to exposure to ambient air pollution are reflected in gene expression levels in peripheral blood mononuclear cells.

Methods: We assessed the association between exposure to ambient air pollution and genome-wide gene expression levels in peripheral blood mononuclear cells collected from 550 healthy subjects participating in cohorts from Italy and Sweden. Annual air pollution estimates of nitrogen oxides (NOx) at time of blood collection (1990-2006) were available from the ESCAPE study. In addition to univariate analysis and two variable selection methods to investigate the association between expression and exposure to NOx, we applied gene set enrichment analysis to assess overlap between our most perturbed genes and gene sets hypothesized to be related to air pollution and cigarette smoking. Finally, we assessed associations between NOx and CpG island methylation at the identified genes.

Results: Annual average NOx exposure in the Italian and Swedish cohorts was 94.2 and 6.7 µg/m, respectively. Long-term exposure to NOx was associated with seven probes in the Italian cohort and one probe in the Swedish (and combined) cohorts. For genes AHCYL2 and MTMR2, changes were also seen in the methylome. Genes hypothesized to be downregulated due to cigarette smoking were enriched among the most strongly downregulated genes from our study.

Conclusion: This study provides evidence of subtle changes in gene expression related to exposure to long-term NOx. On a global level, the observed changes in the transcriptome may indicate similarities between air pollution and tobacco induced changes in the transcriptome.
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http://dx.doi.org/10.1097/EDE.0000000000000628DOI Listing
May 2017

DTNI: a novel toxicogenomics data analysis tool for identifying the molecular mechanisms underlying the adverse effects of toxic compounds.

Arch Toxicol 2017 Jun 28;91(6):2343-2352. Epub 2016 Dec 28.

Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, The Netherlands.

Unravelling gene regulatory networks (GRNs) influenced by chemicals is a major challenge in systems toxicology. Because toxicant-induced GRNs evolve over time and dose, the analysis of global gene expression data measured at multiple time points and doses will provide insight in the adverse effects of compounds. Therefore, there is a need for mathematical methods for GRN identification from time-over-dose-dependent data. One of the current approaches for GRN inference is Time Series Network Identification (TSNI). TSNI is based on ordinary differential equations (ODE), describing the time evolution of the expression of each gene, which is assumed to be dependent on the expression of other genes and an external perturbation (i.e. chemical exposure). Here, we present Dose-Time Network Identification (DTNI), a method extending TSNI by including ODE describing how the expression of each gene evolves with dose, which is supposed to depend on the expression of other genes and the exposure time. We also adapted TSNI in order to enable inclusion of time-over-dose-dependent data from multiple compounds. Here, we show that DTNI outperforms TSNI in inferring a toxicant-induced GRN. Moreover, we show that DTNI is a suitable method to infer a GRN dose- and time-dependently induced by a group of compounds influencing a common biological process. Applying DTNI on experimental data from TG-GATEs, we demonstrate that DTNI provides in-depth information on the mode of action of compounds, in particular key events and potential molecular initiating events. Furthermore, DTNI also discloses several unknown interactions which have to be verified experimentally.
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http://dx.doi.org/10.1007/s00204-016-1922-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429357PMC
June 2017
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