Deconvoluting gene and environment interactions to develop an "epigenetic score meter" of disease.

Human health is determined both by genetics (G) and environment (E). This is clearly illustrated in groups of individuals who are exposed to the same environmental factor showing differential responses. A quantitative measure of the gene-environment interactions (GxE) effects has not been developed and in some instances, a clear consensus on the concept has not even been reached; for example, whether cancer is predominantly emerging from "bad luck" or "bad lifestyle" is still debated. In this article, we provide a panel of examples of GxE interaction as drivers of pathogenesis. We highlight how epigenetic regulations can represent a common connecting aspect of the molecular bases. Our argument converges on the concept that the GxE is recorded in the cellular epigenome, which might represent the key to deconvolute these multidimensional intricated layers of regulation. Developing a key to decode this epigenetic information would provide quantitative measures of disease risk. Analogously to the epigenetic clock introduced to estimate biological age, we provocatively propose the theoretical concept of an "epigenetic score-meter" to estimate disease risk.

Gene-Environment Interactions in Developmental Neurotoxicity: a Case Study of Synergy between Chlorpyrifos and CHD8 Knockout in Human BrainSpheres.

BACKGROUND: Autism spectrum disorder (ASD) is a major public health concern caused by complex genetic and environmental components. Mechanisms of gene-environment (G×E) interactions and reliable biomarkers associated with ASD are mostly unknown or controversial. Induced pluripotent stem cells (iPSCs) from patients or with clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9)-introduced mutations in candidate ASD genes provide an opportunity to study (G×E) interactions. OBJECTIVES: In this study, we aimed to identify a potential synergy between mutation in the high-risk autism gene encoding chromodomain helicase DNA binding protein 8 (CHD8) and environmental exposure to an organophosphate pesticide (chlorpyrifos; CPF) in an iPSC-derived human three-dimensional (3D) brain model. METHODS: This study employed human iPSC-derived 3D brain organoids (BrainSpheres) carrying a heterozygote CRISPR/Cas9-introduced inactivating mutation in CHD8 and exposed to CPF or its oxon-metabolite (CPO). Neural differentiation, viability, oxidative stress, and neurite outgrowth were assessed, and levels of main neurotransmitters and selected metabolites were validated against human data on ASD metabolic derangements. RESULTS: Expression of CHD8 protein was significantly lower in CHD8 heterozygous knockout (CHD8+/-) BrainSpheres compared with CHD8+/+ ones. Exposure to CPF/CPO treatment further reduced CHD8 protein levels, showing the potential (G×E) interaction synergy. A novel approach for validation of the model was chosen: from the literature, we identified a panel of metabolic biomarkers in patients and assessed them by targeted metabolomics in vitro. A synergistic effect was observed on the cholinergic system, S-adenosylmethionine, S-adenosylhomocysteine, lactic acid, tryptophan, kynurenic acid, and α-hydroxyglutaric acid levels. Neurite outgrowth was perturbed by CPF/CPO exposure. Heterozygous knockout of CHD8 in BrainSpheres led to an imbalance of excitatory/inhibitory neurotransmitters and lower levels of dopamine. DISCUSSION: This study pioneered (G×E) interaction in iPSC-derived organoids. The experimental strategy enables biomonitoring and environmental risk assessment for ASD. Our findings reflected some metabolic perturbations and disruption of neurotransmitter systems involved in ASD. The increased susceptibility of CHD8+/- BrainSpheres to chemical insult establishes a possibly broader role of (G×E) interaction in ASD. https://doi.org/10.1289/EHP8580.

Effect of sub-chronic exposure to cigarette smoke, electronic cigarette and waterpipe on human lung epithelial barrier function.

BACKGROUND: Taking into consideration a recent surge of a lung injury condition associated with electronic cigarette use, we devised an in vitro model of sub-chronic exposure of human bronchial epithelial cells (HBECs) in air-liquid interface, to determine deterioration of epithelial cell barrier from sub-chronic exposure to cigarette smoke (CS), e-cigarette aerosol (EC), and tobacco waterpipe exposures (TW). METHODS: Products analyzed include commercially available e-liquid, with 0% or 1.2% concentration of nicotine, tobacco blend (shisha), and reference-grade cigarette (3R4F). In one set of experiments, HBECs were exposed to EC (0 and 1.2%), CS or control air for 10 days using 1 cigarette/day. In the second set of experiments, exposure of pseudostratified primary epithelial tissue to TW or control air exposure was performed 1-h/day, every other day, until 3 exposures were performed. After 16-18 h of last exposure, we investigated barrier function/structural integrity of the epithelial monolayer with fluorescein isothiocyanate-dextran flux assay (FITC-Dextran), measurements of trans-electrical epithelial resistance (TEER), assessment of the percentage of moving cilia, cilia beat frequency (CBF), cell motion, and quantification of E-cadherin gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: When compared to air control, CS increased fluorescence (FITC-Dextran assay) by 5.6 times, whereby CS and EC (1.2%) reduced TEER to 49 and 60% respectively. CS and EC (1.2%) exposure reduced CBF to 62 and 59%, and cilia moving to 47 and 52%, respectively, when compared to control air. CS and EC (1.2%) increased cell velocity compared to air control by 2.5 and 2.6 times, respectively. The expression of E-cadherin reduced to 39% of control air levels by CS exposure shows an insight into a plausible molecular mechanism. Altogether, EC (0%) and TW exposures resulted in more moderate decreases in epithelial integrity, while EC (1.2%) substantially decreased airway epithelial barrier function comparable with CS exposure. CONCLUSIONS: The results support a toxic effect of sub-chronic exposure to EC (1.2%) as evident by disruption of the bronchial epithelial cell barrier integrity, whereas further research is needed to address the molecular mechanism of this observation as well as TW and EC (0%) toxicity in chronic exposures.

Toxicity, recovery, and resilience in a 3D dopaminergic neuronal in vitro model exposed to rotenone.

To date, most in vitro toxicity testing has focused on acute effects of compounds at high concentrations. This testing strategy does not reflect real-life exposures, which might contribute to long-term disease outcome. We used a 3D-human dopaminergic in vitro LUHMES cell line model to determine whether effects of short-term rotenone exposure (100 nM, 24 h) are permanent or reversible. A decrease in complex I activity, ATP, mitochondrial diameter, and neurite outgrowth were observed acutely. After compound removal, complex I activity was still inhibited; however, ATP levels were increased, cells were electrically active and aggregates restored neurite outgrowth integrity and mitochondrial morphology. We identified significant transcriptomic changes after 24 h which were not present 7 days after wash-out. Our results suggest that testing short-term exposures in vitro may capture many acute effects which cells can overcome, missing adaptive processes, and long-term mechanisms. In addition, to study cellular resilience, cells were re-exposed to rotenone after wash-out and recovery period. Pre-exposed cells maintained higher metabolic activity than controls and presented a different expression pattern in genes previously shown to be altered by rotenone. NEF2L2, ATF4, and EAAC1 were downregulated upon single hit on day 14, but unchanged in pre-exposed aggregates. DAT and CASP3 were only altered after re-exposure to rotenone, while TYMS and MLF1IP were downregulated in both single-exposed and pre-exposed aggregates. In summary, our study shows that a human cell-based 3D model can be used to assess cellular adaptation, resilience, and long-term mechanisms relevant to neurodegenerative research.

3S - Systematic, systemic, and systems biology and toxicology.

A biological system is more than the sum of its parts - it accomplishes many functions via synergy. Deconstructing the system down to the molecular mechanism level necessitates the complement of reconstructing functions on all levels, i.e., in our conceptualization of biology and its perturbations, our experimental models and computer modelling. Toxicology contains the somewhat arbitrary subclass "systemic toxicities"; however, there is no relevant toxic insult or general disease that is not systemic. At least inflammation and repair are involved that require coordinated signaling mechanisms across the organism. However, the more body components involved, the greater the challenge to reca-pitulate such toxicities using non-animal models. Here, the shortcomings of current systemic testing and the development of alternative approaches are summarized. We argue that we need a systematic approach to integrating existing knowledge as exemplified by systematic reviews and other evidence-based approaches. Such knowledge can guide us in modelling these systems using bioengineering and virtual computer models, i.e., via systems biology or systems toxicology approaches. Experimental multi-organ-on-chip and microphysiological systems (MPS) provide a more physiological view of the organism, facilitating more comprehensive coverage of systemic toxicities, i.e., the perturbation on organism level, without using substitute organisms (animals). The next challenge is to establish disease models, i.e., micropathophysiological systems (MPPS), to expand their utility to encompass biomedicine. Combining computational and experimental systems approaches and the chal-lenges of validating them are discussed. The suggested 3S approach promises to leverage 21st century technology and systematic thinking to achieve a paradigm change in studying systemic effects.

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd.

On the role of low-dose effects and epigenetics in toxicology.

For a long time, scientists considered genotoxic effects as the major issue concerning the influence of environmental chemicals on human health. Over the last decades, a new layer superimposed the genome, i.e., the epigenome, tremendously changing this point of view. The term "epigenetics" comprises stable alterations in gene expression potential arising from variations in DNA methylation and a variety of histone modifications, without changing the underlying DNA sequence. Recently, also gene silencing by small noncoding RNAs (ncRNAs), in particular by microRNAs, was included in the list of epigenetic mechanisms. Multiple studies in vivo as well as in vitro have shown that a multitude of different environmental factors are capable of changing the epigenetic pattern as well as miRNA expression in certain cell types, leading to aberrant gene expression profiles in cells and tissues. These changes may have extensive effects concerning the proper gene expression necessary in a specified cell type and can even lead into a state of disease. Especially the roles of epigenetic modifications and miRNA alterations in tumorigenesis have been a major focus in research over the last years. This chapter will give an overview on epigenetic features and on the spectrum of epigenetic changes observed after exposure against environmental chemicals and pollutants.

The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2.

The let-7 miRNA and its target gene Lin-28 interact in a regulatory circuit controlling pluripotency. We investigated an additional let-7 target, mLin41 (mouse homologue of lin-41), as a potential contributor to this circuit. We demonstrate the presence of mLin41 protein in several stem cell niches, including the embryonic ectoderm, epidermis and male germ line. mLin41 colocalized to cytoplasmic foci with P-body markers and the miRNA pathway proteins Ago2, Mov10 and Tnrc6b. In co-precipitation assays, mLin41 interacted with Dicer and the Argonaute proteins Ago1, Ago2 and Ago4. Moreover, we show that mLin41 acts as an E3 ubiquitin ligase in an auto-ubiquitylation assay and that mLin41 mediates ubiquitylation of Ago2 in vitro and in vivo. Overexpression and depletion of mLin41 led to inverse changes in the level of Ago2 protein, implicating mLin41 in the regulation of Ago2 turnover. mLin41 interfered with silencing of target mRNAs for let-7 and miR-124, at least in part by antagonizing Ago2. Furthermore, mLin41 cooperated with the pluripotency factor Lin-28 in suppressing let-7 activity, revealing a dual control mechanism regulating let-7 in stem cells.

Cancer-associated alteration in fatty acid binding to albumin studied by spin-label electron spin resonance.

Human serum albumin accumulates low molecular weight biomarkers related to cancer. This accumulation can lead to allosteric modification of albumin and change its ability to bind essential fatty acids. Using 16-doxyl-stearic acid spin probe, which is specific for albumin, the serum samples of 98 patients with a variety of cancer types and 86 cancer free individuals were analysed by electron spin resonance (ESR) spectroscopy in order to evaluate cancer-induced modifications that occur to albumin. The ESR spectra showed significant differences between the investigated groups. These differences were most apparent in the intensities and widths of the spectral lines corresponding to the different types of albumin binding sites.

Post-transcriptional regulation of the let-7 microRNA during neural cell specification.

The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons.