Etablierung eines zellulären genetischen Screeningverfahrens zur Suche nach Regulatoren des Glucocorticoidrezeptors und Identifizierung von Cofilin 1 als Glucocorticoidrezeptor-Inhibitor

Glucocorticoidresistenz ist ein Phänomen, das bei vielen Krankheiten eine wichtige Bedeutung hat. Insbesondere wird vermutet, dass ihr eine kausale Rolle bei depressiven Erkrankungen zukommt. In den meisten Fällen kommt die Resistenz durch eine Fehlfunktion des Glucocorticoidrezeptors zustande. Deswegen ist es von grossem Interesse, den Signalweg dieses Rezeptors im Detail zu verstehen. In der Vergangenheit wurde viel zum Verständnis beigetragen, unter anderem indem eine Reihe von GR-Regulatoren identifiziert und deren Wirkungsmechanismus aufgeklärt wurde. Das Ziel der vorliegenden Arbeit war es, neue Faktoren zu finden, die in die GR-Signaltransduktion verwickelt sind. Dazu wurde ein funktioneller Screen durchgeführt, der darauf beruhte, GC-resistente Zellen herzustellen, diese mit responsiven Zellen zu vergleichen und damit Kandidaten zu identifizieren, die möglicherweise die GR-Funktion regulieren. Für die Herstellung hormonresistenter Zellen wurde eine humane Zelllinie hergestellt, die in Anwesenheit von Hormon nicht überleben kann; diese wurde zufallsverteilt mutiert und in GC-haltigem Medium selektiert. Drei hormonresistente Klone konnten die Selektion überleben, einer davon wurde im Detail charakterisiert, dessen Proteinexpressionsmuster mittels 2D-Gelektrophorese mit derjenigen der Ausgangszelllinie verglichen und die unterschiedlich exprimierten Faktoren mittels Tandem-Massenspektrometrie analysiert. Dies führte zur Identifikation von vier Kandidaten: Thioredoxin, hsp27, Reticulocalbin und Cofilin 1, deren Wirkung auf den GR in verschiedenen Zelllinien getestet wurde. Während die ersten drei keinen Einfluss auf den GR hatten, konnte Cofilin als neuer GR-Inhibitor etabliert werden. Cofilin ist gut untersucht als Depolymerisierungsfaktor des Actin-Cytoskeletts, eine Rolle im Signalweg des GRs oder eines anderen Transkriptionsfaktors war bis jetzt jedoch nicht bekannt. Es zeigte sich, dass seine inhibitorische Wirkung auf den GR von seiner Funktion in der Actin-Regulation abhängig war, und ausserdem, dass Cofilin eine Veränderung der intrazellulären Rezeptorverteilung vor Hormongabe bewirkte. In nachfolgenden Experimenten wurde gefunden, dass sowohl die chemische Zerstörung des Actin-Cytoskeletts wie auch die direkte Erhöhung des Anteils an freiem Actin zur GR-Inhibierung und veränderten Rezeptorverteilung führt. Des Weiteren wurde entdeckt, dass erhöhte Mengen an freiem Actin den bekannten GR-Inhibitor c-Jun induzieren, wodurch folgendes Modell aufgestellt wurde: Cofilin erhöht durch seine Actin-Depolymerisierungsfunktion freies Actin, damit wird über einen noch unbekannten Mechanismus c-Jun induziert, welches wiederum den GR inhibiert. Damit wurde über einen zellulären genetischen Screen Cofilin 1 als ein neuer GR-Regulator identifiziert und nachfolgend der inhibierende Wirkungsmechanismus von Cofilin aufgeklärt

Bovine oocyte exposure to perfluorohexane sulfonate (PFHxS) induces phenotypic, transcriptomic, and DNA methylation changes in resulting embryos in vitro

Knowledge on the effects of perfluorohexane sulfonate (PFHxS) on ovarian function is limited. In the current study, we investigated the sensitivity of oocytes to PFHxS during in vitro maturation (IVM), including conse-quences on embryo development at the morphological, transcriptomic, and epigenomic levels. Bovine cumulus-oocyte complexes (COCs) were exposed to PFHxS during 22 h IVM. Following fertilisation, developmental competence was recorded until day 8 of culture. Two experiments were conducted: 1) exposure of COCs to 0.01 mu g mL(-1) -100 mu g mL(-1) PFHxS followed by confocal imaging to detect neutral lipids and nuclei, and 2) exposure of COCs to 0.1 mu g mL(-1) PFHxS followed by analysis of transcriptomic and DNA methylation changes in blastocysts. Decreased oocyte developmental competence was observed upon exposure to & nbsp;>= 40 mu g mL(-1) PFHxS and altered lipid distribution was observed in the blastocysts upon exposure to 1-10 mu g mL(-1) PFHxS (not observed at lower or higher concentrations). Transcriptomic data showed that genes affected by 0.1 mu g mL(-1) PFHxS were enriched for pathways related to increased synthesis and production of reactive oxygen species. Enrichment for peroxisome proliferator-activated receptor-gamma and oestrogen pathways was also observed. Genes linked to DNA methylation changes were enriched for similar pathways. In conclusion, exposure of the bovine oocyte to PFHxS during the narrow window of IVM affected subsequent embryonic development, as reflected by morphological and mo- lecular changes. This suggests that PFHxS interferes with the final nuclear and cytoplasmic maturation of the oocyte leading to decreased developmental competence to blastocyst stage

The biosocial genome? : Interdisciplinary perspectives on environmental epigenetics, health and society

In recent years, research on how the human environment and life-style influence gene expression has generated considerable scientific and public interest. Articles in prominent international newspapers with headlines such as “Why your DNA isn’t your destiny” (Time Magazine in 2010) or “Poverty leaves traces in children’s genome” (Süddeutsche Zeitung in 2016) have drawn public interest to the emerging field of environmental epigenetics. It is a sub-division of the much more heterogeneous research field of epigenetics, which aims to understand how interactions between the environment and the genome can lead to epigenetic modifications that affect gene expression. Environmental epigenetics is often heralded as providing a revolutionary perspective on disease etiology, particularly with regard to so-called ‘life-style diseases’ such as cardiovascular disease or diabetes. It is also often presented as a vital new framework for understanding differences in the susceptibility and resilience to mental illness and the long-term damaging effects of a wide variety of environmental factors. Environmental epigenetics engages with the social context of both individuals and populations. Studies investigate, for example, how socio-economic status, exercise habits, diet or experiences of trauma might influence biological processes at the molecular level. This has created great interest among social scientists and scholars in the humanities as it raises a number of questions at the intersection of the natural sciences, the social sciences and the humanities: for example, how to conceptualize the social environment in a laboratory context. To explore research areas at these intersections and assess the potential social and political implications of environmental epigenetics, international scholars from the life sciences, social sciences and humanities met in January 2017 in Munich, Germany. This article presents some of the main findings from these interdisciplinary discussions. We conclude that environmental epigenetics has great potential for elucidating how human society affects human biology, but we caution against over-simplified translations from social structures to biological processes and vice versa

Statement on Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment

The number of anthropogenic chemicals, manufactured, by-products, metabolites and abiotically formed transformation products, counts to hundreds of thousands, at present. Thus, humans and wildlife are exposed to complex mixtures, never one chemical at a time and rarely with only one dominating effect. Hence there is an urgent need to develop strategies on how exposure to multiple hazardous chemicals and the combination of their effects can be assessed. A workshop, “Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment” was organized in May 2018 together with Joint Research Center in Ispra, EU-funded research projects and Commission Services and relevant EU agencies. This forum for researchers and policy-makers was created to discuss and identify gaps in risk assessment and governance of chemical mixtures as well as to discuss state of the art science and future research needs. Based on the presentations and discussions at this workshop we want to bring forward the following Key Messages: We are at a turning point: multiple exposures and their combined effects require better management to protect public health and the environment from hazardous chemical mixtures. Regulatory initiatives should be launched to investigate the opportunities for all relevant regulatory frameworks to include prospective mixture risk assessment and consider combined exposures to (real-life) chemical mixtures to humans and wildlife, across sectors. Precautionary approaches and intermediate measures (e.g. Mixture Assessment Factor) can already be applied, although, definitive mixture risk assessments cannot be routinely conducted due to significant knowledge and data gaps. A European strategy needs to be set, through stakeholder engagement, for the governance of combined exposure to multiple chemicals and mixtures. The strategy would include research aimed at scientific advancement in mechanistic understanding and modelling techniques, as well as research to address regulatory and policy needs. Without such a clear strategy, specific objectives and common priorities, research, and policies to address mixtures will likely remain scattered and insufficient

Cofilin 1 Is Revealed as an Inhibitor of Glucocorticoid Receptor by Analysis of Hormone-Resistant Cells

Significant knowledge about glucocorticoid signaling has accumulated, yet many aspects remain unknown. We aimed to discover novel factors involved in glucocorticoid receptor regulation that do not necessarily require direct receptor interaction. We achieved this by using a functional genetic screen: a stable cell line which cannot survive hormone treatment was engineered, randomly mutated, and selected in the presence of glucocorticoid. A hormone-resistant clone was analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified and tested as candidates for regulation of the glucocorticoid receptor. An unexpected candidate, cofilin 1, inhibited receptor activity. Cofilin is known to promote actin depolymerization and filament severing. Several experiments suggest that this feature of cofilin is involved in its inhibitory action. Both its actin depolymerization activity and its inhibitory action on the receptor are dependent on its phosphorylation state. Treatment of cells with a cytoskeleton-disrupting agent decreased receptor activity, as did overexpression of actin, particularly a mutant actin that does not polymerize. In addition, overexpression of cofilin and actin as well as chemical cytoskeleton disruption changed the subcellular receptor distribution and upregulated c-Jun, which could constitute the inhibitory mechanism of cofilin. In summary, cofilin represents a novel factor that can cause glucocorticoid resistance

Epigenetics of methylmercury

Purpose of review: Methylmercury (MeHg) is neurotoxic at high levels and particularly affects the developing brain. One proposed mechanism of MeHg neurotoxicity is alteration of the epigenetic programming. In this review, we summarise the experimental and epidemiological literature on MeHg-associated epigenetic changes. Recent findings: Experimental and epidemiological studies have identified changes in DNA methylation following in utero exposure to MeHg, and some of the changes appear to be persistent. A few studies have evaluated associations between MeHg-related changes in DNA methylation and neurodevelopmental outcomes. Experimental studies reveal changes in histone modifications after MeHg exposure, but we lack epidemiological studies supporting such changes in humans. Experimental and epidemiological studies have identified microRNA-related changes associated with MeHg; however, more research is needed to conclude if these changes lead to persistent and toxic effects. Summary: MeHg appears to interfere with epigenetic processes, potentially leading to persistent changes. However, observed associations of mercury with epigenetic changes are as of yet of unknown relevance to neurodevelopmental outcomes

Childhood adversity increases methylation in the GRIN2B gene

Childhood adversity is an early life stressor associated with increased risk of several psychiatric disorders such as depression. Epigenetic changes, primarily DNA methylation, can be affected by early life stress, which in turn might contribute to altered disease susceptibility later in life. One plausible biomarker of early life stress is methylation of the ionotropic glutamate receptor NMDA type subunit 2B (GRIN2B) gene, which has been previously shown to be epigenetically affected by prenatal environmental stressors. Here, we set out to investigate if stress-inducing adversity during childhood is associated with changes in methylation of GRIN2B in adulthood. We studied 186 individuals from a Swedish naturalistic population-based cohort who had provided saliva samples (DNA) as well as information regarding both childhood adversity (CA) and depressive symptoms (dep) (nCA,dep = 41, nCA,no-dep = 56, nno-CA,dep = 40, Nno-CA,no-dep = 49). Methylation at four CpG sites in a regulatory region of GRIN2B was analysed using bisulfite pyrosequencing. Associations for methylation status to childhood adversity and to depression status were investigated using linear regression models. Our study shows that childhood adversity is associated with increased methylation levels of GRIN2B in adulthood, for three of the measured CpGs (p = 0.007, 0.006 and 5 × 10−14). This indicates that GRIN2B methylation is susceptible to early life stress, and that methylation at this gene is persistent over time. No association was found between GRIN2B methylation and depression status. Yet, this does not rule out a role for alterations in GRIN2B methylation for other neuropsychological outcomes not studied here

Prenatal exposure to phthalates and gender-specific play behavior at seven years of age in the SELMA study

Background: A growing body of evidence shows that prenatal exposure to phthalates affects child development. Since many phthalates have been shown to alter endocrine signaling, they may influence reproductive development, neurodevelopment, and child behavior. Indeed, a few studies reported associations between prenatal phthalate exposure and gender-specific play behavior. However, evidence for this relationship is limited, and previous findings are based on single phthalates, while human exposure entails mixtures of chemicals. Objective: We aimed to investigate the associations between prenatal exposure to single phthalates, as well as a phthalate mixture, and gender-specific play behavior. Methods: A total of 715 mother-child pairs from the Swedish Environmental Longitudinal, Mother and Child, Asthma and Allergy (SELMA) study were included. In the median week 10 of pregnancy, phthalate metabolites were measured in urine. Gender-specific play behavior was measured with Preschool Activities Inventory at the age of seven years. Linear and weighted quantile sum regressions were used; data was stratified by sex. Models were adjusted for child and maternal age, maternal education, parental attitudes toward play behavior, and urinary creatinine concentration. Results: For boys, single compound analyses revealed negative associations of prenatal exposure to di-isononyl phthalate (DINP) concentrations with masculine (13 =-1.44; 95% CI =-2.72,-0.16) and composite (13 =-1.43; 95% CI =-2.72,-0.13) scores. Suggestive associations were also observed with a mixture approach identifying DINP as the main contributor of the association of decreased masculine play. Among girls, higher urinary concentrations of 2,4-methyl-7-oxyooctyl-oxycarbonyl-cyclohexane carboxylic acid (MOiNCH) was associated with decreased feminine (13 =-1.59; 95% CI =-2.62,-0.57) and masculine scores (13 =-1.22; 95% CI =-2.14,-0.29), whereas the mixture analyses did not yield conclusive results for girls. Conclusion: Our findings suggest associations of prenatal exposure to DINP with decreased masculine play behavior in boys while the results for girls were not fully conclusive

Impact of endocrine disrupting chemicals on neurodevelopment : the need for better testing strategies for endocrine disruption-induced developmental neurotoxicity

Introduction: Brain development is highly dependent on hormonal regulation. Exposure to chemicals disrupting endocrine signaling has been associated with neurodevelopmental impairment. This raises concern about exposure to the suspected thousands of endocrine disruptors, and has resulted in efforts to improve regulation of these chemicals. Yet, the causal links between endocrine disruption and developmental neurotoxicity, which would be required for regulatory action, are still largely missing. Areas covered: In this review, we illustrate the importance of two endocrine systems, thyroid hormone and retinoic acid pathways, for neurodevelopment. We place special emphasis on TH and RA synthesis, metabolism, and how endocrine disrupting chemicals known or suspected to affect these systems are associated with developmental neurotoxicity. Expert opinion: While it is clear that neurodevelopment is dependent on proper hormonal functioning, and evidence is increasing for developmental neurotoxicity induced by endocrine disrupting chemicals, this is not grasped by current chemical testing. Thus, there is an urgent need to develop test methods detecting endocrine disruption in the context of neurodevelopment. Key to this development is further mechanistic insights on the involvement of endocrine signaling in neurodevelopment as well as increased support to develop and validate new test methods for the regulatory context