Ellen Fritsche

Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmatic target for ultraviolet B radiation

UVB radiation-induced signaling in mammalian cells involves two major pathways: one that is initiated through the generation of DNA photoproducts in the nucleus and a second one that occurs independently of DNA damage and is characterized by cell surface receptor activation. The chromophore for the latter one has been unknown. Here, we report that the UVB response involves tryptophan as a chromophore. We show that through the intracellular generation of photoproducts, such as the arylhydrocarbon receptor (AhR) ligand 6-formylindolo[3,2-b]carbazole, signaling events are initiated, which are transferred to the nucleus and the cell membrane via activation of the cytoplasmatic AhR. Specifically, AhR activation by UVB leads to (i) transcriptional induction of cytochrome P450 1A1 and (ii) EGF receptor internalization with activation of the EGF receptor downstream target ERK1/2 and subsequent induction of cyclooxygenase-2. The role of the AhR in the UVB stress response was confirmed in vivo by studies employing AhR KO mice.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Polybrominated diphenyl ethers induce developmental neurotoxicity in a human in vitro model: evidence for endocrine disruption.

Background Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative flame retardants, which are found in rising concentrations in human tissues. They are of concern for human health because animal studies have shown that they possess the potential to be developmentally neurotoxic. Objective Because there is little knowledge of the effects of PBDEs on human brain cells, we investigated their toxic potential for human neural development in vitro. Moreover, we studied the involvement of thyroid hormone (TH) disruption in the effects caused by PBDEs. Methods We used the two PBDE congeners BDE-47 and BDE-99 (0.1–10 μM), which are most prominent in human tissues. As a model of neural development, we employed primary fetal human neural progenitor cells (hNPCs), which are cultured as neurospheres and mimic basic processes of brain development in vitro: proliferation, migration, and differentiation. Results PBDEs do not disturb hNPC proliferation but decrease migration distance of hNPCs. Moreover, they cause a reduction of differentiation into neurons and oligodendrocytes. Simultaneous exposure with the TH receptor (THR) agonist triiodothyronine rescues these effects on migration and differentiation, whereas the THR antagonist NH-3 does not exert an additive effect. Conclusion PBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

Human neurospheres as three-dimensional cellular systems for developmental neurotoxicity testing.

Background Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the “3 Rs” (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. Objectives The goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells. Methods We established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells. Results We found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress. Conclusions Human neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.

Polychlorinated biphenyls disturb differentiation of normal human neural progenitor cells: clue for involvement of thyroid hormone receptors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental chemicals that accumulate in adipose tissues over the food chain. Epidemiologic studies have indicated that PCBs influence brain development. Children who are exposed to PCBs during development suffer from neuropsychologic deficits such as a lower full-scale IQ (intelligence quotient), reduced visual recognition memory, and attention and motor deficits. The mechanisms leading to these effects are not fully understood. It has been speculated that PCBs may affect brain development by interfering with thyroid hormone (TH) signaling. Because most of the data are from animal studies, we established a model using primary normal human neural progenitor (NHNP) cells to determine if PCBs interfere with TH-dependent neural differentiation. NHNP cells differentiate into neurons, astrocytes, and oligodendrocytes in culture, and they express a variety of drug metabolism enzymes and nuclear receptors. Like triiodothyronine (T3), treatment with the mono-ortho-substituted PCB-118 (2,3′,4,4′,5-pentachlorobiphenyl; 0.01–1 μM) leads to a dose-dependent increase of oligodendrocyte formation. This effect was congener specific, because the coplanar PCB-126 (3,3′,4,4′,5-pentachlorobiphenyl) had no effect. Similar to the T3 response, the PCB-mediated effect on oligodendrocyte formation was blocked by retinoic acid and the thyroid hormone receptor antagonist NH-3. These results suggest that PCB-118 mimics T3 action via the TH pathway.

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy, predictivity and sensitivity of the screening assays. In recent years neuroprogenitor cells from rodents and humans have become more widely available and may offer useful models having advantages over primary neuronal cultures and/or transformed cell lines. To date, these models have been utilized in only a limited number of toxicity studies. This review summarizes the state of the science regarding stem and neuroprogenitor models that could be used for screening assays, provides researchers in this field with examples of how these cells have been utilized to date, and discusses the advantages, limitations and knowledge gaps regarding these models. Data are available from both rodent and human stem and neuroprogenitor cell models that indicate that these models will be a valid and useful tool for developmental neurotoxicity testing. Full potential of these models will only be achieved following advances in neurobiology that elucidate differentiation pathways more clearly, and following further evaluation of larger sets of developmentally neurotoxic and non-toxic chemicals to define the sensitivity and predictivity of assays based on stem or progenitor cell models.

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I)

Abstract:  The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first‐pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic‐metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S‐transferase, UDP‐glucuronosyltransferase and N‐acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI‐200), immortalized keratinocyte‐based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI‐200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

Advancing the science of developmental neurotoxicity (DNT): Testing for better safety evaluation

test Guidelines OPPtS 8706300 on DNt (US ePA, 1998) and in 2007 the Organization for economic Cooperation and Development (OeCD) endorsed a new OeCD DNt test Guideline 426 (OeCD, 2007). these guidelines are largely based on animal studies and are used as higher tiered, triggered tests based on structure activity relationships or evidence of neurotoxicity in standard adult, developmental, or reproduction studies (Makris et al., 2009). experts at the conference stated that these in vivo tests are unsuitable for screening large numbers of chemicals for many reasons including low throughput, high cost, and questions regarding reliability. there was also consensus that new, reliable, and efficient screening and assessment tools are needed for better identification, prioritization, and evaluation of chemicals with the potential to induce developmental neurotoxicity. the information obtained from these screening studies will likely also help to refine animal tests and to inform epidemiological studies.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

Abstract A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Analysis of the Transcriptional Regulation and Molecular Function of the Aryl Hydrocarbon Receptor Repressor in Human Cell Lines

The aryl hydrocarbon receptor repressor (AhRR) is a member of the aryl hydrocarbon receptor (AhR) signaling cascade, which mediates dioxin toxicity and is involved in regulation of cell growth and differentiation. The AhRR was described as a feedback modulator, which counteracts AhR-dependent gene expression. We investigated the molecular mechanisms of transcriptional regulation of the human AhRR by cloning its regulatory DNA region located in intron I of the AhRR. By means of reporter gene analyses and generation of deletion variants, we identified a functional, 3-methylcholanthrene-sensitive xenobiotic response element (XRE) site. Chromatin immunoprecipitation analyses revealed that the AhRR binds to this XRE, displaying an autoregulatory loop of AhRR expression. In addition we show that an adjacent GC-box is of functional relevance for AhRR transcription, since blocking of this GC-box resulted in a decrease of constitutive and inducible AhRR gene activity. The differences in constitutive AhRR mRNA level observed in HepG2, primary fibroblast, and HeLa cells are directly correlated with CYP1A1 inducibility. We show that the nonresponsiveness of high AhRR-expressing cells toward AhR-agonists is associated with a constitutive binding of the AhRR to XRE sites of CYP1A1. Treatment with the histone deacetylase inhibitor sodium butyrate restored the responsiveness of CYP1A1 in these cell lines, due to the dissociation of AhRR from the XREs. Furthermore, transient AhRR mRNA silencing in untreated HeLa cells was accompanied by an increase of basal CYP1A1 expression, pointing to a constitutive role of the AhRR in regulation of CYP1A1. The functional relevance of the AhRR in high AhRR-expressing primary fibroblasts is discussed.