Comprehensive mapping of the AOP-Wiki database: identifying biological and disease gaps.

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development.

Innovative tools and methods for toxicity testing within PARC work package 5 on hazard assessment.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC's proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical's Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 'Innovative Tools and methods for Toxicity Testing,' with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD's and PARC's AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

A human iPSC-based in vitro neural network formation assay to investigate neurodevelopmental toxicity of pesticides.

Proper brain development is based on the orchestration of key neurodevelopmental processes (KNDP), including the for­mation and function of neural networks. If at least one KNDP is affected by a chemical, an adverse outcome is expected. To enable a higher testing throughput than the guideline animal experiments, a developmental neurotoxicity (DNT) in vitro testing battery (DNT IVB) comprising a variety of assays that model several KNDPs was set up. Gap analysis revealed the need for a human-based assay to assess neural network formation and function (NNF). Therefore, we established the human NNF (hNNF) assay. A co-culture comprised of human induced pluripotent stem cell (hiPSC)-derived excitatory and inhibitory neurons as well as primary human astroglia was differentiated for 35 days on microelectrode arrays (MEA), and spontaneous electrical activity, together with cytotoxicity, was assessed on a weekly basis after washout of the compounds 24 h prior to measurements. In addition to the characterization of the test system, the assay was challenged with 28 com­pounds, mainly pesticides, identifying their DNT potential by evaluating specific spike-, burst-, and network parameters. This approach confirmed the suitability of the assay for screening environmental chemicals. Comparison of benchmark con­centrations (BMC) with an NNF in vitro assay (rNNF) based on primary rat cortical cells revealed differences in sensitivity. Together with the successful implementation of hNNF data into a postulated stressor-specific adverse outcome pathway (AOP) network associated with a plausible molecular initiating event for deltamethrin, this study suggests the hNNF assay as a useful complement to the DNT IVB.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Progenitor cells derived from gene-engineered human induced pluripotent stem cells as synthetic cancer cell alternatives for in vitro pharmacology.

Limitations in genetic stability and recapitulating accurate physiological disease properties challenge the utility of patient-derived (PD) cancer models for reproducible and translational research. A portfolio of isogenic human induced pluripotent stem cells (hiPSCs) with different pan-cancer relevant oncoprotein signatures followed by differentiation into lineage-committed progenitor cells was genetically engineered. Characterization on molecular and biological level validated successful stable genetic alterations in pluripotency state as well as upon differentiation to prove the functionality of our approach. Meanwhile proposing core molecular networks possibly involved in early dysregulation of stem cell homeostasis, the application of our cell systems in comparative substance testing indicates the potential for cancer research such as identification of augmented therapy resistance of stem cells in response to activation of distinct oncogenic signatures.

Interaction between magnesium and methylglyoxal in diabetic polyneuropathy and neuronal models.

OBJECTIVE: The lack of effective treatments against diabetic sensorimotor polyneuropathy demands the search for new strategies to combat or prevent the condition. Because reduced magnesium and increased methylglyoxal levels have been implicated in the development of both type 2 diabetes and neuropathic pain, we aimed to assess the putative interplay of both molecules with diabetic sensorimotor polyneuropathy. METHODS: In a cross-sectional study, serum magnesium and plasma methylglyoxal levels were measured in recently diagnosed type 2 diabetes patients with (n = 51) and without (n = 184) diabetic sensorimotor polyneuropathy from the German Diabetes Study baseline cohort. Peripheral nerve function was assessed using nerve conduction velocity and quantitative sensory testing. Human neuroblastoma cells (SH-SY5Y) and mouse dorsal root ganglia cells were used to characterize the neurotoxic effect of methylglyoxal and/or neuroprotective effect of magnesium. RESULTS: Here, we demonstrate that serum magnesium concentration was reduced in recently diagnosed type 2 diabetes patients with diabetic sensorimotor polyneuropathy and inversely associated with plasma methylglyoxal concentration. Magnesium, methylglyoxal, and, importantly, their interaction were strongly interrelated with methylglyoxal-dependent nerve dysfunction and were predictive of changes in nerve function. Magnesium supplementation prevented methylglyoxal neurotoxicity in differentiated SH-SY5Y neuron-like cells due to reduction of intracellular methylglyoxal formation, while supplementation with the divalent cations zinc and manganese had no effect on methylglyoxal neurotoxicity. Furthermore, the downregulation of mitochondrial activity in mouse dorsal root ganglia cells and consequently the enrichment of triosephosphates, the primary source of methylglyoxal, resulted in neurite degeneration, which was completely prevented through magnesium supplementation. CONCLUSIONS: These multifaceted findings reveal a novel putative pathophysiological pathway of hypomagnesemia-induced carbonyl stress leading to neuronal damage and merit further investigations not only for diabetic sensorimotor polyneuropathy but also other neurodegenerative diseases associated with magnesium deficiency and impaired energy metabolism.

Different Calculation Strategies Are Congruent in Determining Chemotherapy Resistance of Brain Tumors In Vitro.

In cancer pharmacology, a drug candidate's therapeutic potential is typically expressed as its ability to suppress cell growth. Different methods in assessing the cell phenotype and calculating the drug effect have been established. However, inconsistencies in drug response outcomes have been reported, and it is still unclear whether and to what extent the choice of data post-processing methods is responsible for that. Studies that systematically examine these questions are rare. Here, we compare three established calculation methods on a collection of nine in vitro models of glioblastoma, exposed to a library of 231 clinical drugs. The therapeutic potential of the drugs is determined on the growth curves, using growth inhibition 50% (GI50) and point-of-departure (PoD) as the criteria. An effect is detected on 36% of the drugs when relying on GI50 and on 27% when using PoD. For the area under the curve (AUC), a threshold of 9.5 or 10 could be set to discriminate between the drugs with and without an effect. GI50, PoD, and AUC are highly correlated. The ranking of substances by different criteria varies somewhat, but the group of the top 20 substances according to one criterion typically includes 17-19 top candidates according to another. In addition to generating preclinical values with high clinical potential, we present off-target appreciation of top substance predictions by interrogating the drug response data of non-cancer cells in our calculation technology.

Rapalink-1 Targets Glioblastoma Stem Cells and Acts Synergistically with Tumor Treating Fields to Reduce Resistance against Temozolomide.

Glioblastoma (GBM) is a lethal disease with limited clinical treatment options available. Recently, a new inhibitor targeting the prominent cancer signaling pathway mTOR was discovered (Rapalink-1), but its therapeutic potential on stem cell populations of GBM is unknown. We applied a collection of physiological relevant organoid-like stem cell models of GBM and studied the effect of RL1 exposure on various cellular features as well as on the expression of mTOR signaling targets and stem cell molecules. We also undertook combination treatments with this agent and clinical GBM treatments tumor treating fields (TTFields) and the standard-of-care drug temozolomide, TMZ. Low nanomolar (nM) RL1 treatment significantly reduced cell growth, proliferation, migration, and clonogenic potential of our stem cell models. It acted synergistically to reduce cell growth when applied in combination with TMZ and TTFields. We performed an in silico analysis from the molecular data of diverse patient samples to probe for a relationship between the expression of mTOR genes, and mesenchymal markers in different GBM cohorts. We supported the in silico results with correlative protein data retrieved from tumor specimens. Our study further validates mTOR signaling as a druggable target in GBM and supports RL1, representing a promising therapeutic target in brain oncology.

Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells.

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection. Differentiation of transduced hiPSCs gives insight into better understanding cancer formation in different tissues and may be a useful tool for genetic or pharmacologic screening assays. © 2019 The Authors. Basic Protocol 1: Production and concentration of third-generation lentivirus Support Protocol 1: Cloning of gene of interest into modulation vector Support Protocol 2: Preparation of DMEM GlutaMAX™ with 10% fetal bovine serum and 1% penicillin-streptomycin Basic Protocol 2: Transduction of human induced pluripotent stem cells and selection of positively transfected cells Support Protocol 3: Preparation of Matrigel® -coated plates Support Protocol 4: Preparation of mTeSR™1 medium.

Arsenite interrupts neurodevelopmental processes of human and rat neural progenitor cells: The role of reactive oxygen species and species-specific antioxidative defense.

Arsenic exposure disturbs brain development in humans. Although developmental neurotoxicity (DNT) of arsenic has been studied in vivo and in vitro, its mode-of-action (MoA) is not completely understood. Here, we characterize the adverse neurodevelopmental effects of sodium arsenite on developing human and rat neural progenitor cells (hNPC, rNPC). Moreover, we analyze the involvement of reactive oxygen species (ROS) and the role of the glutathione (GSH)-dependent antioxidative defense for arsenite-induced DNT in a species-specific manner. We determined IC50 values for sodium arsenite-dependent (0.1-10 µM) inhibition of hNPC and rNPC migration (6.0 µM; >10 µM), neuronal (2.7 µM; 4.4 µM) and oligodendrocyte (1.1 µM; 2.0 µM) differentiation. ROS involvement was studied by quantifying the expression of ROS-regulated genes, measuring glutathione (GSH) levels, inhibiting GSH synthesis and co-exposing cells to the antioxidant N-acetylcysteine. Arsenite reduces NPC migration, neurogenesis and oligodendrogenesis of differentiating hNPC and rNPC at sub-cytotoxic concentrations. Species-specific arsenite cytotoxicity and induction of antioxidative gene expression is inversely related to GSH levels with rNPC possessing >3-fold the amount of GSH than hNPC. Inhibition of GSH synthesis increased the sensitivity towards arsenite in rNPC > hNPC. N-acetylcysteine antagonized arsenite-mediated induction of HMOX1 expression as well as reduction of neuronal and oligodendrocyte differentiation in hNPC suggesting involvement of oxidative stress in arsenite DNT. hNPC are more sensitive towards arsenite-induced neurodevelopmental toxicity than rNPC, probably due to their lower antioxidative defense capacities. This species-specific MoA data might be useful for adverse outcome pathway generation and future integrated risk assessment strategies concerning DNT.

Comparison of migration disturbance potency of epigallocatechin gallate (EGCG) synthetic analogs and EGCG PEGylated PLGA nanoparticles in rat neurospheres.

Epigallocatechin gallate (EGCG), the main catechin of green tea, is described to have potential health benefits in several fields like oncology, neurology or cardiology. Currently, it is also under pre-clinical investigation as a potential therapeutic or preventive treatment during pregnancy against developmental adverse effects induced by toxic substances. However, the safety of EGCG during pregnancy is unclear due to its proven adverse effects on neural progenitor cells' (NPCs) migration. As lately several strategies have arisen to generate new therapeutic agents derived from EGCG, we have used the rat 'Neurosphere Assay' to characterize and compare the effects of EGCG structurally related compounds and EGCG PEGylated PLGA nanoparticles on a neurodevelopmental key event: NPCs migration. Compounds structurally-related to EGCG induce the same pattern of NPCs migration alterations (decreased migration distance, decreased formation of migration corona, chaotic orientation of cellular processes and decreased migration of neurons at higher concentrations). The potency of the compounds does not depend on the number of galloyl groups, and small structure variations can imply large potency differences. Due to their lower toxicity observed in vitro in NPCs, 4,4'-bis[(3,4,5-trihydroxybenzoyl)oxy]-1,1'-biphenyl and EGCG PEGylated PLGA nanoparticles are suggested as potential future therapeutic or preventive alternatives to EGCG during prenatal period.

Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia.

In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP's informed Integrated Approach for Testing and Assessment (IATA).

Differential cellular metabolite alterations in HaCaT cells caused by exposure to the aryl hydrocarbon receptor-binding polycyclic aromatic hydrocarbons chrysene, benzo[a]pyrene and dibenzo[a,l]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the human environment. Since they are present in crude oilfractions used for the production of rubber and plastics, consumers may come into direct dermal contacts with these compounds (e.g., via tool handles) on a daily basis. Some individual PAHs are identified as genotoxic mutagens thereby prompting particular toxicological and environmental concern. Among this group, benzo[a]pyrene (BAP) constitutes a model carcinogen which is also used as reference compound for risk assessment purposes. It acts as a strong agonist of the aryl hydrocarbon receptor (AHR) and becomes metabolically activated toward mutagenic and carcinogenic intermediates by cytochrome P450-dependent monooxygenases (CYPs). While BAP has been exhaustively characterized with regard to its toxicological properties, there is much less information available for other PAHs. We treated an AHR-proficient immortal human keratinocyte cell line (i.e., HaCaT) with three selected PAHs: BAP, chrysene (CRY) and dibenzo[a,l]pyrene (DALP). Compound-mediated alterations of endogenous metabolites were investigated by an LC-MS/MS-based targeted approach. To examine AHR-dependent changes of the measured metabolites, AHR-deficient HaCaT knockdown cells (AHR-KD) were used for comparison. Our results reveal that 24 metabolites are sufficient to separate the PAH-exposed cells from untreated controls by application of a multivariate model. Alterations in the metabolomics profiles caused by each PAH show influences on the energy and lipid metabolism of the cells indicating reduced tricarboxylic acid (TCA) cycle activity and ß-oxidation. Up-regulation of sphingomyelin levels after exposure to BAP and DALP point to pro-apoptotic processes caused by these two potent PAHs. Our results suggest that in vitro metabolomics can serve as tool to develop bioassays for application in hazard assessment.

Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

Activation of the aryl hydrocarbon receptor by the widely used Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2).

Small molecular weight protein kinase inhibitors are frequently used tools to unravel the complex network of cellular signal transduction under certain physiological and pathophysiological conditions. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) is a widely used compound to block the activity of Src family kinases, the major group of non-receptor tyrosine kinases, which trigger multiple cellular signaling pathways. Here, we show that PP2 induces cytochrome P450 1A1 mRNA expression and enzyme activity in a dose-dependent manner in human HepG2 hepatoma cells and NCTC 2544 keratinocytes. By means of reporter gene assays, RNA interference, electrophoretic mobility shift assay, and competitive ligand-binding assay, we further demonstrate that PP2 is a ligand for the aryl hydrocarbon receptor (AHR), an intracellular chemosensor that regulates xenobiotic metabolism, environmental stress responses, and immune functions. Upon ligand-dependent activation, the AHR translocates into the nucleus and dimerizes with the AHR nuclear translocator (ARNT) to modulate the expression of its target genes. In addition, AHR activation is frequently accompanied by an activation of the tyrosine kinase c-Src, resulting in stimulation of cell-surface receptors and downstream signal transduction. As PP2 activates the AHR/ARNT pathway by simultaneously blocking c-Src-mediated alternative signaling routes, this compound may be a suitable tool to study the contribution of the different AHR-dependent signaling pathways to biological processes and adverse outcomes. On the other hand, the unexpected property of PP2 to stimulate AHR/ARNT signaling should be carefully taken into account in future investigations in order to avoid a false interpretation of experimental results and molecular interrelations.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

The Aryl hydrocarbon receptor is involved in UVR-induced immunosuppression.

UVR suppresses the immune system through the induction of regulatory T cells (Tregs). UVR-induced DNA damage has been recognized as the major molecular trigger involved, as reduction of DNA damage by enhanced repair prevents the compromise to the immune system by UVR. Nevertheless, other signaling events may also be involved. The aryl hydrocarbon receptor (AhR) was identified as another target for UVR, as UVR activates the AhR and certain UVR effects were not detected in AhR-deficient cells. We studied whether the AhR is involved in UVR-induced local immunosuppression and whether similar effects can be induced by AhR agonists. The AhR antagonist 3-methoxy-4-nitroflavone reduced UVR-mediated immunosuppression and the induction of Tregs in murine contact hypersensitivity (CHS). Conversely, activation of the AhR by the agonist 4-n-nonylphenol (NP) suppressed the induction of CHS and induced antigen-specific Tregs similar to UVR. This was further confirmed in AhR knockout mice in which UVR- and NP-induced immunosuppression were significantly reduced. Together, this indicates that the AhR is involved in mediating UVR-induced immunosuppression. Activation of the AhR might represent an alternative to modulate the immune system in a similar manner like UVR but without causing the adverse effects of UVR, including DNA damage.

Role of the aryl hydrocarbon receptor in tobacco smoke extract-induced matrix metalloproteinase-1 expression.

Findings from large epidemiologic studies indicate that there is a link between smoking and extrinsic skin ageing. We previously reported that matrix metalloproteinases (MMPs) mediate connective tissue damage in skin exposed to tobacco smoke extracts. Tobacco smoke contains more than 3800 constituents, including numerous water-insoluble polycyclic aromatic hydrocarbons (PAHs) that trigger aryl hydrocarbon receptor (AhR) signalling pathways. To analyse the molecular mechanisms involved in tobacco smoke-induced skin ageing, we exposed primary human fibroblasts and keratinocytes to tobacco smoke extracts. Hexane- and water-soluble tobacco smoke extracts significantly induced MMP-1 mRNA in both human cultured fibroblasts and keratinocytes in a dose-dependent manner. To clarify the involvement of the AhR pathway, we used a stable AhR-knockdown HaCaT cell line. AhR knockdown abolished the increased transcription of the AhR-dependent genes CYP1A1/CYP1B1 and MMP-1 induced by either of the tobacco smoke extracts. Furthermore, the tobacco smoke extracts induced 7-ethoxyresorufin-O-deethylase activity, which was almost completely abolished by AhR knockdown. Likewise, treating fibroblasts with AhR pathway inhibitors, that is, the flavonoids 3-methoxy-4-nitroflavone and α-naphthoflavone, blocked the expression of CYP1B1 and MMP-1. These findings suggest that the tobacco smoke extracts induce MMP-1 expression in human fibroblasts and keratinocytes via activation of the AhR pathway. Thus, the AhR pathway may be pathogenetically involved in extrinsic skin ageing.

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes.

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.

The AhR-Nrf2 pathway in keratinocytes: on the road to chemoprevention?

The ligand-activated transcription factor AhR mediates the cutaneous stress response toward a variety of environmental noxae and is therefore currently of interest for modern preventive medicine. In this issue, Tsuji et al. identify the antifungal agent ketoconazole as an inducer of AhR signaling and the Nrf2 antioxidant response in human keratinocytes. Ketoconazole-stimulated nuclear translocation of Nrf2 and its cytoprotective effects against oxidative stress strongly depend on a functional AhR. This newly identified AhR-Nrf2 pathway opens up new opportunities to prevent and treat inflammatory skin diseases.