An adverse outcome pathway based in vitro characterization of novel flame retardants-induced hepatic steatosis

A wide range of novel replacement flame retardants (nFRs) is consistently detected in increasing concentrations in the environment and human matrices. Evidence suggests that nFRs exposure may be associated with disruption of the endocrine system, which has been linked with the etiology of various metabolic disorders, including nonalcoholic fatty liver disease (NAFLD). NAFLD is a multifactorial disease characterized by the uncontrolled accumulation of fats (lipids) in the hepatocytes and involves multiple-hit pathogenesis, including exposure to occupational and environmental chemicals such as organophosphate flame retardants (OPFRs). In the present study we aimed to investigate the potential mechanisms of the nFRs-induced hepatic steatosis in the human liver cells. In this study, we employed an in vitro bioassay toolbox to assess the key events (KEs) in the proposed adverse outcome pathways (AOP) (s) for hepatic steatosis. We examined nine nFRs using AOP- based in vitro assays measuring KEs such as lipid accumulation, mitochondrial dysfunction, gene expression, and in silico approach to identify the putative molecular initiating events (MIEs). Our findings suggest that several tested OPFRs induced lipid accumulation in human liver cell culture. Tricresyl phosphate (TMPP), triphenyl phosphate (TPHP), tris(1,3-dichloropropyl) phosphate (TDCIPP), and 2-ethylhexyl diphenyl phosphate (EHDPP) induced the highest lipid accumulation by altering the expression of genes encoding hepatic de novo lipogenesis and mitochondrial dysfunction depicted by decreased cellular ATP production. Available in vitro data from ToxCast and in silico molecular docking suggests that pregnane X receptor (PXR) and peroxisome proliferator-activated receptor gamma (PPAR gamma) could be the molecular targets for the tested nFRs. The study identifies several nFRs, such as TMPP and EHDPP, TPHP, and TDCIPP, as potential risk factor for NAFLD and advances our understanding of the mechanisms involved, demonstrating the utility of an AOP-based strategy for screening and prioritizing chemicals and elucidating the molecular mechanisms of toxicity

Flame Retardants-Mediated Interferon Signaling in the Pathogenesis of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a growing concern worldwide, affecting 25% of the global population. NAFLD is a multifactorial disease with a broad spectrum of pathology includes steatosis, which gradually progresses to a more severe condition such as nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and eventually leads to hepatic cancer. Several risk factors, including exposure to environmental toxicants, are involved in the development and progression of NAFLD. Environmental factors may promote the development and progression of NAFLD by various biological alterations, including mitochondrial dysfunction, reactive oxygen species production, nuclear receptors dysregulation, and interference in inflammatory and immune-mediated signaling. Moreover, environmental contaminants can influence immune responses by impairing the immune system's components and, ultimately, disease susceptibility. Flame retardants (FRs) are anthropogenic chemicals or mixtures that are being used to inhibit or delay the spread of fire. FRs have been employed in several household and outdoor products; therefore, human exposure is unavoidable. In this review, we summarized the potential mechanisms of FRs-associated immune and inflammatory signaling and their possible contribution to the development and progression of NAFLD, with an emphasis on FRs-mediated interferon signaling. Knowledge gaps are identified, and emerging pharmacotherapeutic molecules targeting the immune and inflammatory signaling for NAFLD are also discussed

Application of AOPs to assist regulatory assessment of chemical risks – Case studies, needs and recommendations

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.European Commission 733032 857560 101057014Ministry of Education, Youth and Sports by the RECETOX Research Infrastructure LM2018121OP RDE project CETOCOEN Excellence CZ.02.1.01/0.0/0.0/17_043/0009632Japan Agency for Medical Research and Development (AMED) JP21mk0101216 JP22mk0101216Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) 21K1213

Endocrine disrupting potential of replacement flame retardants-Review of current knowledge for nuclear receptors associated with reproductive outcomes

Background and aim: Endocrine disrupting chemicals (EDCs) constitute a major public health concern because they can induce a large spectrum of adverse effects by interfering with the hormonal system. Rapid identification of potential EDCs using in vitro screenings is therefore critical, particularly for chemicals of emerging concerns such as replacement flame retardants (FRs). The review aimed at identifying (1) data gaps and research needs regarding endocrine disrupting (ED) properties of replacement FRs and (2) potential EDCs among these emerging chemicals. Methods: A systematic search was performed from open literature and ToxCast/Tox21 programs, and results from in vitro tests on the activities of 52 replacement FRs towards five hormone nuclear receptors (NRs) associated with reproductive outcomes (estrogen, androgen, glucocorticoid, progesterone, and aryl hydrocarbon receptors) were compiled and organized into tables. Findings were complemented with information from structure-based in silico model predictions and in vivo information when relevant. Results: For the majority of the 52 replacement FRs, experimental in vitro data on activities towards these five NRs were either incomplete (15 FRs) or not found (24 FRs). Within the replacement FRs for which effect data were found, some appeared as candidate EDCs, such as triphenyl phosphate (TPhP) and tris(1,3-dichloropropyl) phosphate (TDCIPP). The search also revealed shared ED profiles. For example, anti-androgenic activity was reported for 19 FRs and predicted for another 21 FRs. Discussion: This comprehensive review points to critical gaps in knowledge on ED potential for many replacement FRs, including chemicals to which the general population is likely exposed. Although this review does not cover all possible characteristics of ED, it allowed the identification of potential EDCs associated with reproductive outcomes, calling for deeper evaluation and possibly future regulation of these chemicals. By identifying shared ED profiles, this work also raises concerns for mixture effects since the population is co-exposed to several FRs and other chemicals

Analysis of a key regulatory region upstream of the Myf5 gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.This work in M.B.'s laboratory was supported by the Pasteur Institute and the CNRS and by grants from the ACI Integrative Biology Programme of the MJER, the AFM and the European Community (QLK3-CT-99/02). J.H. benefited from fellowships from ARC and the AFM, L.B. from funding from the MJER, and T.C. from fellowships from NIH and the AFM. The work in P.R.'s laboratory was supported by a grant from The Institute of Cancer Research.Peer reviewe

lessons learned and future trends

Funding Information: Thanks are due to CESAM (UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020), and ToxOmics (UIDB/00009/2020; UIDP/00009/2020), through national funds. L.B. thanks Research Infrastructure RECETOX RI (No. LM2018121), financed by the Ministry of Education, Youth and Sports, and the project CETOCOEN EXCELLENCE (No. CZ.02.1.01/0.0/0.0/17_043/0009632) for a supportive background of the European Union’s Horizon 2020 research and innovation program under grant agreement No. 857560, and to the HBM4EU, under grant agreement No. 733032. This publication reflects only the authors’ view and the European Commission is not responsible for any use that may be made of the information it contains. Funding Information: This study was co-funded by the HBM4EU, which has received funding from the European Union’s research and innovation programme Horizon 2020 under grant agreement No. 733032, and from the authors’ institutions. Publisher Copyright: © 2022 by the authors.Mycotoxins are natural metabolites produced by fungi that contaminate food and feed worldwide. They can pose a threat to human and animal health, mainly causing chronic effects, e.g., immunotoxic and carcinogenic. Due to climate change, an increase in European population exposure to mycotoxins is expected to occur, raising public health concerns. This urges us to assess the current human exposure to mycotoxins in Europe to allow monitoring exposure and prevent future health impacts. The mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1) were considered as priority substances to be studied within the European Human Biomonitoring Initiative (HBM4EU) to generate knowledge on internal exposure and their potential health impacts. Several policy questions were addressed concerning hazard characterization, exposure and risk assessment. The present article presents the current advances attained under the HBM4EU, research needs and gaps. Overall, the knowledge on the European population risk from exposure to DON was improved by using new harmonised data and a newly derived reference value. In addition, mechanistic information on FB1 was, for the first time, organized into an adverse outcome pathway for a congenital anomaly. It is expected that this knowledge will support policy making and contribute to driving new Human Biomonitoring (HBM) studies on mycotoxin exposure in Europe.publishersversionpublishe

Current Advances, Research Needs and Gaps in Mycotoxins Biomonitoring under the HBM4EU-Lessons Learned and Future Trends

ReviewThis article belongs to the Special Issue Human Biomonitoring and Risk Assessment of Mycotoxins.Mycotoxins are natural metabolites produced by fungi that contaminate food and feed worldwide. They can pose a threat to human and animal health, mainly causing chronic effects, e.g., immunotoxic and carcinogenic. Due to climate change, an increase in European population exposure to mycotoxins is expected to occur, raising public health concerns. This urges us to assess the current human exposure to mycotoxins in Europe to allow monitoring exposure and prevent future health impacts. The mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1) were considered as priority substances to be studied within the European Human Biomonitoring Initiative (HBM4EU) to generate knowledge on internal exposure and their potential health impacts. Several policy questions were addressed concerning hazard characterization, exposure and risk assessment. The present article presents the current advances attained under the HBM4EU, research needs and gaps. Overall, the knowledge on the European population risk from exposure to DON was improved by using new harmonised data and a newly derived reference value. In addition, mechanistic information on FB1 was, for the first time, organized into an adverse outcome pathway for a congenital anomaly. It is expected that this knowledge will support policy making and contribute to driving new Human Biomonitoring (HBM) studies on mycotoxin exposure in Europe.This study was co-funded by the HBM4EU, which has received funding from the European Union’s research and innovation programme Horizon 2020 under grant agreement No. 733032, and from the authors’ institutions. Thanks are due to CESAM(UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020), and ToxOmics (UIDB/00009/2020; UIDP/00009/2020), through national funds. L.B. thanks Research Infrastructure RECETOX RI (No. LM2018121), financed by the Ministry of Education, Youth and Sports, and the project CETOCOEN EXCELLENCE (No. CZ.02.1.01/0.0/0.0/17_043/0009632) for a supportive background of the European Union’s Horizon 2020 research and innovation program under grant agreement No. 857560, and to the HBM4EU, under grant agreement No. 733032.info:eu-repo/semantics/publishedVersio

Mycotoxins human biomonitoring under HBM4EU: the case study of deoxynivalenol and fumonisin B1

Baseado no artigo publicado: Alvito P, A ssunção RM, Bajard L, Mar tins C, Mengelers M, Hans Mol, Van den Brand A, Namorado S, Vasco E, Viegas S, Silva M. Current advances, research needs and gaps in Mycotoxins Biomonitoring under HBM4EU – lessons learned and future t rends. Toxins (Basel ). 2022;14(12):826. https://doi.org/10.3390/toxins14120826As micotoxinas são toxinas naturais produzidas por fungos, apresentando efeitos tóxicos para o homem e para os animais. Reconhece-se, atualmente, que as alterações climáticas terão impacto na distribuição geográfica de algumas espécies de fungos produtores de micotoxinas o que se traduzirá, previsivelmente, num aumento da exposição humana a estes compostos. Pelas razões descritas, urge conhecer a atual exposição a micotoxinas na Europa, com vista à sua futura monitorização e à prevenção/redução do seu impacto na saúde. No âmbito da Iniciativa Europeia em Biomonitorização Humana (HBM4EU) consideraram-se as micotoxinas desoxinivalenol (DON) e fumonisina B1 (FB1) como substâncias prioritárias, tendo sido abordadas várias questões relativas à avaliação da exposição humana e o potencial risco para a saúde. No presente artigo, apresentam-se as questões identificadas como mais importantes, respostas obtidas e perspetivas futuras. Os resultados confirmaram a exposição humana a DON, tendo sido obtidos, pela primeira vez, dados harmonizados de exposição ao nível europeu e derivado um valor de referência para essa exposição. Foi ainda proposto, pela primeira vez no HBM4EU, uma sucessão de eventos biológicos baseados no mecanismo de ação da FB1 que permitiu associar a exposição durante a gravidez ao desenvolvimento de defeitos do tubo neural no feto. Espera-se que estes resultados possam contribuir para uma futura monitorização da exposição a micotoxinas na Europa e para melhorar a avaliação de risco destas substâncias.Mycotoxins are natural low-molecular-weight toxins produced by fungal species that can be toxic for humans and animals. Under the climate change scenario, some fungal species might shift their geographical distribution in response to global warming, leading to changes in the pattern of mycotoxin occurrence and, thus, increasing the risk of human mycotoxin exposure. For this reason, it urges to assess the current human exposure to mycotoxins in Europe, to monitor internal exposure and prevent future health impact. The European Human Biomonitoring Initiative (HBM4EU), running from 2017 to 2022, was set to generate knowledge on internal exposure and their potential health impacts. Within this initiative, the mycotoxins deoxynivalenol (DON) and fumonisin B1 ( FB1) were considered as priority substances to be studied and several policy questions were addressed concerning their risk assessment. The present paper presents policy questions identified within HBM4EU for these mycotoxins, answers obtained and future perspectives. The exposure of the general European population to DON was confirmed using new harmonized data and a reference guidance value for human biomonitoring was set for the first time. Additionaly, an adverse outcome pathway for neural tube defects was proposed for FB1 for the first time. Hopefully these findings may contribute to a more accurate risk assessment of European population´s exposure to mycotoxins.O projeto HBM4EU recebeu financiamento do programa de investigação e inovação da União Europeia Horizonte 2020 sob o contrato de concessão n.º 733032 e das instituições em que os autores desenvolvem as suas atividades. Os autores agradecem à FCT/MCTES pelo financiamento com fundos nacionais relativos ao CESAM (UIDP/50017/2020+UIDB/ DB/50017/2020) e ToxOmics (UIDB/00009/2020; UIDP/00009/ 2020).info:eu-repo/semantics/publishedVersio

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge

ReviewHuman biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2′-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.Supported by the European Union's Horizon 2020 research and innovation program projects HBM4EU [grant number 733032]info:eu-repo/semantics/publishedVersio

A systematic approach synergizing toxicological and epidemiological knowledge

Funding Information: This work was supported by the European Union's Horizon 2020 research and innovation program projects HBM4EU [grant number 733032 ]. Authors acknowledge the editorial assistance of Richard Davies. Publisher Copyright: © 2023 The Author(s)Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2′-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.publishersversionpublishe