Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context

A mode-of-action ontology model for safety evaluation of chemicals: outcome of a series of workshops on repeated dose toxicity

Repeated dose toxicity evaluation aims at assessing the occurrence of adverse effects following chronic or repeated exposure to chemicals. Non-animal approaches have gained importance in the last decades because of ethical considerations as well as due to scientific reasons calling for more human-based strategies. A critical aspect of this challenge is linked to the capacity to cover a comprehensive set of interdependent mechanisms of action, link them to adverse effects and interpret their probability to be triggered in the light of the exposure at the (sub)cellular level. Inherent to its structured nature, an ontology addressing repeated dose toxicity could be a scientific and transparent way to achieve this goal. Additionally, repeated dose toxicity evaluation through the use of a harmonized ontology should be performed in a reproducible and consistent manner, while mimicking as accurately as possible human physiology and adaptivity. In this paper, the outcome of a series of workshops organized by Cosmetics Europe on this topic is reported. As such, this manuscript shows how experts set critical elements and ways of establishing a mode-of-action ontology model as a support to risk assessors aiming to perform animal-free safety evaluation of chemicals based on repeated dose toxicity data

t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies

[EN] Quantitative multinuclear high-resolution magic angle spinning (HRMAS) was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D 1 H and 31P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. 1 H–1 H homonuclear and 1 H–31P heteronuclear correlation experiments enabled the direct assessment of the 1 H–31P spin systems for signals that suffered from overlapping in the 1D 1 H spectra, and linked the information present in the 1D 1 H and 31P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the 31P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from 31P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH3)3 signals of phosphocholine and choline in 1 H spectra of the tissue in these tumour samples.The authors acknowledge the SCSIE-University of Valencia Microscopy Service for the histological preparations. They also acknowledge Martial Piotto (Bruker BioSpin, France) for providing the ERETIC synthetic signal. Furthermore, they acknowledge financial support from the Spanish Government project SAF2007-6547, the Generalitat Valenciana project GVACOMP2009-303, and the E.U.'s VI Framework Programme via the project "Web accessible MR decision support system for brain tumor diagnosis and prognosis, incorporating in vivo and ex vivo genomic and metabolomic data" (FP6-2002-LSH 503094). CIBER-BBN is an initiative funded by the VI National R&D&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Esteve Moya, V.; Celda, B.; Martínez Bisbal, MC. (2012). Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies. 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Internationalizationof Read-Across as a Validated New Approach Method (NAM) for Regulatory Toxicology

Read-across (RAx) translates available information from well-characterized chemicals tothe substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g. EU-REACH) already allow this procedure to be used to waive new in vivotests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimisation to allowa higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target, in the RAx procedure, and (iii) how to deal with issues of ADMEthat may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluatethe RAx process were identified. Here, in particular the RAx guidance, being worked out by the European Commission’s H2020 project EU-ToxRisk, together with many external partners with regulatory experience, is given

Finding synergies for the 3Rs – Repeated dose toxicity testing: Report from an EPAA partners' forum

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum on repeated dose toxicity (RDT) testing to identify synergies between industrial sectors and stakeholders along with opportunities to progress these in existing research frameworks. Although RTD testing is not performed across all industrial sectors, the OECD accepted tests can provide a rich source of information and play a pivotal role for safety decisions relating to the use of chemicals. Currently there are no validated alternatives to repeated dose testing and a direct one-to-one replacement is not appropriate. However, there are many projects and initiatives at the international level which aim to implement various aspects of replacement, reduction and refinement (the 3Rs) in RDT testing. Improved definition of use, through better problem formulation, aligned to harmonisation of regulations is a key area, as is the more rapid implementation of alternatives into the legislative framework. Existing test designs can be optimised to reduce animal use and increase information content. Greater use of exposure-led decisions and improvements in dose selection will be beneficial. In addition, EPAA facilitates sharing of case studies demonstrating the use of Next Generation Risk Assessment applying various New Approach Methodologies to assess RDT

Deciphering the mechanisms underlying the pathogenesis of heart failure with preserved ejection fraction (HFpEF) with multiple comorbidities

Heart failure with preserved ejection fraction (HFpEF) has become as a major public health problem worldwide, with increasing prevalence due to the aging of the population and the epidemic in obesity, hypertension and diabetes mellitus. HFpEF is a syndrome defined by the presence of clinical signs and/or symptoms of left heart failure, diastolic dysfunction leading to increased left ventricular (LV) filling pressure, LV hypertrophy and elevated blood levels of natriuretic peptides, associated with a preserved LV ejection fraction (>50%). HFpEF accounts for about half of heart failure cases and frequently leads to pulmonary hypertension (PH) and right ventricular (RV) failure, both known to increase morbidity-mortality in these patients. The presence of renal dysfunction is also frequently described in these patients and associated with a poor prognosis. Despite recent findings showing beneficial effects of gliflozins inhibitors of the sodium/glucose co-transporter (SGLT-2) in patients with HFpEF, no curative therapeutic strategy has shown beneficial effects on mortality in these patients, making this issue an urgent need in cardiology. The lack of preclinical experimental models recapitulating the complex pathophysiology of HFpEF as observed in patients represents a real obstacle to therapeutic innovation. Moreover, the mechanisms underlying the pathogenesis of HFpEF, as well as PH, RV dysfunction and renal failure associated with HFpEF, remain largely unknown. In this context, the aim of the present work was to develop a model of HFpEF associated with multiple comorbidities, by subjecting a strain of rats prone to develop obesity (obesity-prone rats or OP rats) to a high-fat diet and to characterize the development of HFpEF over time (after 4 and 12 months on specific dieting) (1) to study the pathogenesis of PH and RV-pulmonary artery (un)coupling (2), as well as renal function (3) in these rats, by comparing them to control rats (obesity-resistant rats on standard chow diet). After 4 and 12 months of high-fat diet, OP rats developed a metabolic syndrome characterized by increased total body weight and abdominal fat weight, glucose intolerance, and dyslipidemia (characterized by hypercholesterolemia and hypertriglyceridemia), as well as arterial hypertension (characterized by an increase in LV systolic pressure measured by catheterization, which was only present after 12 months of diet). After 12 months of diet, these rats with metabolic syndrome developed a HFpEF characterized by the presence of diastolic dysfunction (confirmed by an increase in LV end-diastolic pressure measured by catheterization) associated with concentric LV hypertrophy (assessed by echocardiography and histology showing cardiomyocyte hypertrophy) and a preserved LV ejection fraction. Circulating plasma levels of soluble ST2 were increased in these HFpEF rats, while plasma NT-proBNP levels were decreased. In these HFpEF rats, myocardial RNA sequencing showed clusters of upregulated genes involved in lipid metabolism and calcium-dependent contraction. These rats with HFpEF developed PH (assessed by increased RV systolic pressure and pulmonary vascular resistance), associated with pulmonary artery (PA) dilatation (evaluated by echocardiography). In the pulmonary arteries, no differences were observed in terms of pulmonary vascular remodeling and density, or PA reactivity assessed ex vivo in response to acetylcholine and endothelin-1. These lungs did not show inflammatory foci or fibrosis. However, the balance between vasodilator and vasoconstrictor production was altered in HFpEF rats, with increased PA nitric oxide release and decreased endothelin-1 precursor expression. In HFpEF rats, RV cavity dilatation and RV systolic dysfunction characterized by a decrease in RV fractional area change (FAC) were present, as well as impaired RV-pulmonary artery coupling assessed by the ratio of tricuspid annular plane systolic excursion (TAPSE) and systolic PA pressure (sPAP); TAPSE/sPAP. Cardiomyocyte hypertrophy and activation of apoptotic processes were shown in both the LV and RV. An inverse correlation between the TAPSE-to-sPAP ratio and the rate of apoptosis in the RV was demonstrated. In addition, increased circulating plasma levels of various pro-inflammatory cytokines were found for interleukin (IL)-1, -6 and -17a. Circulating levels of monocyte chemoattractant protein (MCP)-1 and IL-18 were unchanged.HFpEF rats also developed renal dysfunction, characterized by elevated circulating levels of cystatin C, associated with diffuse structural damages to the kidney characterized by the presence of glomerular damage (with global glomerular sclerosis), tubular lesions (with extensive necrosis), interstitial lesions (with inflammation), vascular and medullary involvement (medullary tubular necrosis), as well as interstitial fibrosis and tubular atrophy. Renal expression of inflammatory markers IL-1b, tumor necrosis factor (TNF)-a, intercellular adhesion molecule (ICAM)1, vascular adhesion molecule (VCAM)1 and macrophage-specific marker CD68 were increased in HFpEF rats, with an increase in ICAM1 and VCAM1 already present after 4 months of diet. SGLT-2 expression was increased in HFpEF rats after 4 and 12 months of diet. RNA sequencing of renal tissue also showed a clear clustering of genes involved in inflammatory signaling pathways.In conclusion, OP rats fed with a high-fat diet for 12 months developed HFpEF associated with a metabolic syndrome. This experimental model mimics the natural evolution of the disease as observed in patients. It allowed us to demonstrate the presence of VD-pulmonary artery uncoupling disproportionate to the rise in PA pressure, before the development of PA remodeling, suggesting cardiac global myocardial damage in these rats. In addition, diffuse and pronounced renal damage was demonstrated in this early model of HFpEF.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe