A trial to evaluate the effect of the sodium–glucose co‐transporter 2 inhibitor dapagliflozin on morbidity and mortality in patients with heart failure and reduced left ventricular ejection fraction (DAPA‐HF)

Background: Sodium–glucose co‐transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of incident heart failure hospitalization in individuals with type 2 diabetes who have, or are at high risk of, cardiovascular disease. Most patients in these trials did not have heart failure at baseline and the effect of SGLT2 inhibitors on outcomes in individuals with established heart failure (with or without diabetes) is unknown. Design and methods: The Dapagliflozin And Prevention of Adverse‐outcomes in Heart Failure trial (DAPA‐HF) is an international, multicentre, parallel group, randomized, double‐blind, study in patients with chronic heart failure, evaluating the effect of dapagliflozin 10 mg, compared with placebo, given once daily, in addition to standard care, on the primary composite outcome of a worsening heart failure event (hospitalization or equivalent event, i.e. an urgent heart failure visit) or cardiovascular death. Patients with and without diabetes are eligible and must have a left ventricular ejection fraction ≤ 40%, a moderately elevated N‐terminal pro B‐type natriuretic peptide level, and an estimated glomerular filtration rate ≥ 30 mL/min/1.73 m2. The trial is event‐driven, with a target of 844 primary outcomes. Secondary outcomes include the composite of total heart failure hospitalizations (including repeat episodes), and cardiovascular death and patient‐reported outcomes. A total of 4744 patients have been randomized. Conclusions: DAPA‐HF will determine the efficacy and safety of the SGLT2 inhibitor dapagliflozin, added to conventional therapy, in a broad spectrum of patients with heart failure and reduced ejection fraction

Adverse outcome pathways:opportunities, limitations and open questions

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field

Way forward for using in vitro neurotoxicity models in testing strategy: update of FP6 integrated Project "ACUTETOX"

[Purpose]: To develop a strategy in which general cytotoxicity, together with e.g. organspecific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. This presentation summarizes published results from the ACuteTox Project (Forsby et al., in press; Suñol et al., 2009).[Methods]: A set of reference compounds were tested against approximately 50 neuronal endpoints using neural cell lines, and primary neuronal and reaggregate cultures. In vitro data were compared with acute human lethal blood concentration (LC50).[Results]: The testing of a subset of 20 reference compounds revealed that GABAA receptor (GABAAR) function, acetylcholine esterase activity, cell membrane potencial (CMP), glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints. Thirty-six additional chemicals were analyzed against this combined battery of tests. No single endpoint was shown to give a perfect improvement in the in vitro- in vivo correlation. The functional neuronal endpoints GABAAR and cell membrane potential measured in primary neuronal cultures and in the SH-SY5Y cell line identified a high number of chemicals as neurotoxic compounds. A combined analysis of NF-H, GFAP, MBP and HSP32 mRNA expression, glucose uptake and total RNA synthesis measured in aggregated embryonic brain cells was the most sensitive assay. Alteration of caspase-3 mRNA expression in cultured cerebellar granule cells gave a good estimate of the LC50 for a small set of referente chemicals.[Conclusions]: All outliers in the correlation analysis between basal cytotoxicity and human LC50, except atropine and acetonitrile, were identified as alerts by at least one of the endpoints in the test battery. The genomic biomarkers NF-H, GFAP, MBA, HSP32 and caspase-3 were very sensitive endpoints. They showed the possibilities to use high throughput quantitative microarray analyses for toxicological screening. The combined CMP and GABAAR assays correctly identified outliers.EU FP6-512051, FIS PI06-1212 (Spain),The Animal Welfare Agency and The Foundation for research without animal experiments (Sweden

GABAA receptor and cell membrane potential as functional endpoints in cultured neurons to evaluate chemicals for human acute toxicity

El pdf es la versión pre-print.Toxicity risk assessment for chemical-induced human health hazards relies mainly on data obtained from animal experimentation, human studies and epidemiology. In vitro testing for acute toxicity based on cytotoxicity assays predicts 70–80% of rodent and human toxicity. The nervous system is particularly vulnerable to chemical exposure which may result in different toxicity features. Acute human toxicity related to adverse neuronal function is usually a result of over-excitation or depression of the nervous system. The major molecular and cellular mechanisms involved in such reactions include GABAergic, glutamatergic and cholinergic neurotransmission, regulation of cell and mitochondrial membrane potential, and those critical for maintaining central nervous system functionality, such as controlling cell energy. In this work, a set of chemicals that are used in pharmacy, industry, biocide treatments or are often abused by drug users are tested for their effects on GABAA receptor activity, GABA and glutamate transport, cell membrane potential and cell viability in primary neuronal cultures. GABAA receptor function was inhibited by compounds for which seizures have been observed after severe human poisoning. Commonly abused drugs inhibit GABA uptake but not glutamate uptake. Most neurotoxins altered membrane potential. The GABAA receptor, GABA uptake and cell membrane potential assays were those that identified the highest number of chemicals as toxic at low concentrations. These results show that in vitro cell assays may identify compounds that produce acute neurotoxicity in humans, provided that in vitro models expressing neuronal targets relevant for acute neural dysfunctions are used.This work was supported by the European Commission contract LSHB-CT-2004-512051, and projects funded by the Spanish FIS IP 06/1212 and 2005-SGR-00826 from the Generalitat de Catalunya. Daniel García was the recipient of a postdoctoral fellowship from the Fundación Carolina, Spain

Nutritional intake and determinants of nutritional quality changes from pregnancy to postpartum—a longitudinal study

Nutrient requirements vary across the reproductive cycle, but research on changes in nutritional intake and quality from pregnancy to beyond the lactation period is limited. Thus, we aimed to study nutritional intake and quality changes, among Swedish pregnant participants from late pregnancy to 18 months postpartum and to study the determinants of nutritional quality changes. Participants (n = 72) were studied longitudinally from the third trimester of pregnancy and postpartum (2 weeks 4, 12, and 18 months postpartum). At each visit, participant characteristics and 4-day food diaries were collected. Nutritional quality was assessed by energy adjusted Nutrient Rich Food Index 11.3. Linear mixed models were used to analyze the determinants of change in nutritional quality. Intakes of carbohydrate energy percentage (E%), fiber, vitamin A, vitamin C, and potassium were higher in the third trimester compared to postpartum, whereas intakes of E% protein and monounsaturated fat were lower. Adherence to recommended intakes was low at all study visits for saturated fat (4%–11%), fiber (15%–39%), vitamin D (8%–14%), folate (0%–2%), and iron (6%–21%). Overall, nutritional quality did not differ significantly from third trimester to postpartum. Shorter duration (<4 months) of lactation was negatively related to nutritional quality changes, whereas higher age was positively related to changes. In conclusion, nutritional intake from pregnancy to postpartum changed, whereas quality remained relatively stable, with age and lactation duration as determinants. Identification of people at risk of adverse dietary changes from pregnancy to the postpartum period should be further addressed in future larger and more diverse study populations

Neuronal in vitro models for the estimation of acute systemic toxicity

et al.The objective of the EU funded integrated project “ACuteTox” is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABAA receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro–in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.The project was financed by the EU-FP6 Grant (FP6-LIFESCIHEALTH-2004-512051, the Swedish Animal Welfare Agency, the Swedish Fund for Research without Animal Experiments, Spain’s Ministerio de Educación y Ciencia (SAF2005-01604, SAF2006-13092) and the Spanish PI061212 project from Ministry of Health.Peer reviewe

Whole genome microarray analysis of neural progenitor C17.2 cells during differentiation and validation of 30 neural mRNA biomarkers for estimation of developmental neurotoxicity

<div><p>Despite its high relevance, developmental neurotoxicity (DNT) is one of the least studied forms of toxicity. Current guidelines for DNT testing are based on <i>in vivo</i> testing and they require extensive resources. Transcriptomic approaches using relevant <i>in vitro</i> models have been suggested as a useful tool for identifying possible DNT-generating compounds. In this study, we performed whole genome microarray analysis on the murine progenitor cell line C17.2 following 5 and 10 days of differentiation. We identified 30 genes that are strongly associated with neural differentiation. The C17.2 cell line can be differentiated into a co-culture of both neurons and neuroglial cells, giving a more relevant picture of the brain than using neuronal cells alone. Among the most highly upregulated genes were genes involved in neurogenesis (CHRDL1), axonal guidance (BMP4), neuronal connectivity (PLXDC2), axonogenesis (RTN4R) and astrocyte differentiation (S100B). The 30 biomarkers were further validated by exposure to non-cytotoxic concentrations of two DNT-inducing compounds (valproic acid and methylmercury) and one neurotoxic chemical possessing a possible DNT activity (acrylamide). Twenty-eight of the 30 biomarkers were altered by at least one of the neurotoxic substances, proving the importance of these biomarkers during differentiation. These results suggest that gene expression profiling using a predefined set of biomarkers could be used as a sensitive tool for initial DNT screening of chemicals. Using a predefined set of mRNA biomarkers, instead of the whole genome, makes this model affordable and high-throughput. The use of such models could help speed up the initial screening of substances, possibly indicating alerts that need to be further studied in more sophisticated models.</p></div

PCA plot of independent experimental seed-outs.

<p>The data clusters according to the different contrasts, i.e. 10 days vs 5 days of differentiation, 10 days vs undifferentiated, 5 days vs undifferentiated, showing robustness of the cell model as well as technical reproducibility. The first two principal components explained 72.5% of the information (variation) of the dataset (for PC1: 55.7%, for PC2: 16.8%).</p