Genomic and Phenotypic Alterations of the Neuronal-Like Cells Derived from Human Embryonal Carcinoma Stem Cells (NT2) Caused by Exposure to Organophosphorus Compounds Paraoxon and Mipafox

Organophosphorus compounds (OPs) are pesticides of worldwide use due to the acute insecticidal effects mediated by the inhibition of esterases of the central nervous system (mainly acetylcholinesterase and neuropathy target esterase (NTE)). OPs need to inhibit acetylcholinesterase to be effective insecticides, but not NTE since its inhibition might cause progressive, irreversible delayed neuropathy in humans and other species. Additionally, other neurological and neurodevelopmental toxic effects with unknown targets have been reported in humans or animals chronically exposed to OPs. We used a mixed neuronal/glia culture derived from well-characterised human embryonal carcinoma stem cells (hNT2) to determine if neuropathic OP mipafox and non-neuropathic OP paraoxon are able to alter the neuronal differentiation process evaluated by gene expression studies, neuronal electrical activity measurements and neural cell morphology quantification. Exposure to paraoxon at non-cytotoxic concentrations altered the expression of different genes involved mainly in signalling pathways related to chromatin assembly and nucleosome integrity, generating cultures with a larger number of differentiated neurons-like cells and branching points than in the control. Moreover, these paraoxon-exposed neuronal-like cells displayed reduced electrical activity when compared with the control neurons as measured by Micro Electrode Array Chips. Similarly, exposure to mipafox, a known inhibitor of NTE activity, also reduced the electrical activity of hNT2 cultures differentiated into neurons-like cells, but no significant changes in cell morphology or gene expression were detected. Therefore, we conclude that paraoxon is able to strongly disturb in vitro neurodifferentiation, while the absence of morphological and transcriptional alterations did not allow us to conclude if the electrophysiological alterations detected in mipafox-exposed neurons are due to neurodevelopmental toxicity or to effects on mature neurons.JRC.I.2-Public Health Policy Suppor

Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles

Abstract Higher efficacy and safety of nano gold therapeutics require examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells exposed to AuNP (5 and 30 nm). Cellular internalization was dose and time-dependent for both AuNPs. The toxicity was observed by colony forming efficiency (CFE) and not by Trypan blue assay, and exclusively for 5 nm AuNPs, starting at the concentration of 200 μM (24 and 72 h of exposure). The most pronounced changes in gene expression (Agilent microarrays) were detected at 72 h (300 μM) of exposure to AuNPs (5 nm). The biological processes affected by smaller AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins were also differentially expressed. Our findings indicate that exposure to high concentration of AuNPs (5 nm) induces metal exposure, oxidative stress signaling pathways, and might influence selenium homeostasis. Some of detected cellular responses might be explored as potential enhancers of anti-cancer properties of AuNPs based nanomedicines

Advanced Non-animal Models in Biomedical Research: Respiratory Tract Diseases

A study was initiated at the JRC to develop a current overview of available and emerging non-animal models in the field of Respiratory Tract Diseases. In a literature review, over 21,000 abstracts (11,636 non-cancer and 9,421 cancer) were scanned for relevant non-animal methods of respiratory disease. From this, a total of 284 publications were finally identified as being promising candidate methods according to a set of inclusion/exclusion criteria. In vitro cell and tissue cultures, human ex vivo, in silico approaches were chiefly considered. These methods have been collated into a catalogue of biomedical disease models that will form a key knowledge source for researchers, educators and national ethics and funding authorities. The availability of a centralised source of reviewed methods will contribute to extend the requirements of EU Directive 2010/63/EU on the protection of animals used for scientific purposes to biomedical science. Simple cell culture models using immortalised cell lines are long-established, are inexpensive and quick, however they poorly reflect complex disease mechanism observed in vivo. The emerging use of more physiologically-relevant models of disease, such a 3D human tissue cultures, spheroids, organoids, and microfluidic /’lung on a chip’ based systems shows immense promise for the development of in vitro model systems that can more accurately mimic human respiratory diseases. This review shows that, while simple models are still prominent and have their uses, research focus has, in the past 5 years, shifting towards increasingly sophisticated bioengineering approaches that recapitulate lung development, anatomy and physiologic functions in vitro. Such approaches hold the promise of more human-relevant disease models that can be used to elucidate mechanism of disease and aid in the development of new therapies.JRC.F.3-Chemicals Safety and Alternative Method

Genetic Testing in Emerging Economies (GenTEE)

Drivers, barriers and opportunities for genetic testing services in emerging economies: the GenTEE (Genetic Testing in Emerging Economies) project Background: Due to the epidemiological transition in the emerging economies of China, East Asia, India, Latin America, the Middle East and South Africa, these economies are facing (i) an increasing proportion of morbidity and mortality due to congenital and genetic conditions, (ii) a rising need for genetic services to improve patient outcomes and overall population health. These economies are facing the challenge how: (i) to ensure the successful translation of genetic/genomics laboratory and academic research into quality assured pathways, (ii) to develop a service delivery infrastructure that leads to equitable and affordable access to high quality genetic/genomic testing services. Objectives: (i) to document and compare current practices and the state of genetic service provision in eight emerging economies: Argentina, Brazil, China, Egypt, India, Oman, Philippines and South Africa, (ii) to identify current knowledge gaps and unmet service needs. The GenTEE international project is intended to inform policy decisions for the challenges of delivering equitable high quality genetic services and to promote international collaboration for capacity building. Methods: (i) a standardized survey that is the first of its worldwide that allows comparison of services internationally across a number of key dimensions by using a core set of indicators, selected by the GenTEE consortium for their relevance and comparability, (ii) capacity building demonstration projects. To date, the GenTEE project has completed its survey that maps the current state of genetic services in the participating countries and identifies current drivers, barriers and opportunities for genetic services development. Results: There is no equitable access to genetic services in all countries mainly due to financial barriers (underfunded fragmented public services, out-of-pocket expenses tend to be the norm for genetic testing services), geographical barriers (concentration of services in main cities) and skill gaps, resulting in inequitable services or delayed access. The development of services in the private sector is opportunistic and mostly technology and market driven. There is a marked lack of standard operating procedures and agreed quality assessment processes for new technologies. Discussion: International collaborative networks can provide support for capacity building and help to strengthen the provision of quality genetic/genomic services in emerging economies.JRC.I.1-Chemical Assessment and Testin

Clonogenicity and gene expression modulation in the bone marrow of mice chronically exposed to arsenic and atrazine.

The clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1185 cancer-related genes by DNA-macroarrays in bone marrow were used to investigate in male and female mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. In male mice, the exposure to arsenate or to atrazine alone and the combined exposure did not change the clonogenicity of the progenitors. In females the percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined exposure to the two chemicals. Results from microarrays indicate that atrazine alone didn\u2019t stimulate the expression of any of the cancer genes analyzed in both male and female. Arsenic induced gene expression modulation only in female and had no effects on male. Major significant changes on the gene expression in bone marrow cells resulted following the co-exposure to arsenic and atrazine in both male and female. These results indicate that co-exposure of mice to atrazine and arsenate induces significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

Combined in-utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates the gene expression and clonogenicity of myeloid progenitors in bone marrow.

Increasing evidence proves that human fetuses are exposed to multiple risk factors and major concerns have been expressed towards exposure to potential endocrine modulating chemicals at early stage of life and during growth. Understanding that exposures occur as mixture of chemicals and that they converge on other inherent and environmental risk-modulating factors, there is a need to develop experimental models to assess the effects of exposure to multiple chemicals during different stage of life. In the present study, the clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1197 cancer-related genes (DNA macroarrays) in bone marrow were used to investigate in male and female young mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. Female adult mice were treated with arsenate in drinking water (1 mg As/L) for 10 days before mating and during the gestation. Offspring were randomly put into separate groups of males and females. One group of arsenic exposed offspring were exposed for 4 months to atrazine (1mg Atr/L) and arsenate (1 mg As/L) in drinking water (As+Atr). One group of each of arsenic unexposed offspring were exposed for 4 months to atrazine (1mg Atr/L) in drinking water (Atr). Additional arsenate (1 mg As/L) was given to one group of arsenic exposed offspring (As). Control mice without any treatment were also analysed (Ctrl). In male mice the exposure to arsenate or to atrazine alone did not result in significant changes on the gene expression in bone marrow cells, whereas, co-exposure to arsenic and atrazine (As+Atr) resulted in a significant up-modulation of gene expression. The percentage of CFU-GM weakly decreased after exposure to individual compounds, while the co-exposure did not change the clonogenicity of the progenitors. In female mice, the co-exposure to both chemicals resulted in a drastic up-modulation of gene expression, while in these cells the single treatments showed a up-modulation of few genes as well. The percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined administration. These results indicate that in-utero and juvenile co-exposure of mice to atrazine and arsenate induce significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

analysis of non animal methods and models for research in cardiovascular disease

Cardiovascular diseases (CVD) are disorders of the heart and blood vessels and represent 31% of all global deaths. In the contest of CVD, the use of animal experiments has been a contentious subject for many years. In recent years, in vitro and in silico models and methods have been proposed according to the 3Rs statement. However, an exhaustive report regarding the state of art in terms of in vitro and in silico experiments has not been reported yet. This work is focused on providing a collection of non-animal models and methods in use for basic and applied CVD research. The standardized descriptions of such studies will ultimately feed into EURL ECVAM database on alternative methods. Two are the research main phases. Firstly, the exclusion/ inclusion criteria and the list of relevant information resources of the research have been defined. The second phase regards the search, selection and detailed description of the literature papers by analysing records on Scopus and Pubmed databases