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Biomarker Quantification by Multiplexing: Analyses of hormone and zytokine level after exposure of blood and skin samples to active substances(Endpoints Endocrine effects, Immunotoxicity)

Project


Project code: BfR-SiP-08-1322-
Contract period: 01.01.2018 - 31.12.2018
Purpose of research: Applied research

In this project biomarkers should be measured quantitatively by a multiplexing-system. A fast and practicable detection method will be established, that is capable of measuring inflammatory and hormone responses in human and rodent blood and plasma. For that reason, the new purchased MAGPIX system will be used that enables detection of the concentrations of even small samples in 25 µl and in up to 50 analytes. The method is economic in different ways, as it on the one hand generates equal data per detection to a total of fifty ELISA analyses. This can be performed with a relativly limited experimental effort, as "customized" antibody- panels for particular sets of hormones and cytokines are already available (a total of more than 125 (will be updated) MILLIPLEX® MAP magnetic bead-based assay kits). The panel - Immunology I for instance detects the inflammatory markers sCD40L, EGF, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, PDGF-AB/BB, RANTES, TGF-α, TNF-α, TNF-β, VEGF, Eotaxin/CCL11, PDGF-AA all together within one sample. With respect to the hormones the Endocrine Multiplex assay detects cortisol, estradiol, progesterone, T3 und T4, the Metabolism Multiplex assay detects amylin, C-peptide 2, ghrelin, GIP, GLP-1, glucagon, IL-6, insulin, leptin, MCP-1, PP, PYY und TNF-α. Furthermore, the panels Toxicity, Cancer, Early Apoptosis and Cell Cycle Multiplex Assays for rat, mouse and man are available. The idea of this project is rather not to develop new animal experiments for hormone and inflammatory analyses, but to establish a refinement of the existing OECD test methods such as for example the 28- and 90- day studies. Even more for the laborious Cancer- and Extended One-Generation Reproduction Toxicity studies, the implementation of hormone- and cytokine-assays from blood samples at different time points will be a feasible improvement concerning the data even more than the customary clinical picture. However, this SFP project should provide a proof-of-principle for the relevant species human beings and in order to avoid not necessary animal experiments, human samples shall be analyzed. Representative for human blood, detections of supernatants derived from blood monocytes, for human skin derived from keratinocytes will be performed. The approach uses for reasons of practicability and reproducebility for both primary cell types the availability of cell lines, THP-1 and HaCaT cells, respectively. These will be treated and stimulated by active substances derived from pesticidal products. A preliminary selection of active substances was made, which was listed in the EFSA analysis (1) for immunotoxicity of pesticides in Tab. 3.4.2 (Compounds with one or more studies having only an immunotoxicity marker driving the NOAEL in a study) and for which effects on cellular level were reported: Active Immunotox-Endpoint Copper compounds Reduced T-and B-cell response Lenacil Reduced WBC counts Metamitron Decreased WBC counts Pirimicarb Increased myeloid to erythroid ratio Propamocarb Reduced lymphocytes in bone marrow

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Subjects

Framework programme

BMEL - research cluster

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