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Berlin-Brandenburg Research Platform BB3R with Integrated Graduate Education 'Innovations in the 3R Research - Genetic Engineering, Tissue Engineering and Bioinformatics' - Subproject 4: 'Individualized pain suppression in mice' (Work program Dept. 9) and 'Development of a nanotoxicology test platform for testing the inhalation toxicity of nanoparticles in vitro' (work program Dept. 7) (BB3R)

Project

Food and consumer protection

This project contributes to the research aim 'Food and consumer protection'. Which funding institutions are active for this aim? What are the sub-aims? Take a look:
Food and consumer protection


Project code: BfR-TOX-08-1334-216
Contract period: 01.04.2014 - 31.03.2018
Purpose of research: Networks and research co-ordination

The establishment of the Berlin-Brandenburg research platform BB3R with integrated graduate education will strengthen the 3R expertise of the region Berlin-Brandenburg. Intensive systematic research will provide substantial progress in 3R-alternatives and excellent young scientists will be trained and highly qualified in alternative methods to animal experiments. Work program Dept. 9 (Prof. Schönfelder): Article 4 of the European Directive 2010/63/EU on the protection of animals used for scientific purposes requires the use of methods for breeding, housing and care that avoid or at least minimize pain, suffering, distress or lasting harm to animals (refinement). Since the mouse is currently the most widely used animal in experiments (70%), a high percentage of experimental animals would benefit from effective pain management. Systemic analgesia for post-operative care in mice is usually performed by subcutaneous injection of opioids. Current recommendations for opioid dosing in mice range widely and do not take strain-specific differences into account. However, studies on the efficacy of the opioid fentanyl in mice have shown that the analgesic effect may vary from strain to strain. This may be due to differences in opioid metabolism. The aim of this project is to investigate the efficacy of buprenorphine in three commonly used mouse inbred strains (C57BL/6J, Balb/cJ and 129S1/SvImJ). In addition, it will be determined whether differences in the efficacy of buprenorphine are due to strain-specific differences in the metabolism of the animals. In vivo, in vitro and in silico methods are used. Based on the findings, improved recommendations for the dosage of buprenorphine for different mouse strains should be given in the future in order to minimize pain during the experiment (refinement).

Work program Dept. 9 (Prof. Schönfelder): Basal pain sensitivity differed considerably between the mouse strains with Balb/cJ mice being the most and 129S1/SvImJ mice the least sensitive strain. The application of buprenorphine led to dose and strain dependent analgesic differences. While the analgesic effect of buprenorphine increased in C57BL/6J and Balb/cJ mice with the higher administered dose, the analgesic effect remained stable in 129S1/SvImJ mice. Serum and blood concentrations of buprenorphine and its metabolites were dose and partly strain dependent (4.0 mg/kg dose group). The metabolic ratio in blood and distribution between brain and blood showed no dose and only slight strain dependent variations, which did not correspond with the analgesic effect of buprenorphine. Additionally, no strain dependent differences for CYP3A activity and protein content could be detected in vitro, whereas at mRNA expression level slight strain differences were present. However, the mRNA expression differences of some CYP3A isozymes were not in accordance with activity data. Thus, administration of buprenorphine for optimal pain management should be adapted to the respective mouse strain to avoid pain or adverse side effects. However, strain differences in the analgesic effect of buprenorphine could not be explained by differences in pharmacokinetics. Hence, differences in pharmacodynamic parameters are more likely and should be investigated in further studies. In summary, the results of this study should sensitize the scientific community to optimize pain management in order to contribute to laboratory animal welfare in life sciences. Publication: J. Rudeck, B. Bert, P. Marx-Stoelting, G. Schönfelder, S. Vogl. Liver lobe and strain differences in the activity of murine cytochrome P450 enzymes. Toxicology, (2018). Jul 1; 404-405:76-85. doi: 10.1016/j.tox.2018.06.001.

Work program Dept. 7 (Prof. Luch): Due to the increasing use of nanoparticles (NP) in consumer products, the determination of a possible toxicity of these NP is an important task for the risk assessment of these materials. Currently, the toxicity of nanoparticles and inhalable chemicals is still being tested in animals. In order to reduce these tests to a minimum, the development of alternative methods to determine the short-term toxicity of NP is of crucial importance. The aim of the study is to develop a cell model that best represents the in vivo situation in order to determine the toxicity of NP. For this, different lung cell lines will be investigated concerning the formation of an intact cell-cell barrier function in order to determine the translocation of NP after inhalation, which represents a decisive contribution to the toxicity of NP. The obtained data were compared with an established alveolar cell model, which is often used to evaluate the toxicity of NP, but is inadequate due to the absence of an intact barrier function. Based on these findings, the translocation of NP and the improved in vitro models should contribute to predicting the toxicity of NP in the future and thus reduce animal experiments to the indispensable level (refinement).

Work program Dept. 7 (Prof. Luch): Immunohistochemical experiments and the determination of transepithelial resistance (TEER) were performed. Further, the toxicity of CeO2-NP (NM-212) and ZnO-NP was determined. In addition, the intracellular uptake of NM-212 was characterized more precisely. It was shown that both new cell models developed a barrier function corresponding to that of primary cells. NM-212 showed no impairment of cell viability and no cytotoxicity in any of the models. However, ZnO-NP showed significant cytotoxicity and reduced viability in all models used. It was also shown that all cell lines were able to uptake NP, but to different degrees. A translocation of NP through the epithelium could not be detected for any of the models. In conclusion, it can be stated that the new cell lines have a clear advantage for toxicity testing of NP due to the formation of an intact barrier function and should therefore be used in the future.

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Subjects

Framework programme

BMEL Frameworkprogramme 2008

Participating institutions

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