Pinho 2019 MiPschool Coimbra
Event: MiPschool Coimbra 2019
Unmasking selective mitochondrial toxicity is crucial for toxicological studies . The correct identification of mitochondrial stressors is known to be influenced by cell culture conditions. Stimulation of oxidative phosphorylation (OXPHOS) in cultured cells can unmask specific mitochondrial toxicants and has gained increased interest for the study of mitochondrial health in Toxicology and Biomedicine . Fibroblasts are an interesting cell model for Toxicology and Biomedicine , allowing for the validation of ingredients for cosmetics and also diagnostic and therapeutic development for personalized medicine. Here, we aimed to identify the best cell culture conditions to study different mitochondrial stressors in Normal Human Dermal Fibroblasts (NHDF).
NHDF were cultured in DMEM containing 25 mM Glucose (HGm) and gradually adapted to DMEM containing 5 mM Glucose (LGm) or to glucose-free galactose-containing DMEM (OXPHOSm). In these cells, we performed functional and molecular metabolic measurements of oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) using a Seahorse XFe96 Analyzer and quantified mitochondrial network extension and polarization through fluorescence microscopy imaging. The expression of several key genes was also quantified by qRTPCR. Furthermore, we evaluated the relevance of galactose, glutamine, pyruvate and uridine as medium components for OXPHOS stimulation using Seahorse XFe96 Analyzer, by comparing respiratory parameters in cells cultured in the presence or absence of these substrates. In addition, we assessed the susceptibility of cells to different mitochondrial stressors by following ATP content and resazurin reduction in the presence of increasing concentrations of OXPHOS system inhibitors (rotenone, dimethylmalonate, antimycin A, oligomycin, TTFA), oxidant agents (tBHP and H2O2) and a glycolytic inhibitor (2-DG).
Our results suggest that culture of NHDF in OXPHOSm increased their OXPHOS-reliance, sensitizing them to the toxic effects of mitochondrial poisons, and interestingly also to tBHP. The promotion of an OXPHOS-based cellular phenotype was accompanied by changes in the expression of several genes and proteins, as well as in the mitochondrial network morphology, without affecting the cellular global energetic state. Moreover, we found that the reduction of glucose concentration is not equivalent to its replacement by galactose; galactose and pyruvate seem to be dispensable to promote OXPHOS, whereas glutamine is essential; and uridine could replace galactose. We concluded that the toxicity of different mitochondrial stressors requires particular culture conditions to be evidenced.
• Bioblast editor: Plangger M
Labels: MiParea: Respiration, Pharmacology;toxicology
Organism: Human Tissue;cell: Fibroblast
Affiliations and support
- CNC, Center Neuroscience Cell Biology, Univ Coimbra, UC-Biotech, Biocant Park, Cantanhede, Portugal. – [email protected]
- This work was financed by the European Regional Development Fund (ERDF), through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, under project[s] POCI-01-0145-FEDER-029297, SFRH/PD/BD/143055/2018 (to Pinho SA and UID/NEU/04539/2019)
- Meyer JN, Hartman JH, Mello DF (2018) Mitochondrial Toxicity. Toxicol Sci 162:15-23.
- Will Y, Dykens J (2014) Mitochondrial toxicity assessment in industry – a decade of technology development and insight. Expert Opin Drug Metab Toxicol 10:1061-7.
- Wieckowski MR, Danese M, Morciano G, Pronicki M, Duszynski J, Pinton P, Karkucinska-Wieckowska A (2018) Recovering Mitochondrial Function in Patients’ Fibroblasts. Mitochondrial Biology and Experimental Therapeutics 359-78.