Effect of titanium tetrafluoride varnish on fibroblasts (NIH3T3): viability assay, morphological analysis and cell signals for apoptotic pathway

• Grant number: 15/05009-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: October 01, 2015
• End date: January 31, 2016
• Field of knowledge: Health Sciences - Dentistry - Dental Materials
• Principal Investigator: Ana Carolina Magalhães
• Grantee: Priscila Maria Aranda Salomão
• Supervisor: John Michael Edwardson
• Host Institution: Faculdade de Odontologia de Bauru (FOB). Universidade de São Paulo (USP). Bauru , SP, Brazil
• Institution abroad: University of Cambridge, England
• Associated to the scholarship: 13/13484-6 - Effect of titanium tetrafluoride varnish on fibroblasts (NIH3T3): viability assay, morphological analysis and cell signals for apoptotic pathways, BP.DR

Abstract

The aim of the project is to evaluate the citoxicity of titanium tetrafluoride (TiF4) compared with sodium fluoride (NaF) varnishes, regardless the fluoride concentration and time of treatment, by analyzing the cell viability, morphology and the death type (apoptosis or necrosis). NIH3T3 will be cultured in DMEM supplemented with 10% fetal bovine serum, at 37oC in a humidified atmosphere of 5% CO2. The cells will be treated with DMEM containing TiF4 or NaF varnishes (0.95%, 1.9% and 2.45% F) in 24-well microplates. After 6, 12 and 24h, the cells will be subjected to viability assay (n=6, MTT) and to morphological analysis (n=3, HE images). Furthermore, the cell signs involved in apoptosis will be analyzed (n=3): mitochondria- and death receptor-mediated apoptotic pathways. The cells will be stained with Hoechst 33342 and iodate to differentiate necrosis and apoptosis, as well as they will be examined by the TUNEL method, using confocal microscopy. The caspase-3, -8 and -9 activities will be assessed by an ELISA reader using assay kit. The RNAm will be isolated, submitted to reverse transcription and cDNA for cytocrome c, Bax, Bad (pro-apoptotic proteins), Bcl-2 (anti-apoptotic protein), PARP (poli(ADP-Ribose) polimerase), VDAC (voltage dependent anionic channel) and Fas-L (ligand to the death receptor) amplified by quantitative PCR (qPCR). Finally, the expression of cytocrome c, Bax, Bad (pro-apoptotic proteins), Bcl-2 (anti-apoptotic protein), PARP (poli(ADP-Ribose) polimerase), VDAC and Fas-L will be detected by western blot. The BEPE project involves a new analysis, where cells will be treated with TiF4, NaF (2.45% F) or placebo varnishes (for 6, 12 and 24h) in Petri dishes. The individual cells will be identified by phase contrast microscopy, and their stiffness will be measured using Atomic Force Microscopy-AFM, in the University of Cambridge, UK, under supervision of Prof. Dr. John Michael Edwardson, who is expert in imaging of biomolecules using AFM. The stiffness of the cells will be deduced from the linear slope curve and the data will be subjected to statistical analysis considering the level of significance of 5%. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

ARANDA SALOMAO, PRISCILA MARIA; DE OLIVEIRA, FLAVIA AMADEU; RODRIGUES, PAULA DANIELLE; AL-AHJ, LUANA POLIONI; DA SILVA GASQUE, KELLEN CRISTINA; JEGGLE, PIA; RABELO BUZALAF, MARILIA AFONSO; DE OLIVEIRA, RODRIGO CARDOSO; EDWARDSON, JOHN MICHAEL; MAGALHAES, ANA CAROLINA. The cytotoxic effect of TiF4 and NaF on fibroblasts is influenced by the experimental model, fluoride concentration and exposure time. PLoS One, v. 12, n. 6, JUN 14 2017. (13/20372-0, 14/16415-8, 09/17360-4, 15/05009-1, 10/14984-4, 13/13484-6)