ECTS Abstracts (2015) 1 P185

Toxicity and Metabolic Evaluation of Oestradiol, Coumestrol and Resveratrol on Osteoblast-like MLO-A5 and Osteocyte-like MLO-Y4 Cell Lines

Ana M. Silva1,2 & Vilma A. Sardão1

1CNC – Center for Neuroscience and Cell Biology, Coimbra, Portugal, 2Department of Life Sciences, University of Coimbra, Coimbra, Portugal.

Phytoestrogens have been widely used by women as alternative hormone replacement therapy (HRT) during menopause. However, despite of several studies performed with phytoestrogens (PE), their risks and benefits are not yet well established. The identification of the molecular mechanisms of PE effects and their toxicity are required for a safe and appropriate therapeutic administration. We propose here to assess the effects of two distinct PEs coumestrol (COU) and resveratrol (RESV), when compared with Oestradiol (E2) on cell bioenergetics. We hypothesise that mitochondrial bioenergetics impairment in osteoblasts and/or osteocytes may be associated with the osteoporotic profile during E2 reduction, and can be recovered after HRT. Additionally, we tested the toxicity of those compounds on two murine osteoblast- and osteocyte-like cell lines. MLO-Y4 and MLO-A5 cell lines were seeded and screened in terms of cell viability, cell cycle, as well as extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) by using a XFe96 Extracellular Flux Analyzer. 24 hours prior to each assay cells were incubated with oestradiol, coumestrol and resveratrol at 1 μM concentration. Oestradiol, coumestrol and resveratrol had no toxicity in both cell types, and did not alter cell cycle. Interestingly, when we reduced the serum in the medium (from 10% to 2%) resveratrol decreased the apoptotic peak in MLO-A5 (compared with the control). MLO-A5 presented a notorious glycolytic profile, and in increasing order, E2, COU and RESV increased the ECAR parameter after the addition of glucose. The oxygen consumption analysis during mitochondrial stress tests in MLO-Y4 showed a slight increase after FCCP addition. No changes in media acidification were observed. The putative protective effect exerted by E2 and PEs is probably related to a positive influence on mitochondrial capacity and bioenergetics. MLO-A5 cell line showed a clear glycolytic profile, slightly increased in the presence of those PEs. MLO-Y4 cells seemed less susceptible to the effects of the compounds. At the selected concentrations, no toxic effects were observed. Although preliminary, the results contribute to understanding how the three selected PEs interfere with osteoblast and osteocyte-like cells.

Disclosure: The authors declared no competing interests. This Work was supported by PTDC/AGR-ALI/108326/2008 and PEst-C/SAU/LA0001/2013-2014 grants from Fundação para a Ciência e Tecnologia (FCT), co-funded by FEDER/Compete/National Funds. FCT PhD and Post-doc fellowships (SFRH/BD/33892/2009, SFRH/BD/76086/2011 and SFRH/BPD/31549/2006, respectively). The work was also supported by QREN project #4832 ”Stemcell-based platforms for Regenerative and Therapeutic Medicine”, reference CENTRO-07-ST24-FEDER-002008.

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