Environmental pollutants and osteoarthritis: Effects of non-dioxin-like polychlorinated biphenyls on cultured chondrocytes

Abstract

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S173 used in experiments up to the 2nd passage. Two experimental protocols were used: a membrane water permeability assay and measurement of intracellular calcium using Fura-2AM. Results: Using an established permeability assay, whereby a hypotonic solution is applied to the cells and cell volume recorded, aquaporin permeability was measured. In healthy cells permeability was found to be 31 3x104cm.s (n 1⁄4 5). Stimulated cells did not show a significant change in aquaporin permeability (n 1⁄4 12). Calcium measurements showed that healthy cells responded to the same hypotonic challenge with an intracellular calcium increase of 115 15nM (n 1⁄4 16). This calcium increase was inhibited by the TRP channel antagonist, PYR3 (n 1⁄4 69). When applied to cells from the in vitro model of arthritis the same osmotic challenge caused a significantly greater calcium increase of 328 45nM (n 1⁄4 11; p 0.01). Conclusions: We have investigated changes to two ion channels in healthy chondrocytes and those from an in vitro model of arthritis. Aquaporin function appeared unchanged in our cytokine arthritis model, possibly suggesting that this change in gene expression occurs as a result of OA, rather than contributing to OA development. Previous work has shown that intracellular calcium increases greater than :300nM can lead to cell apoptosis and therefore the changes we observe here could implicate important pathological changes in cytokinestimulated chondrocytes. Further work is necessary to identify the exact mechanism by which this calcium increase occurs. 291 ENVIRONMENTAL POLLUTANTS AND OSTEOARTHRITIS: EFFECTS OF NON-DIOXIN-LIKE POLYCHLORINATED BIPHENYLS ON CULTURED CHONDROCYTES V. Abella yz, M. Scotece y, J. Conde y, V. Lopez y, V. Lazzaro yx, J. Pino y, J.J. Gomez-Reino y, R. Meli k, O. Gualillo y. y SERGAS-IDIS Santiago Univ. Clinical Hosp., Santiago de Compostela, SPAIN; zUniv. of A Coru~ na, A Coru~ na, Spain; xUniversit a della Magna Grecia, Catanzaro, Italy; kUniv. of Naples Federico II, Napoli, Italy a. Purpose: Non-dioxin-like polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in fatty tissues causing immune suppression and endocrine disruption. Several studies suggest that PCBs may be involved in pathogenesis of osteoarthritis (OA). Alterations in the mechanisms of programmed cell death (apoptosis) are strongly related to the degradation of extracellular matrix (ECM) in the cartilage of OA subjects. Identification of apoptosis inducers is of paramount relevance to understand the pathogenesis and/or progression of OA. Thus, the aim of the present study was to assess the effect of several PCBs on chondrocytes viability and apoptosis induction. b. Methods: The murine chondrogenic cell line ATDC5 and human juvenile costal chondrocyte cell line T/C-28a2 were treated with several doses of PCB 101, 153 and 180, alone and in combination. Cell viability was examined using a colorimetric assay based on the MTT labeling reagent. Apoptosis was evaluated by Annexin V flow cytometric assay and by the involvement of apoptotic related proteins, such as caspase-3 and Bcl-2/Bax ratio, using western blot analysis. Finally, to evaluate whether PCBs exert necrotic effect, apart from apoptosis pathways, we have also assessed lactate dehydrogenase (LDH) levels in culture supernatants. c. Results: ATDC5 and T/C-28a2 cell lines treated with PCBs, alone and in combination, showed a significant reduction of cell viability rate in a concentration-dependent manner. Neither synergisms nor additive effects were observed on cell viability with the combined treatment. Data from annexin V assays suggested that PCBs clearly induced apoptotic pathways, as well as, a certain rate of necrosis. Actually, this effect was confirmed by evaluating LDH levels that were strongly increased in supernatants of PCBs-treated cells, suggesting that necrotic mechanisms are at play too. PCBs also induced caspase-3 activation by increasing its proteolytic cleavage in a concentration-dependent manner. Finally, the Bcl2/Bax ratio was also altered. d. Conclusions: The viability of murine and human chondrocytes was reduced in presence of PCBs. The activity of PCBs on cell viability is likely to be mediated by alterations in the mechanisms of regulation of apoptosis and necrosis. Overall, this work highlights a novel role of environmental pollutants in the pathophysiology of chondrocytes. 292 IL-1b MEDIATES MMP SECRETION AND IL-1B NEOSYNTHESIS VIA UPREGULATION OF P22PHOX AND NOX4 ACTIVITY IN HUMAN ARTICULAR CHONDROCYTES F. Rousset y, F. Hazane Puch z, L. Grange yz, M. Nguyen y, C. Pinosa y, C. Combaz Lair z, B. Burroni z, B. Rubens-Duval z, F. Morel y, B. Lardy yz. yUniversit e Joseph Fourier, Grenoble, France; zCHU Grenoble, Grenoble,