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Nitric oxide-mediated cytotoxic effect induced by zoledronic acid treatment on human gingival fibroblasts

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Abstract

Objectives

The aim of our work was to evaluate the role of nitric oxide (NO) in the in vitro response of human gingival fibroblasts (HGFs) to 1, 5, 10, and 100 μM doses of zoledronic acid (ZA), a bisphosphonate largely used in the clinical practice and for which several adverse effects are reported.

Materials and methods

Phase contrast microscopy and live/dead staining were used to evaluate HGFs morphology; cell viability, collagen type I and interleukin 6 (IL-6) secretion were evaluated by 3-[4,5-dimethyl-thiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA) assays. Reactive oxygen species (ROS) production and mitochondrial membrane potential were evaluated by flow cytometry; NO production and NOS activity by spectrophotometric analysis; endothelial NOS (eNOS) and neuronal NOS (nNOS) expression by immunofluorescence.

Results

Viable fibroblasts are evidenced in control sample while floating dead cells and cells close to detachment phase in ZA-treated sample, in agreement with decreased level of collagen type I. Control sample shows higher number of viable cells respect to ZA-treated one and ROS production increases when ZA is added. Released NO in ZA-treated sample appears higher and NO overproduction is related to increased nNOS activity. IL-6 secretion level is higher in ZA-treated sample than in control one.

Conclusions

Our results suggest ROS involvement in NO overproduction, due to nNOS recruitment, both at low and high doses. In turn, NO release seems to be able to trigger the inflammatory response only when high doses are administered, thus confirming the ZA cytotoxic effect on HGFs.

Clinical relevance

The knowledge of ZA-mediated cytotoxicity mechanisms on HGFs allows to better understand drug pharmacological activity.

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Acknowledgments

The authors would like to thank Dott.ssa Piera Sozio for drug purity determinations, Dott.ssa Alessandra di Paolo for the kind gift of Zometa, and MIUR FIRB-Accordi di Programma 2010 Project “Processi degenerativi dei tessuti mineralizzati del cavo orale, impiego di biomateriali e controllo delle interazioni con i microrganismi dell’ambiente” for the fellowships attributed to Dr. V.L. Zizzari and Dr. M. De Colli.

Conflict of interest

The authors declare that there is no conflict of interest.

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Authors and Affiliations

  1. Section of Human Anatomy, Department of Pharmacy, University “G. d’Annunzio”, Chieti Pescara, Italy

    Marianna De Colli, Susi Zara, Viviana di Giacomo, Guya Diletta Marconi, Marialucia Gallorini, Vincenzo Luca Zizzari, Giulia Tetè & Amelia Cataldi

  2. Department of Medicine and Sciences of Aging, University “G. d’Annunzio”, Chieti Pescara, Italy

    Antonia Patruno

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  1. Marianna De Colli
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  2. Susi Zara
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Correspondence to Susi Zara.

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De Colli, M., Zara, S., di Giacomo, V. et al. Nitric oxide-mediated cytotoxic effect induced by zoledronic acid treatment on human gingival fibroblasts. Clin Oral Invest 19, 1269–1277 (2015). https://doi.org/10.1007/s00784-014-1344-9

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  • DOI: https://doi.org/10.1007/s00784-014-1344-9

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