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Cytotoxic effects of a chlorhexidine mouthwash and of an enzymatic mouthwash on human gingival fibroblasts

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Abstract

The aim of this study was to evaluate the cytotoxic effects of an enzymatic mouthwash and of a chlorhexidine mouthwash on human gingival fibroblasts. The metabolic activity of the fibroblasts exposed to each mouthwash was assessed by the MTT assay and the protein content was assessed by the SRB assay. The flow cytometry was used to evaluate the cell cycle and the types of cell death. The oxidative status was evaluated through the DCF and the DHE probes and the intracellular GSH concentration and the mitochondrial membrane potential through JC-1. The cytotoxicity of both mouthwashes was found to be dependent on the exposure time and on the concentration. However, the cytotoxicity of the enzymatic mouthwash was found to be lower than that of the chlorhexidine mouthwash. A trend towards increased oxidative stress was observed for both mouthwashes. After exposing the fibroblasts to the mouthwashes, a G2/M phase block was observed and cell death occurred predominantly by necrosis. The effects of chlorhexidine on fibroblasts were identified at lower concentrations than those used in clinical practice. Therefore, the use of chlorhexidine as an antiseptic in surgical and postoperative situations should be limited. In order to clarify the clinical significance of the enzymatic mouthwash cytotoxicity new clinical studies will be necessary.

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References

  1. Tenovuo J. Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral Dis. 2002;8(1):23–9.

    Article  Google Scholar 

  2. Soukka T, Lumikari M, Tenovuo J. Combined bactericidal effect of human lactoferrin and lysozyme against Streptococcus mutans serotype c. Microb Ecol Health Dis. 1991;4:259–64.

    Google Scholar 

  3. Lenander-Lumikari M, Mansson-Rahemtulla B, Rahemtulla F. Lysozyme enhances the inhibitory effects of the peroxidase system on glucose metabolism of Streptococcus mutans. J Dent Res. 1992;71(3):484–90. https://doi.org/10.1177/00220345920710031201.

    Article  PubMed  Google Scholar 

  4. Lynge Pedersen AM, Belstrom D. The role of natural salivary defences in maintaining a healthy oral microbiota. J Dent. 2019;80(Suppl 1):S3–12. https://doi.org/10.1016/j.jdent.2018.08.010.

    Article  PubMed  Google Scholar 

  5. Kirstila V, Lenander-Lumikari M, Soderling E, Tenovuo J. Effects of oral hygiene products containing lactoperoxidase, lysozyme, and lactoferrin on the composition of whole saliva and on subjective oral symptoms in patients with xerostomia. Acta Odontol Scand. 1996;54(6):391–7.

    Article  Google Scholar 

  6. Lenander-Lumikari M, Tenovuo J, Mikola H. Effects of a lactoperoxidase system-containing toothpaste on levels of hypothiocyanite and bacteria in saliva. Caries Res. 1993;27(4):285–91. https://doi.org/10.1159/000261552.

    Article  PubMed  Google Scholar 

  7. Tenovuo J, Mansson-Rahemtulla B, Pruitt KM, Arnold R. Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system. Infect Immun. 1981;34(1):208–14.

    Article  Google Scholar 

  8. Supranoto SC, Slot DE, Addy M, Weijden GA. The effect of chlorhexidine dentifrice or gel versus chlorhexidine mouthwash on plaque, gingivitis, bleeding and tooth discoloration: a systematic review. Int J Dent Hyg. 2015;13(2):83–92. https://doi.org/10.1111/idh.12078.

    Article  PubMed  Google Scholar 

  9. Coelho A, Paula ABP, Carrilho TMP, da Silva M, Botelho M, Carrilho E. Chlorhexidine mouthwash as an anticaries agent: a systematic review. Quintessence Int. 2017;48(7):585–91. https://doi.org/10.3290/j.qi.a38353.

    Article  PubMed  Google Scholar 

  10. Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A. Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. Toxicol In Vitro. 2008;22(2):308–17. https://doi.org/10.1016/j.tiv.2007.09.012.

    Article  PubMed  Google Scholar 

  11. Oncag O, Hosgor M, Hilmioglu S, Zekioglu O, Eronat C, Burhanoglu D. Comparison of antibacterial and toxic effects of various root canal irrigants. Int Endod J. 2003;36(6):423–32.

    Article  Google Scholar 

  12. Pucher JJ, Daniel JC. The effects of chlorhexidine digluconate on human fibroblasts in vitro. J Periodontol. 1992;63(6):526–32. https://doi.org/10.1902/jop.1992.63.6.526.

    Article  PubMed  Google Scholar 

  13. Babich H, Wurzburger BJ, Rubin YL, Sinensky MC, Blau L. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol. 1995;11(2):79–88.

    Article  Google Scholar 

  14. Chang YC, Huang FM, Tai KW, Chou MY. The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;92(4):446–50. https://doi.org/10.1067/moe.2001.116812.

    Article  PubMed  Google Scholar 

  15. Damour O, Hua SZ, Lasne F, Villain M, Rousselle P, Collombel C. Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes. Burns. 1992;18(6):479–85.

    Article  Google Scholar 

  16. Tatnall FM, Leigh IM, Gibson JR. Comparative study of antiseptic toxicity on basal keratinocytes, transformed human keratinocytes and fibroblasts. Skin Pharmacol. 1990;3(3):157–63.

    Article  Google Scholar 

  17. Segura JJ, Jimenez-Rubio A, Guerrero JM, Calvo JR. Comparative effects of two endodontic irrigants, chlorhexidine digluconate and sodium hypochlorite, on macrophage adhesion to plastic surfaces. J Endod. 1999;25(4):243–6. https://doi.org/10.1016/S0099-2399(99)80151-4.

    Article  PubMed  Google Scholar 

  18. Cabral CT, Fernandes MH. In vitro comparison of chlorhexidine and povidone-iodine on the long-term proliferation and functional activity of human alveolar bone cells. Clin Oral Investig. 2007;11(2):155–64. https://doi.org/10.1007/s00784-006-0094-8.

    Article  PubMed  Google Scholar 

  19. Bhandari M, Adili A, Schemitsch EH. The efficacy of low-pressure lavage with different irrigating solutions to remove adherent bacteria from bone. J Bone Joint Surg Am. 2001;83-A(3):412–9.

    Article  Google Scholar 

  20. Helgeland K, Heyden G, Rolla G. Effect of chlorhexidine on animal cells in vitro. Scand J Dent Res. 1971;79(3):209–15.

    PubMed  Google Scholar 

  21. Gabler WL, Roberts D, Harold W. The effect of chlorhexidine on blood cells. J Periodontal Res. 1987;22(2):150–5.

    Article  Google Scholar 

  22. Goldschmidt P, Cogen R, Taubman S. Cytopathologic effects of chlorhexidine on human cells. J Periodontol. 1977;48(4):212–5. https://doi.org/10.1902/jop.1977.48.4.212.

    Article  PubMed  Google Scholar 

  23. Alleyn CD, O’Neal RB, Strong SL, Scheidt MJ, Dyke TE, McPherson JC. The effect of chlorhexidine treatment of root surfaces on the attachment of human gingival fibroblasts in vitro. J Periodontol. 1991;62(7):434–8. https://doi.org/10.1902/jop.1991.62.7.434.

    Article  PubMed  Google Scholar 

  24. Balloni S, Locci P, Lumare A, Marinucci L. Cytotoxicity of three commercial mouthrinses on extracellular matrix metabolism and human gingival cell behaviour. Toxicol In Vitro. 2016;34:88–96. https://doi.org/10.1016/j.tiv.2016.03.015.

    Article  PubMed  Google Scholar 

  25. Mariotti AJ, Rumpf DA. Chlorhexidine-induced changes to human gingival fibroblast collagen and non-collagen protein production. J Periodontol. 1999;70(12):1443–8. https://doi.org/10.1902/jop.1999.70.12.1443.

    Article  PubMed  Google Scholar 

  26. Eren K, Ozmeric N, Sardas S. Monitoring of buccal epithelial cells by alkaline comet assay (single cell gel electrophoresis technique) in cytogenetic evaluation of chlorhexidine. Clin Oral Investig. 2002;6(3):150–4. https://doi.org/10.1007/s00784-002-0168-1.

    Article  PubMed  Google Scholar 

  27. Wyganowska-Swiatkowska M, Kotwicka M, Urbaniak P, Nowak A, Skrzypczak-Jankun E, Jankun J. Clinical implications of the growth-suppressive effects of chlorhexidine at low and high concentrations on human gingival fibroblasts and changes in morphology. Int J Mol Med. 2016;37(6):1594–600. https://doi.org/10.3892/ijmm.2016.2550.

    Article  PubMed  Google Scholar 

  28. Flemingson, Emmadi P, Ambalavanan N, Ramakrishnan T, Vijayalakshmi R. Effect of three commercial mouth rinses on cultured human gingival fibroblast: an in vitro study. Indian J Dent Res. 2008;19(1):29–35.

    Article  Google Scholar 

  29. Hidalgo E, Dominguez C. Mechanisms underlying chlorhexidine-induced cytotoxicity. Toxicol In Vitro. 2001;15(4–5):271–6.

    Article  Google Scholar 

  30. Rajabalian S, Mohammadi M, Mozaffari B. Cytotoxicity evaluation of Persica mouthwash on cultured human and mouse cell lines in the presence and absence of fetal calf serum. Indian J Dent Res. 2009;20(2):169–73. https://doi.org/10.4103/0970-9290.52894.

    Article  PubMed  Google Scholar 

  31. Verma UP, Dixit J. Development of a human gingival fibroblast (HGF) cell line for the evaluation of a novel mouthwash from azadirachta indica vis-à-vis chlorhexidine. Int J Pharm Pharm Sci. 2012;4(2):217–21.

    Google Scholar 

  32. Cline NV, Layman DL. The effects of chlorhexidine on the attachment and growth of cultured human periodontal cells. J Periodontol. 1992;63(7):598–602. https://doi.org/10.1902/jop.1992.63.7.598.

    Article  PubMed  Google Scholar 

  33. Tsourounakis I, Palaiologou-Gallis AA, Stoute D, Maney P, Lallier TE. Effect of essential oil and chlorhexidine mouthwashes on gingival fibroblast survival and migration. J Periodontol. 2013;84(8):1211–20. https://doi.org/10.1902/jop.2012.120312.

    Article  PubMed  Google Scholar 

  34. Pisoschi AM, Pop A. The role of antioxidants in the chemistry of oxidative stress: a review. Eur J Med Chem. 2015;97:55–74. https://doi.org/10.1016/j.ejmech.2015.04.040.

    Article  PubMed  Google Scholar 

  35. Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol. 2014;24(10):R453–62. https://doi.org/10.1016/j.cub.2014.03.034.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Aliko A, Alushi A, Tafaj A, Isufi R. Evaluation of the clinical efficacy of Biotene Oral Balance in patients with secondary Sjogren’s syndrome: a pilot study. Rheumatol Int. 2012;32(9):2877–81. https://doi.org/10.1007/s00296-011-2085-6.

    Article  PubMed  Google Scholar 

  37. Shahdad SA, Taylor C, Barclay SC, Steen IN, Preshaw PM. A double-blind, crossover study of Biotene Oralbalance and BioXtra systems as salivary substitutes in patients with post-radiotherapy xerostomia. Eur J Cancer Care (Engl). 2005;14(4):319–26. https://doi.org/10.1111/j.1365-2354.2005.00587.x.

    Article  Google Scholar 

  38. Jose A, Siddiqi M, Cronin M, DiLauro TS, Bosma ML. A randomized clinical trial in subjects with dry mouth evaluating subjective perceptions of an experimental oral gel, an oral rinse and a mouth spray compared to water. Am J Dent. 2016;29(1):58–64.

    PubMed  Google Scholar 

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Funding

This work was supported by a grant from Portuguese Diabetes Association (“Bolsa de Estudo Pedro Eurico Lisboa SPD/BAYER”, 2015), CNC.IBILI UID/NEU/04539/2013, PT2020 - COMPETE 2020 and (POCI) POCI-01-0145-FEDER-007440.

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Authors and Affiliations

  1. Institute of Integrated Clinical Practice, Faculty of Medicine, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Ana Sofia Coelho, Anabela Paula, Siri Paulo, Manuel Marques Ferreira & Eunice Carrilho

  2. Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Ana Sofia Coelho, Mafalda Laranjo, Ana Cristina Gonçalves, Anabela Paula, Siri Paulo, Ana Margarida Abrantes, Francisco Caramelo, Manuel Marques Ferreira, Eunice Carrilho & Maria Filomena Botelho

  3. Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Ana Sofia Coelho, Mafalda Laranjo, Ana Cristina Gonçalves, Anabela Paula, Siri Paulo, Ana Margarida Abrantes, Francisco Caramelo, Manuel Marques Ferreira, Eunice Carrilho & Maria Filomena Botelho

  4. CIMAGO – Center of Investigation on Enviromnent, Genetics and Oncobiology, Faculty of Medicine, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Ana Sofia Coelho, Mafalda Laranjo, Ana Cristina Gonçalves, Anabela Paula, Siri Paulo, Ana Margarida Abrantes, Francisco Caramelo, Manuel Marques Ferreira, Eunice Carrilho & Maria Filomena Botelho

  5. CNC.IBILI, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Ana Sofia Coelho, Mafalda Laranjo, Ana Cristina Gonçalves, Anabela Paula, Siri Paulo, Ana Margarida Abrantes, Francisco Caramelo, Manuel Marques Ferreira, Eunice Carrilho & Maria Filomena Botelho

  6. Biophysics Institute, Faculty of Medicine, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Mafalda Laranjo, Ana Cristina Gonçalves, Ana Margarida Abrantes & Maria Filomena Botelho

  7. Endodontics Institute, Faculty of Medicine, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Siri Paulo & Manuel Marques Ferreira

  8. Laboratory of Biostatistics and Medical Informatics, Faculty of Medicine, University of Coimbra, Área de Medicina Dentária, Av. Bissaya Barreto, Bloco de Celas, 3000-075, Coimbra, Portugal

    Francisco Caramelo

  9. Faculty of Dental Medicine, University of Oporto, Oporto, Portugal

    Mário Jorge Silva

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  1. Ana Sofia Coelho
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  2. Mafalda Laranjo
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Correspondence to Ana Sofia Coelho.

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Coelho, A.S., Laranjo, M., Gonçalves, A.C. et al. Cytotoxic effects of a chlorhexidine mouthwash and of an enzymatic mouthwash on human gingival fibroblasts. Odontology 108, 260–270 (2020). https://doi.org/10.1007/s10266-019-00465-z

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