Ameliorative Effects of Alpha-tocopherol and Crithmum Martimum L. Against Hydrogen Peroxide-induced Toxicity on Dental Pulp Stem Cells In Vitro

Year 2025, Volume: 26 Issue: 3, 363 - 373, 22.09.2025

Mukaddes Mergen Dalyanoğlu , Mücahit Seçme , Canan Eroğlu Güneş , Vural Küçükatay , Yavuz Dodurga , Ali Çelik , Sebahat Turgut

https://doi.org/10.69601/meandrosmdj.1684694

Abstract

Objective: Tooth-bleaching agents contain peroxide and are asserted to cause cellular damage. The study aims to evaluate the effects of the application of hydrogen peroxide (HP) on dental pulp stem cells (DPSCs) in vitro and whether alpha-tocopherol (AT) and Crithmum maritimum L. (CM) have protective effects against potential damage to occur.
Materials and Methods: HP, AT and CM doses applied in the experiment were determined according to the xtt analysis results. DPSCs were treated with 2 or 6 μg/mL HP, also 100 µM AT or 2 µg/mL CM followed by two doses of HP, DNA damage levels were measured using comet assay in 0, 24, 72 hours.
Results: Tail length, as a indicative of dna damage increased significantly in groups that applied only HP, compared to the control group. In contrast, the tail length of the groups that applied AT and CM was significantly shorter than that only HP-treated groups. The tail intensity and tail moment averages of all groups increased significantly than the control group at all times, except the mean tail moment of CM6HP, AT2HP groups, and mean tail intensity of the CM6HP group on the 72 hour. The tail intensity and tail moment averages of 6HP groups exhibited a significant increase compared to CM and AT applied groups at all times, except the AT6HP group on the 24th hour. The averages of 2HP group demonstrated a significant increase on the 24th hour compared to CM and AT applied groups, while no significance was observed on 0 and 72nd hours.
Conclusion: HP causes genotoxicity. AT and CM, however, reduce DNA damage, significantly.

Keywords

alpha-tocopherol , Crithmum martimum L. essential oil , toxicity , Dental Pulp Stem Cell , hydrogen peroxide

Ethical Statement

This article does not contain any studies involving animals or human participants performed by any of the authors. Since it was an in vitro study, ethics committee approval was not required.

Supporting Institution

This study was supported by grant 2014SBE005 from Pamukkale University Scientific Research Projects Coordination Unit (PAÜBAP)

Project Number

2014SBE005

Thanks

None to declare

Alfa-tokoferol ve Crithmum martimum L.'nin in vitro dental pulpa kök hücre kültüründe hidrojen peroksit kaynaklı toksisiteye karşı iyileştirici etkileri

Abstract

Amaç: Estetik gülüş sağlayan diş beyazlatıcı ajanların peroksit içermesi nedeniyle hücresel hasara neden olduğu iddia edilmektedir. Bu in vitro çalışmada hidrojen peroksit (HP) uygulamasının dental pulpa kök hücreleri (DPSCs) üzerindeki etkileri ve oluşabilecek hasara karşı alfa-tokoferol (AT) ve Crithmum maritimum L. (CM)'nin koruyucu etkilerinin olup olmadığının değerlendirilmesi amaçlanmıştır.
Gereç ve Yöntem: Deneyde uygulanan HP, AT, CM dozları xtt analiz sonuçlarına göre belirlenmiştir. Hücrelere ayrı ayrı olmak üzere hem 2 μg/mL hem de 6 μg/mL HP tek başına, ayrıca hücreler 100 µM AT veya 2 µg/mL CM ile muamele edildikten sonra da hücrelere ayrı ayrı bu iki doz HP uygulandı, DNA hasar seviyeleri 0, 24, 72. saatlerde comet analizi yapılarak ölçüldü.
Sonuçlar: Comet analizine göre kuyruk uzunluğu, kuyruk yoğunluğu ve kuyruk momentinin artışı DNA hasarının göstergesidir. Kuyruk uzunluğu, tek başına HP uygulanan gruplarda kontrol grubuna kıyasla anlamlı olarak artmıştır. Buna karşılık, AT ve CM uygulanan grupların kuyruk uzunluğu, tek başına HP uygulanan gruplara göre anlamlı derecede kısadır. Tüm grupların kuyruk yoğunluğu ve kuyruk momenti ortalamaları, sadece 72. saatte CM6HP, AT2HP gruplarının ortalama kuyruk momenti ve CM6HP grubunun ortalama kuyruk yoğunluğu hariç olmak üzere tüm zamanlarda kontrol grubuna göre önemli ölçüde artmıştır. 6HP gruplarının kuyruk yoğunluğu ve kuyruk momenti ortalamaları, 24. saatteki AT6HP grubu hariç, tüm zamanlarda, CM ve AT uygulanan gruplara kıyasla anlamlı artış göstermiştir. 2HP grubunun ortalamaları CM ve AT uygulanan gruplara kıyasla 24. saatte anlamlı artış gösterirken, 0 ve 72. Saatlerdeki artış anlamlı değildir.
Sonuç: HP genotoksisiteye neden olurken; bununla birlikte AT ve CM, DNA hasarını önemli ölçüde azaltmaktadır.

Keywords

alfa-tokoferol , Crithmum martimum L. uçucu yağı , toksisite , Diş Pulpası Kök Hücresi , hidrojen peroksit

Project Number

2014SBE005

References

• 1. Kwon SR, Wertz PW. Review of the mechanism of tooth whitening. Journal of Esthetic and Restorative Dentistry. 2015;27(5):240-257. https://doi.org/10.1111/jerd.12152
• 2. Roderjan DA, Stanislawczuk R, Hebling J, Costa CA de S, Reis A, Loguercio AD. Response of human pulps to different in-office bleaching techniques: Preliminary findings. Brazilian Dental Journal. 2015;26(3):242-248. https://doi.org/10.1590/0103-6440201302282
• 3. Vaz MM, Lopes LG, Cardoso PC, de Souza JB, Batista AC, Costa NL, Torres ÉM, Estrela C. Inflammatory response of human dental pulp to at-home and in-office tooth bleaching. Journal of Applied Oral Science. 2016;24(5):509-517. https://doi.org/10.1590/1678-775720160137
• 4. Cintra LTA, Benetti F, Da Silva Facundo AC, Ferreira LL, Gomes-Filho JE, Ervolino E, Rahal V, Briso ALF. The number of bleaching sessions influences pulp tissue damage in rat teeth. Journal of Endodontics. 2013;39(12):1576-1580. https://doi.org/10.1016/j.joen.2013.08.007
• 5. Harrison MS, Wang Y, Frick KJ, Moniz J, Walker MP. Effects of Alpha-tocopherol Antioxidant on Dentin-composite Microtensile Bond Strength after Sodium Perborate Bleaching. Journal of Endodontics. 2019;45(8):1053-1059. https://doi.org/10.1016/j.joen.2019.04.013
• 6. Chen C, Huang X, Zhu W, Ding C, Huang P, Li R. H2O2 gel bleaching induces cytotoxicity and pain conduction in dental pulp stem cells via intracellular reactive oxygen species on enamel/dentin disc. PLoS ONE 2021;16(9):1-17. e0257221. https://doi.org/10.1371/journal.pone.0257221.
• 7. Benetti F, Gomes-Filho JE, Ferreira LL, Ervolino E, Briso ALF, Sivieri-Araújo G, Dezan-Júnior E, Cintra LTA. Hydrogen peroxide induces cell proliferation and apoptosis in pulp of rats after dental bleaching in vivo: Effects of the dental bleaching in pulp. Archives of Oral Biology. 2017;81:103-109. https://doi.org/10.1016/j.archoralbio.2017.04.013
• 8. Soares DG, Marcomini N, Duque CC de O, Bordini EAF, Zuta UO, Basso FG, Hebling J, Costa CA de S. Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel. Journal of Applied Oral Science. 2019;27:1-10. https://doi.org/10.1590/1678-7757-2018-0453
• 9. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(25):13625-13630. https://doi.org/10.1073/pnas.240309797
• 10. Da Silveira Vargas F, Soares DG, Basso FG, Hebling J, De Souza Costa CA. Dose-Response and Time-Course of α-Tocoferol Mediating the Cytoprotection of Dental Pulp Cells against Hydrogen Peroxide. Brazilian Dental Journal. 2014;25(5):367-371. https://doi.org/10.1590/0103-6440201302434
• 11. Da Silveira Vargas F, Soares DG, Ribeiro APD, Hebling J, De Souza Costa CA. Protective Effect of Alpha-Tocopherol Isomer from Vitamin Eagainst the H2O2 Induced Toxicity on Dental Pulp Cells. BioMed Research International. Volume 2014, Article ID 895049, 5 pages. http://dx.doi.org/10.1155/2014/895049
• 12. Kızıldağ A, Mergen Dalyanoğlu M, Yazkan B, Kara R. Crithmum Maritimum L.’nin Farklı Konsantrasyonlarının İnsan Periodontal Ligament Fibroblast Hücreleri Üzerine Etkileri. Turkiye Klinikleri J Dental Sci. 2020;26(1):31-5. DOI: 10.5336/dentalsci.2019-65336.
• 13. Atia A, Barhoumi Z, Mokded R, Abdelly C, Smaoui A. Environmental eco-physiology and economical potential of the halophyte Crithmum maritimum L. (Apiaceae). Journal of Medicinal Plants Research. 2011;5(16):3564-3571.
• 14. Dzhoglova V, Ivanova S, Shishmanova-Doseva M and Kremena Saracheva. Crithmum maritimum L.: Phytochemical Profile, Biological Activities, and Therapeutic Potential. Molecules 2025, 30, 2832. https://doi.org/10.3390/molecules30132832.
• 15. Meot-Duros L, Magné C. Antioxidant activity and phenol content of Crithmum maritimum L. leaves. Plant Physiology and Biochemistry. 2009;47(1):37-41. https://doi.org/10.1016/j.plaphy.2008.09.006
• 16. Soares DG, Basso FG, Hebling J, De Souza Costa CA. Concentrations of and application protocols for hydrogen peroxide bleaching gels: Effects on pulp cell viability and whitening efficacy. Journal of Dentistry. 2014;42(2):185-198. https://doi.org/10.1016/j.jdent.2013.10.021
• 17. Celik A, Arslan I, Herken EN, Ermis A. Constituents, Oxidant-Antioxidant Profile, and Antimicrobial Capacity of the Essential Oil Obtained from Ferulago Sandrasica Peşmen and Quézel. International Journal of Food Properties. 2013;16(8):1655-166. http://dx.doi.org/10.1080/10942912.2011.618898
• 18. Araldi RP, de Melo TC, Mendes TB, de Sá Júnior PL, Nozima BHN, Ito ET, de Carvalho RF, de Souza EB, de Cassia Stocco R. Using the comet and micronucleus assays for genotoxicity studies: A review. Biomedicine and Pharmacotherapy. 2015;72:74-82. https://doi.org/10.1016/j.biopha.2015.04.004
• 19. Mincarelli L, Vischetti C, Craft J, Tiano L. DNA damage in different Eisenia andrei coelomocytes sub-populations after in vitro exposure to hydrogen peroxide. SpringerPlus. 2016;5(1):302. https://doi.org/10.1186/s40064-016-1950-x
• 20. Klaric E, Par M, Profeta I, Kopjar N, Rozgaj R, Kasuba V, Zeljezic D, Tarle Z. Genotoxic effect of two bleaching agents on oral mucosa. Cancer Genomics and Proteomics. 2013;10(5):209-215.
• 21. de Souza Costa CA, Riehl H, Kina JF, Sacono NT, Hebling J. Human pulp responses to in-office tooth bleaching. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology. 2010;109(4):e59-e64. https://doi.org/10.1016/j.tripleo.2009.12.002
• 22. Ribeiro DA, Marques MEA, Salvadori DMF. Assessment of genetic damage induced by dental bleaching agents on mouse lymphoma cells by single cell gel (comet) assay. Journal of Oral Rehabilitation. 2005;32(10):766-771. https://doi.org/10.1111/j.1365-2842.2005.01500.x
• 23. Collins AR. The comet assay for DNA damage and repair: Principles, applications, and limitations. Applied Biochemistry and Biotechnology - Part B Molecular Biotechnology. 2004;26(3):249-261. https://doi.org/10.1385/MB:26:3:249
• 24. Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, Smith CC, Štětina R. The comet assay: Topical issues. Mutagenesis. 2008;23(3):143-151. https://doi.org/10.1093/mutage/gem051
• 25. Yedjou CG, Tchounwou HM, Tchounwou PB. DNA damage, cell cycle arrest, and apoptosis induction caused by lead in human leukemia cells. International Journal of Environmental Research and Public Health. 2015;13(1):56. https://doi.org/10.3390/ijerph13010056
• 26. Ribeiro DA, Marques MEA, Salvadori DMF. Study of DNA damage induced by dental bleaching agents in vitro. Brazilian Oral Research. 2006;20(1):47-51. https://doi.org/10.1590/s1806-83242006000100009
• 27. Pligina KL, Rodina IA, Shevchenko TV, Bekchanova ES, Tikhonov VP, Sirota NP. DNA-damaging effects of dental bleaching agents. Bulletin of Experimental Biology and Medicine. 2012;153(1):57-60. https://doi.org/10.1007/s10517-012-1642-7
• 28. Atkinson J, Epand RF, Epand RM. Tocopherols and tocotrienols in membranes: A critical review. Free Radical Biology and Medicine. 2008;44(5):739-764. https://doi.org/10.1016/j.freeradbiomed.2007.11.010
• 29. Kim YJ, Ahn YH, Lim Y, Kim JY, Kim J, Kwon O. Daily nutritional dose supplementation with antioxidant nutrients and phytochemicals improves DNA and LDL stability: A double-blind, randomized, and placebo-controlled trial. Nutrients. 2013;5(12):5218-5232. https://doi.org/10.3390/nu5125218
• 30. Fernández MR, Carvalho RV, Ogliari FA, Beira FA, Etges A, Bueno M. Cytotoxicity and genotoxicity of sodium percarbonate: A comparison with bleaching agents commonly used in discoloured pulpless teeth. International Endodontic Journal. 2010;43(2):102-108. https://doi.org/10.1111/j.1365-2591.2009.01648.x
• 31. Goldberg M, Grootveld M, Lynch E. Undesirable and adverse effects of tooth-whitening products: A review. Clinical Oral Investigations.2010;14(1):1-10. https://doi.org/10.1007/s00784-009-0302-4
• 32. Vinoth KJ, Manikandan J, Sethu S, Balakrishnan L, Heng A, Lu K, Poonepalli A, Hande MP, Cao T. Differential resistance of human embryonic stem cells and somatic cell types to hydrogen peroxide-induced genotoxicity may be dependent on innate basal intracellular ROS levels. Folia Histochemica et Cytobiologica. 2015;53(2):169-174. https://doi.org/10.5603/FHC.a2015.0016
• 33. Squier TC. Oxidative stress and protein aggregation during biological aging. Exp Gerontol. 2001;36(9):1539-1550. https://doi.org/10.1016/s0531-5565(01)00139-5
• 34. Cecarini V, Gee J, Fioretti E, Amici M, Angeletti M, Eleuteri AM, Keller JN. Protein oxidation and cellular homeostasis: Emphasis on metabolism. Biochimica et Biophysica Acta - Molecular Cell Research. 2007;1773(2):93-104. https://doi.org/10.1016/j.bbamcr.2006.08.039
• 35. Azzi A. Molecular mechanism of α-tocopherol action. Free Radical Biology and Medicine. 2007;43(1):16-21. https://doi.org/10.1016/j.freeradbiomed.2007.03.013
• 36. Makpol, S., Zainuddin, A., Rahim, N. A., Yusof, Y. A. M., & Ngah, W. Z. W. (2010). Alpha-tocopherol modulates hydrogen peroxide-induced DNA damage and telomere shortening of human skin fibroblasts derived from differently aged individuals. Planta Medica, 76(9), 869-875. https://doi.org/10.1055/s-0029-1240812
• 37. Yurdakul T, Kulaksizoglu H, Pişkin MM, Avunduk MC, Ertemli E, Barişkaner H, Büyükbaş S, Kocabas V. Combination antioxidant effect of α-tocoferol and erdosteine in ischemia-reperfusion injury in rat model. International Urology and Nephrology. 2010;42(3):647-655. https://doi.org/10.1007/s11255-009-9641-y
• 38. Tahan G, Aytac E, Aytekin H, Gunduz F, Dogusoy G, Aydin S, Tahan V, Uzun H. Vitamin e has a dual effect of anti-inflammatory and antioxidant activities in acetic acid-induced ulcerative colitis in rats. Canadian Journal of Surgery. 2011;54(5):333-338. https://doi.org/10.1503/cjs.013610
• 39. Guney M, Erdemoglu E, Mungan T. Selenium-vitamin e combination and melatonin modulates diabetes-induced blood oxidative damage and fetal outcomes in pregnant rats. Biological Trace Element Research. 2011;143(2):1091-1102. https://doi.org/10.1007/s12011-010-8951-3
• 40. Yilmaz N, Yilmaz M, Altuntas I. Diazinon-induced brain toxicity and protection by vitamins e plus C. Toxicology and Industrial Health. 2012;28(1):51-57. https://doi.org/10.1177/0748233711404035
• 41. Lequeux C, Lhoste A, Rovere MR, Montastier C, Damour O. Model of in vitro healing to test the influence of dedifferentiated crithmum maritimum cells on dermal repair and epidermal regeneration. Skin Pharmacology and Physiology. 2011;24(2):75-80. https://doi.org/10.1159/000321991