The aim of the present study is to investigate protective effects of hydrogen-rich water (HW) against reactive oxygen species (ROS)-induced cellular harmful events and cell death in human gingival fibroblasts (HGF) and three-dimensional (3D-) gingival tissue equivalents. HW was prepared with a magnesium stick in 600-mL double distilled water (DDW) overnight. Dissolved hydrogen was about 1460 ± 50 μg/L versus approximately 1600 μg/L for the saturated hydrogen. Under cell-free conditions, HW, dose-dependently, significantly scavenged peroxyl radicals (ROO·) derived from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). Extract from HW-treated HGF cells scavenged ROO· more markedly than that from DDW-treated cells, suggesting that HW can increase the intracellular antioxidant capacity. Hydrogen peroxide dose-dependently increased the intracellular ROS generation, which was significantly repressed by HW, both in the cytoplasm and nuclei. LIVE/DEAD staining and our original cell viability dye-extraction assay showed that HW significantly protected HGF cells from hydrogen peroxide-induced cell death. Hydrogen peroxide also diminished the contents of intracellular glutathione, which were appreciably relieved by HW-pretreatment. Additionally, HW noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-gingival equivalents. The in vitro scratch assay showed that HW was able to diminish physical injury-induced ROS generation and promote wound healing in HGF cell monolayer sheets. In summary, HW was able to increase intracellular antioxidative capacity and to protect cells and tissue from oxidative damage. Thus, HW might be used for prevention/treatment of oxidative stress-related diseases.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Van Linthout S, Miteva K, Tschöpe C. Crosstalk between fibroblasts and inflammatory cells. Cardiovasc Res. 2014;102:258–69.
Guo S, DiPietro LA. Factors affecting wound healing. J Dent Res. 2010;89:219–29.
Sculley DV, Langley-Evans SC. Salivary antioxidants and periodontal disease status. Proc Nutr Soc. 2002;61:137–43.
Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Beher D, Masters CL, et al. Reactive oxygen species and Alzheimer’s disease. Biochem Pharmacol. 1997;54:533–9.
Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol. 2009;7:65–74.
Hori M, Nishida K. Oxidative stress and left ventricular remodelling after myocardial infarction. Cardiovasc Res. 2009;81:457–64.
Rani V, Deep G, Singh RK, Palle K, Yadav UC. Oxidative stress and metabolic disorders: pathogenesis and therapeutic strategies. Life Sci. 2016;148:183–93.
Colavitti R, Finkel T. Reactive oxygen species as mediators of cellular senescence. IUBMB Life. 2005;57:277–81.
Wikesjö UM, Selvig KA. Periodontal wound healing and regeneration. Periodontol. 2000;1999(19):21–39.
Galasko DR, Peskind E, Clark CM, Quinn JF, Ringman JM, Jicha GA, et al. Alzheimer’s disease cooperative study. Antioxidants for Alzheimer disease: a randomized clinical trial with cerebrospinal fluid biomarker measures. Arch Neurol. 2012;69:836–41.
Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA. 2007;297:842–57.
Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med. 2007;13:688–94.
Lin CL, Huang WN, Li HH, Huang CN, Hsieh S, Lai C, et al. Hydrogen-rich water attenuates amyloid β-induced cytotoxicity through upregulation of Sirt1-FoxO3a by stimulation of AMP-activated protein kinase in SK-N-MC cells. Chem Biol Interact. 2015;240:12–21.
Miyazaki N, Yamaguchi O, Nomiya M, Aikawa K, Kimura J. Preventive effect of hydrogen water on the development of detrusor overactivity in a rat model of bladder outlet obstruction. J Urol. 2016;195:780–7.
Takeuchi S, Nagatani K, Otani N, Nawashiro H, Sugawara T, Wada K, et al. Hydrogen improves neurological function through attenuation of blood-brain barrier disruption in spontaneously hypertensive stroke-prone rats. BMC Neurosci. 2015;16:22.
Zhang JY, Song SD, Pang Q, Zhang RY, Wan Y, Yuan DW, et al. Hydrogen-rich water protects against acetaminophen-induced hepatotoxicity in mice. World J Gastroenterol. 2015;21:4195–209.
Kajiyama S, Hasegawa G, Asano M, Hosoda H, Fukui M, Nakamura N, et al. Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance. Nutr Res. 2008;28:137–43.
Song G, Li M, Sang H, Li X, Yao S, Yu Y, et al. Hydrogen-rich water decreases serum LDL-cholesterol levels and improves HDL function in patients with potential metabolic syndrome. J Lipid Res. 2013;54:1884–93.
Nakayama M, Nakano H, Hamada H, Itami N, Nakazawa R, Ito S. A novel bioactive haemodialysis system using dissolved dihydrogen (H2) produced by water electrolysis: a clinical trial. Nephrol Dial Transplant. 2010;25:3026–33.
Ishibashi T. Molecular hydrogen: new antioxidant and anti-inflammatory therapy for rheumatoid arthritis and related diseases. Curr Pharm Des. 2013;19:6375–81.
Hong Y, Shao A, Wang J, Chen S, Wu H, McBride DW, et al. Neuroprotective effect of hydrogen-rich saline against neurologic damage and apoptosis in early brain injury following subarachnoid hemorrhage: possible role of the Akt/GSK3β signaling pathway. PLoS One. 2014;9:e96212.
Xia C, Liu W, Zeng D, Zhu L, Sun X, Sun X. Effect of hydrogen-rich water on oxidative stress, liver function, and viral load in patients with chronic hepatitis B. Clin Transl Sci. 2013;6:372–5.
Schoenfeld MP, Ansari RR, Zakrajsek JF, Billiar TR, Toyoda Y, Wink DA, et al. Hydrogen therapy may reduce the risks related to radiation-induced oxidative stress in space flight. Med Hypotheses. 2011;76:117–8.
Ohta S. Recent progress toward hydrogen medicine: potential of molecular hydrogen for preventive and therapeutic applications. Curr Pharm Des. 2011;17:2241–52.
Seo T, Kurokawa R, Sato B. Convenient method for determining the concentration of hydrogen in water: use of methylene blue with colloidal platinum. Med Gas Res. 2012;2:1.
Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988;106:761–71.
Burgess A, Vigneron S, Brioudes E, Labbé JC, Lorca T, Castro A. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA. 2010;107:12564–9.
Xiao L, Tsutsui T, Miwa N. The lipophilic vitamin C derivative, 6-opalmitoylascorbate, protects human lymphocytes, preferentially over ascorbate, against X-ray-induced DNA damage, lipid peroxidation, and protein carbonylation. Mol Cell Biochem. 2014;394:247–59.
Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2:329–33.
Ng CF, Schafer FQ, Buettner GR, Rodgers VG. The rate of cellular hydrogen peroxide removal shows dependency on GSH: mathematical insight into in vivo H2O2 and GPx concentrations. Free Radic Res. 2007;41:1201–11.
Li S, Yan T, Yang JQ, Oberley TD, Oberley LW. The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res. 2000;60:3927–39.
Im YS, Ryu YK, Moon EY. Mouse melanoma cell migration is dependent on production of reactive oxygen species under normoxia condition. Biomol Ther (Seoul). 2012;20:165–70.
Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003;552:335–44.
Di Mascio P, Murphy ME, Sies H. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am J Clin Nutr. 1991;53:194S–200S.
Cadet J, Douki T, Ravanat JL. Oxidatively generated base damage to cellular DNA. Free Radic Biol Med. 2010;49:9–21.
Manda K, Ueno M, Anzai K. AFMK, a melatonin metabolite, attenuates X-ray-induced oxidative damage to DNA, proteins and lipids in mice. J Pineal Res. 2007;42:386–93.
Corre I, Niaudet C, Paris F. Plasma membrane signaling induced by ionizing radiation. Mutat Res. 2010;704:61–7.
Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, Leapman RD, et al. Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS Biol. 2007;5:e92.
Haro KJ, Sheth A, Scheinberg DA. Dysregulation of IRP1-mediated iron metabolism causes gamma ray-specific radioresistance in leukemia cells. PLoS One. 2012;7:e48841.
Baraibar MA, Ladouce R, Friguet B. Proteomic quantification and identification of carbonylated proteins upon oxidative stress and during cellular aging. J Proteomics. 2013;92:63–70.
Basu S, Helmersson J, Jarosinska D, Sällsten G, Mazzolai B, Barregård L. Regulatory factors of basal F(2)-isoprostane formation: population, age, gender and smoking habits in humans. Free Radic Res. 2009;43:85–91.
D’Aiuto F, Nibali L, Parkar M, Patel K, Suvan J, Donos N. Oxidative stress, systemic inflammation, and severe periodontitis. J Dent Res. 2010;89:1241–6.
Boyapati L, Wang HL. The role of stress in periodontal disease and wound healing. Periodontol. 2000;2007(44):195–210.
Ara T, Kurata K, Hirai K, Uchihashi T, Uematsu T, Imamura Y, et al. Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease. J Periodontal Res. 2009;44:21–7.
Horton AL, Boggess KA, Moss KL, Beck J, Offenbacher S. Periodontal disease, oxidative stress, and risk for preeclampsia. J Periodontol. 2010;81:199–204.
Pendyala G, Thomas B, Kumari S. The challenge of antioxidants to free radicals in periodontitis. J Indian Soc Periodontol. 2008;12:79–83.
Kato S, Matsuoka D, Miwa N. Antioxidant activities of nano-bubble hydrogen-dissolved water assessed by ESR and 2,2′-bipyridyl methods. Mater Sci Eng C Mater Biol Appl. 2015;53:7–10.
D’Autréaux B, Toledano MB. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol. 2007;8:813–24.
Halliwell B. The antioxidant paradox: less paradoxical now? Br J Clin Pharmacol. 2013;75:637–44.
The present study was supported in part by a Grant-in-Aid for AntiAging Scientific Research #1517 by Japanese Center for AntiAging MedSciences, Hiroshima, Japan to XL.
Conflict of interest
The authors declare no conflicts of interest associated with this manuscript.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Xiao, L., Miwa, N. Hydrogen-rich water achieves cytoprotection from oxidative stress injury in human gingival fibroblasts in culture or 3D-tissue equivalents, and wound-healing promotion, together with ROS-scavenging and relief from glutathione diminishment. Human Cell 30, 72–87 (2017). https://doi.org/10.1007/s13577-016-0150-x