Skip to main content

Advertisement

SpringerLink
Log in

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model

  • Original Paper
  • Published:
Archives of Dermatological Research Aims and scope Submit manuscript

Abstract

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, β-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2′-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with β-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2 · − in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Bech-Thomsen N, Wulf HC (1995) Carcinogenic and melanogenic effects of a filtered metal halide UVA source and a tubular fluorescent UVA tanning source with or without additional solar-simulated UV radiation in hairless mice. Photochem Photobiol 62:773–779

    Article  PubMed  CAS  Google Scholar 

  2. Chen Q, Ames BN (1994) Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci USA 91:4130–4134

    Article  PubMed  CAS  Google Scholar 

  3. Chen Q, Fischer A, Reagan JD, Yan LJ, Ames BN (1995) Oxidative DNA damage and senescence of human diploid fibroblast cells. Proc Natl Acad Sci USA 92:4337–4341

    Article  PubMed  CAS  Google Scholar 

  4. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ (2003) Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem 278:36027–36031

    Article  PubMed  CAS  Google Scholar 

  5. Date A, Hakozaki T, Yoshii T, Yasui H, Sakurai H (2006) Detection and identification of reactive oxygen species and followed free radicals generated in the UVB-exposed 3 dimensional human epidermal cells. In: The 126th annual meeting of pharmaceutical society of Japan, Sendai, Japan, p 28, [R]am-174

  6. Hanson KM, Clegg RM (2002) Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin. Photochem Photobiol 76:57–63

    Article  PubMed  CAS  Google Scholar 

  7. Hattori Y, Nishigori C, Tanaka T, Uchida K, Nikaido O, Osawa T, Hiai H, Imamura S, Toyokuni S (1996) 8-Hydroxy-2′-deoxyguanosine is increased in epidermal cells of hairless mice after chronic UVB exposure. J Invest Dermatol 107:733–737

    Article  PubMed  CAS  Google Scholar 

  8. He G, Kutala VK, Kuppusamy P, Zweier JL (2004) In vivo measurement and mapping of skin redox stress induced by ultraviolet light exposure. Free Radic Biol Med 36:665–672

    Article  PubMed  CAS  Google Scholar 

  9. Heck DE, Vetrano AM, Mariano TM, Laskin JD (2003) UVB light stimulates production of reactive oxygen species: unexpected role for catalase. J Biol Chem 278:22432–22436

    Article  PubMed  CAS  Google Scholar 

  10. Hellemans L, Corstjens H, Neven A, Declercq L, Maes D (2003) Antioxidant enzyme activity in human stratum corneum shows seasonal variation with an age-dependent recovery. J Invest Dermatol 120: 434–439

    Article  PubMed  CAS  Google Scholar 

  11. Ichihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, Tsuru K, Horikawa T (2003) UV-induced skin damage. Toxicology 189:21–39

    Article  PubMed  CAS  Google Scholar 

  12. Jurkiewicz BA, Buettner GR (1996) EPR detection of free radicals in UV-irradiated skin: mouse versus human. Photochem Photobiol 64:918–922

    Article  PubMed  CAS  Google Scholar 

  13. Kligman L (1989) The ultraviolet-irradiated hairless mouse: a model for photoaging. J Am Acad Dermatol 21:623–631

    PubMed  CAS  Google Scholar 

  14. Kripke ML, Cox PA, Alas LG, Yarosh DB (1992) Pyrimidine dimmers in DNA initiate systemic immunosuppression in UV-irradiated mice. Proc Natl Acad Sci USA 89:7516–7520

    Article  PubMed  CAS  Google Scholar 

  15. Lee EH, Faulhaber D, Hanson KM, Ding W, Peters W, Kodali W, Granstein RD (2004) Dietary lutein reduces ultraviolet radiation-induced inflammation and immunosuppression. J Invest Dermatol 122:510–517

    Article  PubMed  CAS  Google Scholar 

  16. Nishimura H, Yasui H, Sakurai H (2006) Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in vivo chemiluminescent detection and tape stripping methods. Exp Dermatol 15:891–899

    Article  PubMed  CAS  Google Scholar 

  17. Ou-Yang H, Stamatas G, Saliou C, Kollias N (2004) A chemiluminescence study of UVA-induced oxidative stress in human skin in vivo. J Invest Dermatol 122:1020–1029

    Article  PubMed  Google Scholar 

  18. Roda A, Pasini P, Musiani M, Girotti S, Baraldini M, Garrea G, Suozzi A (1996) Chemiluminescent low-light imaging of biospecific reactions on macro- and microsamples using a videocamera-based luminograph. Anal Chem 68:1073–1080

    Article  CAS  Google Scholar 

  19. Sander CS, Chang H, Salzmann S, Müller CSL, Mudiyanselage SE, Elsner P, Thiele JJ (2002) Photoaging is associated with protein oxidation in human skin in vivo. J Invest Dermatol 118:618–625

    Article  PubMed  CAS  Google Scholar 

  20. Sasaki H, Akamatsu H, Horio T (2000) Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT. J Invest Dermatol 114:502–507

    Article  PubMed  CAS  Google Scholar 

  21. Sauermann G, Mei WP, Hoppe U, Stäb F (1999) Ultraweak photon emission of human skin in vivo: influence of topically applied antioxidants on human skin. Meth Enzymol 300:419–428

    Article  PubMed  CAS  Google Scholar 

  22. Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T (1997) Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 76:365–374

    PubMed  CAS  Google Scholar 

  23. Toyokuni S, Yasui H, Date A, Hakozaki T, Akatsuka S, Kohda H, Yoshii T, Sakurai H (2006) Novel screening method for ultraviolet protection: combination of a human skin-equivalent model and 8-hydroxy-2′-deoxyguanosine. Pathol Int 56:760–762

    Article  PubMed  Google Scholar 

  24. Yasui H, Hakozaki T, Date A, Yoshii T, Sakurai H (2006) Real-time chemiluminescent imaging and detection of reactive oxygen species in the UVB-exposed human skin equivalent model. Biochem Biophys Res Commun 347:83–88

    Article  PubMed  CAS  Google Scholar 

  25. Yasui H, Sakurai H (2000) Chemiluminescent detection and imaging of reactive oxygen species in live mouse skin exposed to UVA. Biochem Biophys Res Commun 269:131–136

    Article  PubMed  CAS  Google Scholar 

  26. Yasui H, Sakurai H (2003) Age-dependent generation of reactive oxygen species in the skin of live hairless rats exposed to UVA light. Exp Dermatol 12:655–661

    Article  PubMed  CAS  Google Scholar 

Download references

Conflict of interest

StatementNone of the authors has any potential conflict of interest.

Author information

Authors and Affiliations

  1. Kobe Technical Center 7F, Procter & Gamble Japan K.K., Naka 1-17, Koyo-cho, Higashinada-ku, Kobe, 658-0032, Japan

    Tomohiro Hakozaki, Akira Date & Takashi Yoshii

  2. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Shinya Toyokuni

  3. Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan

    Hiroyuki Yasui & Hiromu Sakurai

Authors
  1. Tomohiro Hakozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akira Date
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takashi Yoshii
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shinya Toyokuni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroyuki Yasui
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiromu Sakurai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tomohiro Hakozaki.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hakozaki, T., Date, A., Yoshii, T. et al. Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Arch Dermatol Res 300 (Suppl 1), 51–56 (2008). https://doi.org/10.1007/s00403-007-0804-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00403-007-0804-3

Keywords

Search

Navigation