Skip to main content
SpringerLink
Log in

Quantitative analysis of UV photolesions suggests that cyclobutane pyrimidine dimers produced in mouse skin by UVB are more mutagenic than those produced by UVC

  • Paper
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

The amount of photolesions produced in DNA after exposure to physiological doses of ultraviolet radiation (UVR) can be estimated with high sensitivity and at low cost through an immunological assay, ELISA, which, however, provides only a relative estimate that cannot be used for comparisons between different photolesions such as cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproduct (64PP) or for analysis of the genotoxicity of photolesions on a molecular basis. To solve this drawback of ELISA, we introduced a set of UVR-exposed, calibration DNA whose photolesion amounts were predetermined and estimated the absolute molecular amounts of CPDs and 64PPs produced in mouse skin exposed to UVC and UVB. We confirmed previously reported observations that UVC induced more photolesions in the skin than UVB at the same dose, and that both types of UVR produced more CPDs than 64PPs. The UVR protection abilities of the cornified and epidermal layers for the lower tissues were also evaluated quantitatively. We noticed that the values of absorbance obtained in ELISA were not always proportional to the molecular amounts of the lesion, especially for CPD, cautioning against the direct use of ELISA absorbance data for estimation of the photolesion amounts. We further estimated the mutagenicity of a CPD produced by UVC and UVB in the epidermis and dermis using the mutation data from our previous studies with mouse skin and found that CPDs produced in the epidermis by UVB were more than two-fold mutagenic than those by UVC, which suggests that the properties of CPDs produced by UVC and UVB might be different. The difference may originate from the wavelength-dependent methyl CpG preference of CPD formation. In addition, the mutagenicity of CPDs in the dermis was lower than that in the epidermis irrespective of the UVR source, suggesting a higher efficiency in the dermis to reduce the genotoxicity of CPDs produced within it. We also estimated the minimum amount of photolesions required to induce the mutation induction suppression (MIS) response in the epidermis to be around 15 64PPs or 100 CPDs per million bases in DNA as the mean estimate from UVC and UVB-induced MIS.

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

Similar content being viewed by others

References

  1. R. B. Setlow, The wavelengths in sunlight effective in producing skin cancer: a theoretical analysis, Proc. Natl. Acad. Sci. U. S. A., 1974, 71, 3363–3366.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. D. E. Brash, J. A. Rudolph, J. A. Simon, A. Lin, G. J. McKenna, H. P. Baden, A. J. Halperin and J. Pontén, A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma, Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 10124–10128.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. F. R. de Gruijl and J. C. van der Leun, Estimate of the wavelength dependency of ultraviolet carcinogenesis in humans and its relevance to the risk assessment of a stratospheric ozone depletion, Health Phys., 1994, 67, 319–325.

    Article  PubMed  Google Scholar 

  4. H. Ikehata and T. Ono, The mechanisms of UV mutagenesis, J. Radiat. Res., 2011, 52, 115–125.

    Article  CAS  PubMed  Google Scholar 

  5. J. Cadet, E. Sage and T. Douki, Ultraviolet radiation-mediated damage to cellular DNA, Mutat. Res., 2005, 571, 3–17.

    Article  CAS  PubMed  Google Scholar 

  6. T. Douki, The variety of UV-induced pyrimidine dimeric photoproducts in DNA as shown by chromatographic quantification methods, Photochem. Photobiol. Sci., 2013, 12, 1286–1302.

    Article  CAS  PubMed  Google Scholar 

  7. J. Cadet, A. Grand and T. Douki, Solar UV radiation-induced DNA bipyrimidine photoproducts: formation and mechanistic insights, Top. Curr. Chem., 2015, 356, 249–275.

    Article  CAS  PubMed  Google Scholar 

  8. Y. You, D. Lee, J. Yoon, S. Nakajima, A. Yasui and G. P. Pfeifer, Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells, J. Biol. Chem., 2001, 276, 44688–44694.

    Article  CAS  PubMed  Google Scholar 

  9. D. Perdiz, P. Gróf, M. Mezzina, O. Nikaido, E. Moustacchi and E. Sage, Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells, J. Biol. Chem., 2000, 275, 26732–26742.

    Article  CAS  PubMed  Google Scholar 

  10. T. Douki, A. Reynaud-Angelin, J. Cadet and E. Sage, Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation, Biochemistry, 2003, 42, 9221–9226.

    Article  CAS  PubMed  Google Scholar 

  11. T. Douki, M. Court, S. Sauvaigo, F. Odin and J. Cadet, Formation of the main UV-induced thymine dimeric lesions within isolated and cellular DNA as measured by high performance liquid chromatography-tandem mass spectrometry, J. Biol. Chem., 2000, 275, 11678–11685.

    Article  CAS  PubMed  Google Scholar 

  12. T. Douki and J. Cadet, Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions, Biochemistry, 2001, 40, 2495–2501.

    Article  CAS  PubMed  Google Scholar 

  13. A. P. Schuch, R. da S. Galhardo, K. M. de Lima-Bessa, N. J. Schuch and C. F. M. Menck, Development of a DNAdosimeter system for monitoring the effects of solar-ultraviolet radiation, Photochem. Photobiol. Sci., 2009, 8, 111–120.

    Article  CAS  PubMed  Google Scholar 

  14. N. Kobayashi, S. Katsumi, K. Imoto, A. Nakagawa, S. Miyagawa, M. Furumura and T. Mori, Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology, Pigm. Cell Res., 2001, 14, 94–102.

    Article  CAS  Google Scholar 

  15. D. Mitchell and B. Brooks, Antibodies and DNA photoproducts: applications, milestones and reference guide, Photochem. Photobiol., 2010, 86, 2–17.

    Article  CAS  PubMed  Google Scholar 

  16. T. Mori, M. Nakane, T. Hattori, T. Matsunaga, M. Ihara and O. Nikaido, Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6-4)photoproduct from the same mouse immunized with ultraviolet-irradiated DNA, Photochem. Photobiol., 1991, 54, 225–232.

    Article  CAS  PubMed  Google Scholar 

  17. J. A. Gossen, W. J. F. de Leeuw, C. H. T. Tan, E. C. Zwarthoff, F. Berends, P. H. M. Lohman, D. L. Knook and J. Vijg, Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutationsin vivo, Proc. Natl. Acad. Sci. U. S. A., 1989, 86, 7971–7975.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. H. Ikehata, T. Mori and M. Yamamoto, In vivo spectrum of UVC-induced mutation in mouse skin epidermis may reflect the cytosine deamination propensity of cyclobutane pyrimidine dimers, Photochem. Photobiol., 2015, 91, 1488–1496.

    Article  CAS  PubMed  Google Scholar 

  19. H. Ikehata and T. Ono, Mutation induction with UVB in mouse skin epidermis is suppressed in acute high-dose exposure, Mutat. Res., 2002, 508, 41–47.

    Article  CAS  PubMed  Google Scholar 

  20. H. Ikehata, Y. Chang, M. Yokoi, M. Yamamoto and F. Hanaoka, Remarkable induction of UV-signature mutations at the 3'-cytosine of dipyrimidine sites except at 5'-TCG-3' in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice, DNA Repair, 2014, 22, 112–122.

    Article  CAS  PubMed  Google Scholar 

  21. R. A. Deering and R. B. Setlow, Effects of ultraviolet light on thymidine dinucleotide and polynucleotide, Biochim. Biophys. Acta, 1963, 68, 526–534.

    Article  CAS  PubMed  Google Scholar 

  22. F. Garcès and C. A. Davila, Alterations in DNA irradiated with ultraviolet radiation I. The formation process of cyclobutylpyrimidine dimers: cross sections, action spectra and quantum yields, Photochem. Photobiol., 1982, 35, 9–16.

    Article  PubMed  Google Scholar 

  23. W. A. G. Bruls, H. Slaper, J. C. van der Leun and L. Berrens, Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths, Photochem. Photobiol., 1984, 40, 485–494.

    Article  CAS  PubMed  Google Scholar 

  24. J.-P. Therrien, M. Rouabhia, E. A. Drobetsky and R. Drouin, The multilayered organization of engineered human skin does not influence the formation of sunlight-induced cyclobutane pyrimidine dimers in cellular DNA, Cancer Res., 1999, 59, 285–289.

    CAS  PubMed  Google Scholar 

  25. E. Sage, D. Perdiz, P. Gróf, A. Reynaud-Angelin, T. Douki, J. Cadet, P. Rochette, N. Bastien and R. Drouin, DNA damage induced by UVA radiation: role in solar mutagenesis, in Comprehensive Series in Photochemical and Photobiological Sciences, Royal Society of Chemistry, 2005, ch. 3, vol. 5, pp. 33–47.

    CAS  Google Scholar 

  26. S. Mouret, C. Baudouin, M. Charveron, A. Favier, J. Cadet and T. Douki, Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 13765–13770.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. A. Banyasz, T. Douki, R. Improta, T. Gustavsson, D. Onidas, I. Vayá, M. Perron and D. Markovitsi, Electronic excited states responsible for dimer formation upon UV absorption directly by thymine strands: Joint experimental and theoretical study, J. Am. Chem. Soc., 2012, 134, 14834–14845.

    Article  CAS  PubMed  Google Scholar 

  28. T. Douki, Relative contributions of UVB and UVA to the photoconversion of (6-4) photoproducts into their Dewar valence isomers, Photochem. Photobiol., 2016, 92, 587–594.

    Article  CAS  PubMed  Google Scholar 

  29. P. Mao, J. J. Wyrick, S. A. Roberts and M. J. Smerdon, UV-induced DNA damage and mutagenesis in chromatin, Photochem. Photobiol., 2017, 93, 216–228.

    Article  CAS  PubMed  Google Scholar 

  30. H. Ikehata, R. Okuyama, E. Ogawa, S. Nakamura, A. Usami, T. Mori, K. Tanaka, S. Aiba and T. Ono, Influences of p53 deficiency on the apoptotic response, DNA damage removal and mutagenesis in UVB-exposed mouse skin, Mutagenesis, 2010, 25, 397–405.

    Article  CAS  PubMed  Google Scholar 

  31. H. Ikehata, S. Higashi, S. Nakamura, Y. Daigaku, Y. Furusawa, Y. Kamei, M. Watanabe, K. Yamamoto, K. Hieda, N. Munakata and T. Ono, Action spectrum analysis of UVR genotoxicity for skin: the border wavelengths between UVA and UVB can bring serious mutation loads to skin, J. Invest. Dermatol., 2013, 133, 1850–1856.

    Article  CAS  PubMed  Google Scholar 

  32. J. Jans, W. Schul, Y.-G. Sert, Y. Rijksen, H. Rebel, A. P. M. Eker, S. Nakajima, H. van Steeg, F. R. de Gruijl, A. Yasui, J. H. J. Hoeijmakers and G. T. J. van der Horst, Powerful skin cancer protection by a CPD-photolyase transgene, Curr. Biol., 2005, 15, 105–115.

    Article  CAS  PubMed  Google Scholar 

  33. T. M. Rünger, B. Farahvash, Z. Hatvani and A. Rees, Comparison of DNA damage responses following equi- mutagenic doses of UVA and UVB: a less effective cell cycle arrest with DNA may render UVA-induced pyrimidine dimers more mutagenic than UVB-induced ones, Photochem. Photobiol. Sci., 2012, 11, 207–215.

    Article  PubMed  Google Scholar 

  34. X. Qin, S. Zhang, H. Oda, Y. Nakatsuru, S. Shimizu, Y. Yamazaki, O. Nikaido and T. Ishikawa, Quantitative detection of ultraviolet light-induced photoproducts in mouse skin by immunohistochemistry, Jpn. J. Cancer Res., 1995, 86, 1041–1048.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. M. D’Errico, M. Teson, A. Calcagnile, T. Nardo, N. de Luca, C. Lazzari, S. Soddu, G. Zambruno, M. Stefanini and E. Dogliotti, Differential role of transcription-coupled repair in UVB-induced response of human fibroblasts and keratinocytes, Cancer Res., 2005, 65, 432–438.

    PubMed  Google Scholar 

  36. S. Mouret, M. Charveron, A. Favier, J. Cadet and T. Douki, Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin, DNA Repair, 2008, 7, 704–712.

    Article  CAS  PubMed  Google Scholar 

  37. A. Pines, C. Backendorf, S. Alekseev, J. G. Jansen, F. R. de Gruijl, H. Vrieling and L. H. F. Mullenders, Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer, DNA Repair, 2009, 8, 153–161.

    Article  CAS  PubMed  Google Scholar 

  38. T. von Zglinicki, G. Saretzki, J. Ladhoff, F. d’Adda di Fagagna and S. P. Jackson, Human cell senescence as a DNA damage response, Mech. Ageing Dev., 2005, 126, 111–117.

    Article  CAS  Google Scholar 

  39. P. Caillet-Fauquet, M. Defais and M. Radman, Molecular mechanism of induced mutagenesis. I, in vivo replication of the single-stranded ultraviolet-irradiated 0X174 phage DNA in irradiated host cells, J. Mol. Biol., 1977, 117, 95–112.

    Article  CAS  PubMed  Google Scholar 

  40. M. P. Carty, J. Hauser, A. S. Levine and K. Dixon, Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts, Mol. Cell. Biol., 1993, 13, 533–542.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. D. C. Thomas and T. A. Kunkel, Replication of UV-irradiated DNA in human cell extracts - evidence for mutagenic bypass of pyrimidine dimers, Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 7744–7753.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. N. Bastien, J.-P. Therrien and R. Drouin, Cytosine containing dipyrimidine sites can be hotspots of cyclobutane pyrimidine dimer formation after UVB exposure, Photochem. Photobiol. Sci., 2013, 12, 1544–1554.

    Article  CAS  PubMed  Google Scholar 

  43. R. B. Setlow, W. L. Carrier and F. J. Bollum, Pyrimidine dimers in UV-irradiated poly dI:dC, Proc. Natl. Acad. Sci. U. S. A., 1965, 53, 1111–1118.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. F. Liu and N. C. Yang, Photochemistry of cytosine derivatives. 1. Photochemistry of thymidylyl-(3'→5')-deoxy-cytidine, Biochemistry, 1978, 17, 4865–4876.

    Article  CAS  PubMed  Google Scholar 

  45. D. Lee and G. P. Pfeifer, Deamination of 5-methylcytosines within cyclobutane pyrimidine dimers is an important component of UVB mutagenesis, J. Biol. Chem., 2003, 278, 10314–10321.

    Article  CAS  PubMed  Google Scholar 

  46. Q. Song, S. M. Sherrer, Z. Suo and J.-S. Taylor, Preparation of site-specific T=mCG cis-syn cyclobutane dimer-containing template and its error-free bypass by yeast and human polymerase q, J. Biol. Chem., 2012, 287, 8021–8028.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. V. J. Cannistraro and J.-S. Taylor, Acceleration of 5-methyl-cytosine deamination in cyclobutane dimers by G and its implications for UV-induced C-to-T mutation hotspots, J. Mol. Biol., 2009, 392, 1145–1157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Q. Song, V. J. Cannistraro and J.-S. Taylor, Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle, Nucleic Acids Res., 2014, 42, 131122–113133.

    Google Scholar 

  49. V. J. Cannistraro, S. Pondugula, Q. Song and J.-S. Taylor, Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo, J. Biol. Chem., 2015, 290, 26597–26609.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. S. Grünwald and G. P. Pfeifer, Enzymatic DNA methylation, Prog. Clin. Biochem. Med., 1989, 9, 61–103.

    Article  Google Scholar 

  51. R. Drouin and J.-P. Therrien, UVB-induced cyclobutane pyrimidine dimer frequency correlates with skin cancer mutational hotspots in p53, Photochem. Photobiol., 1997, 66, 719–726.

    Article  CAS  PubMed  Google Scholar 

  52. S. Tommasi, M. F. Denissenko and G. P. Pfeifer, Sunlight induces pyrimidine dimers preferentially at 5-methylcytosine bases, Cancer Res., 1997, 57, 4727–4730.

    CAS  PubMed  Google Scholar 

  53. P. J. Rochette, S. Lacoste, J.-P. Therrien, N. Bastien, D. E. Brash and R. Drouin, Influence of cytosine methylation on ultraviolet-induced cyclobutane pyrimidine dimer formation in genomic DNA, Mutat. Res., 2009, 665, 7–13.

    Article  CAS  PubMed  Google Scholar 

  54. S. Tornaletti, D. Rozek and G. P. Pfeifer, The distribution of UV photoproducts along the human p53 gene and its relation to mutations in skin cancer, Oncogene, 1993, 8, 2051–2057.

    CAS  PubMed  Google Scholar 

  55. P. Monti, A. Inga, G. Scott, A. Aprile, P. Campomenosi, P. Menichini, L. Ottaggio, S. Viaggi, A. Abbondandolo, P. S. Burns and G. Fronza, 5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation, Mutat. Res., 1999, 431, 93–103.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Y. Hasegawa for experimental assistance, and B. Bell for help in editing the manuscript. This study was supported by Japan Society for the Promotion of Science KAKENHI Grant Number JP15H02815 to H. Ikehata.

Author information

Authors and Affiliations

  1. Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan

    Hironobu Ikehata & Masayuki Yamamoto

  2. Radioisotope Research Center, Nara Medical University School of Medicine, Kashihara, Nara, Japan

    Toshio Mori

  3. Université Grenoble Alpes, CEA, CNRS, INAC-SyMMES, 38000, Grenoble, France

    Thierry Douki

  4. University of Sherbrooke, Sherbrooke, Canada

    Jean Cadet

Authors
  1. Hironobu Ikehata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshio Mori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thierry Douki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean Cadet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masayuki Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hironobu Ikehata.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ikehata, H., Mori, T., Douki, T. et al. Quantitative analysis of UV photolesions suggests that cyclobutane pyrimidine dimers produced in mouse skin by UVB are more mutagenic than those produced by UVC. Photochem Photobiol Sci 17, 404–413 (2018). https://doi.org/10.1039/c7pp00348j

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/c7pp00348j

Search

Navigation