J Vet Sci.  2006 Sep;7(3):217-223. 10.4142/jvs.2006.7.3.217.

Protective effect of the isoflavone equol against DNA damage induced by ultraviolet radiation to hairless mouse skin

• Widyarini

• KMID: 1089904
• DOI: http://doi.org/10.4142/jvs.2006.7.3.217

Abstract

Equol, an isoflavonoid metabolite produced from the dietary isoflavone daidzein by the gut microflora in mammals, has been found to protect not only against ultraviolet (UV) radiation-induced cutaneous inflammation and photoimmune suppression, but also have antiphotocarcinogenic properties in mice. Because the state of DNA damage has been correlated with suppression of the immune system and photocarcinogenesis, we have therefore examined the potential of equol to offer protection from solar-simulated UV (SSUV) radiation-induced DNA damage in hairless mice by the immunohistochemical approach using monoclonal antibody specific for cyclobutane pyrimidine dimers (CPDs; H3 antibody). Topical application of 20 micrometer equol lotion, which was applied both before and after SSUV significantly reduced the number of CPDs. This reduction was evident immediately after SSUV exposure, at 1 h after exposure, and at 24 h after exposure, revealing 54%, 50%, and 26% reduction in CPDs, respectively. When the same concentration was applied for 5 consecutive days after SSUV exposure, there was no significant difference in the reduction of CPDs immediately after SSUV irradiation or at 1 hour afterwards, but there were significant reductions of 23% and 42% at 24 and 48 h after SSUV exposure, respectively. Despite apparently reducing the number of CPDs post-SSUV, topically applied equol did not appear to increase the rate of dimer removal. To conclude, equol applied topically prior to SSUV irradiation offers protection against CPD formation in hairless mice, possibly by acting as a suncreen and thus inhibiting DNA photodamage.

Keyword

equol; H3 antibody; pyrimidine dimers; ultraviolet

MeSH Terms

Administration, Topical
Animals
DNA/drug effects/radiation effects
*DNA Damage
Female
Immunohistochemistry
Isoflavones/*pharmacology
Mice
Mice, Inbred HRS
Pyrimidine Dimers/metabolism
Skin/drug effects/metabolism/*radiation effects
Sunlight/adverse effects
Ultraviolet Rays/*adverse effects

Figure

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  Fig. 1 Accumulation of cyclobutane pyrimidine dimers (CPDs) upon repeated irradiation, or following a single dose of irradiation with solar-simulated ultraviolet radiation (SSUV), expressed as the average number CPD (mean ± SE). Mice were irradiated daily with 1.16 kJ/m2 UVB and 18.52 kJ/m2 UVA for three consecutive days (3 × 1 MED of SSUV), or a single dose of 3.48 kJ/m2 UVB and 55.6 kJ/m2 UVA (1 × 3 MED of SSUV), or not irradiated. Mice were killed at 0 (non-SSUV irradiated skin), 1, 24, 48, 72, and 168 h after being exposed to SSUV. 3 MED SSUV single dose vs 1 MED for 3 days at 1 and 48 h post SSUV irradiation was not significant (p > 0.05). Skin samples were taken from 2 mice at each time point.

• 

  Fig. 2 Effect of topical 20 µM equol lotion on the removal of cyclobutane pyrimidine dimers (CPDs) after irradiation with solar-simulated ultraviolet radiation (SSUV), expressed as the average number CPD (mean ± SE). Mice were irradiated with a single dose of 3.48 kJ/m2 UVB and 55.6 kJ/m2 UVA (1 × 3 MED of SSUV), or not irradiated, and then killed immediately (0.5 h post-SSUV exposure) and at 1, 24, 48, 72, and 168 h post-SSUV exposure. Skin samples were taken from 2 mice at each time point. *: Equol was applied for 7 days prior to SSUV and was continued for 5 consecutive days immediately after SSUV exposure. †: Equol was applied immediately after SSUV exposure for 5 consecutive days. SSUV + BL vs SSUV + 20 µM equol † at 0.5 and 1 h post-SSUV irradiation was not significant (p > 0.05).

• 

  Fig. 3 The time course of the removal of cyclobutane pyrimidine dimers (CPDs) following a single dose of 3 MED of solar-simulated ultraviolet radiation (SSUV). All sections were stained using ABC methods. ×200. A; Normal mouse skin (non-SSUV-irradiated). B; Isotype control of CPD staining, immediately after SSUV exposure. C; The appearance of CPDs immediately after SSUV exposure. Most of CPDs were distributed in the basal layer of epidermis. D; The appearance of CPDs 1 h post-SSUV exposure. Most of CPDs were distributed in the basal layer of epidermis. E; The appearance of CPDs 24 h post-SSUV exposure. Most of CPDs were found in the upper layer of epidermis with a few CPDs remaining in the basal layer of epidermis. F; The appearance of CPDs 48 h post-SSUV exposure. Most of CPDs were found in the upper layer of epidermis, and only a few CPDs were found in the basal layer of epidermis. G; The appearance of CPDs 72 h post-SSUV exposure. CPDs were found in the keratinocyte layer. H; 168 h post-SSUV exposure, CPDs had completely disappeared from the epidermal layer.

Reference

1. Ananthaswamy HN, Loughlin SM, Cox P, Evans RL, Ullrich SE, Kripke ML. Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens. Nature Med. 1997. 3:510–514.

2. Applegate LA, Ley RD, Alcalay J, Kripke ML. Identification of the molecular target for the suppression of contact hypersensitivity by ultraviolet radiation. J Exp Med. 1989. 170:1117–1131.

3. Berg RJ, Ruven HJ, Sands AT, de Gruijl FR, Mullenders LH. Defective global genome repair in XPC mice is associated with skin cancer susceptibility but not with sensitivity to UVB induced erythema and edema. J Invest Dermatol. 1998. 110:405–409.

4. Berneburg M, Krutmann J. Photoimmunology, DNA repair and photocarcinogenesis. J Photochem Photobiol B. 2000. 54:87–93.

5. Brash DE, Rudolph JA, Simon JA, Lin A, McKenna GJ, Baden HP, Halperin AJ, Ponten J. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA. 1991. 88:10124–10128.

6. Brash DE, Ziegler A, Jonason AS, Simon JA, Kunala S, Leffell DJ. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion. J Investig Dermatol Symp Proc. 1996. 1:136–142.

7. Caceres-Dittmar G, Ariizumi K, Xu S, Tapia FJ, Bergstresser PR, Takashima A. Hydrogen peroxide mediates UV-induced impairment of antigen presentation in a murine epidermal-derived dendritic cell line. Photochem Photobiol. 1995. 62:176–183.

8. Devary Y, Rosette C, DiDonato JA, Karin M. NF-kappa B activation by ultraviolet light not dependent on a nuclear signal. Science. 1993. 261:1442–1445.

9. Franke AA, Custer LJ, Cerna CM, Narala K. Rapid HPLC analysis of dietary phytoestrogens from legumes and from human urine. Proc Soc Exp Biol Med. 1995. 208:18–26.

10. Garssen J, Vandebriel RJ, van Loveren H. Molecular aspects of UVB-induced immunosuppression. Arch Toxicol Suppl. 1997. 19:97–109.

11. Gibbs NK, Norval M, Traynor NJ, Wolf M, Johnson BE, Crosby J. Action spectra for the trans to cis photoisomerisation of urocanic acid in vitro and in mouse skin. Photochem Photobiol. 1993. 57:584–590.

12. Greinert R, Boguhn O, Harder D, Breitbart EW, Mitchell DL, Volkmer B. The dose dependence of cyclobutane dimer induction and repair in UVB-irradiated human keratinocytes. Photochem Photobiol. 2000. 72:701–708.

13. Kraemer KH. Sunlight and skin cancer: another linked revealed. Proc Natl Acad Sci USA. 1997. 94:11–14.

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

15. Kripke ML. Immunologic mechanisms in UV radiation carcinogenesis. Adv Cancer Res. 1981. 34:69–106.

16. Ley RD, Fourtanier A. Sunscreen protection against ultraviolet radiation-induced pyrimidine dimers in mouse epidermal DNA. Photochem Photobiol. 1997. 65:1007–1011.

17. Ley RD. Photoreactivation of UV-induced pyrimidine dimers and erythema in the marsupial Monodelphis domestica. Proc Natl Acad Sci USA. 1985. 82:2409–2411.

18. Mohammad T, Tessman I, Morrison H, Kennedy MA, Simmonds SW. Photosensitized inactivation of infectious DNA by urocanic acid, indoleacrylic acid and rhodium complexes. Photochem Photobiol. 1994. 59:189–196.

19. Nishigori C, Yarosh DB, Ullrich SE, Vink AA, Bucana CD, Roza L, Kripke ML. Evidence that DNA damage triggers interleukin 10 cytokine production in UV-irradiated murine keratinocytes. Proc Natl Acad Sci USA. 1996. 93:10354–10359.

20. Noonan FP, De Fabo EC. Immunosuppression by ultraviolet B radiation: initiation by urocanic acid. Immunol Today. 1992. 13:250–254.

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

22. Roza L, De Gruijl FR, Bergen Henegouwen JB, Guikers K, Van Weelden H, Van Der Schans GP, Baan RA. Detection of photorepair of UV-induced thymine dimers in human epidermis by immunofluorescence microscopy. J Invest Dermatol. 1991. 96:903–907.

23. Simon MM, Aragane Y, Schwarz A, Luger TA, Schwarz T. UVB light induces nuclear factor kappa B (NF kappa B) activity independently from chromosomal DNA damage in cell-free cytosolic extracts. J Invest Dermatol. 1994. 102:422–427.

24. Strickland PT. Distribution of thymine dimers induced in mouse skin by ultraviolet radiation. Photodermatol. 1988. 5:1–8.

25. Urbach F. Ultraviolet radiation and skin cancer of humans. J Photochem Photobiol B. 1997. 40:3–7.

26. Vink AA, Berg RJ, de Gruijl FR, Roza L, Baan RA. Induction, repair and accumulation of thymine dimers in the skin of UV-B-irradiated hairless mice. Carcinogenesis. 1991. 12:861–864.

27. Widyarini S, Allanson M, Gallagher NL, Pedley J, Boyle GM, Parsons PG, Whiteman DC, Walker C, Reeve VE. Isoflavonoid photoprotection in mouse and human skin is dependent on metallothionein. J Invest Dermatol. 2006. 126:198–204.

28. Widyarini S, Husband AJ, Reeve VE. Protective effect of the isoflavonoid equol against hairless mouse skin carcinogenesis induced by UV radiation alone or with a chemical cocarcinogen. Photochem Photobiol. 2005. 81:32–37.

29. Widyarini S, Spinks N, Husband AJ, Reeve VE. Isoflavonoid compounds from red clover (Trifolium pratense) protect from inflammation and immune suppression induced by UV radiation. Photochem Photobiol. 2001. 74:465–470.

30. Wolf P, Yarosh DB, Kripke ML. Effects of sunscreens and a DNA excision repair enzyme on ultraviolet radiation-induced inflammation, immune suppression, and cyclobutane pyrimidine dimer formation in mice. J Invest Dermatol. 1993. 101:523–527.

31. Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T, Brash DE. Sunburn and p53 in the onset of skin cancer. Nature. 1994. 372:773–776.