ENDOGENOUS DNA OXIDATION CORRELATES TO INCREASED IRON LEVELS IN MELANOCYTES 283 (±3 SEM) ferritin/mg protein. Intriguingly, even HepG2 cells, hepatocarcinoma cells derived from iron-enriched liver tissue, were signifi cantly lower than melanocytes at 33.2 ng (±2 SEM) ferritin/mg protein. These results demonstrate that melanocytes appear to have higher levels of iron in comparison to other cell types and support the concept of a higher oxidative environment in melanocytes. DISCUSSION Previously, we reported a fourfold higher level of H2O2 in NHEKs than in melanocytes (6), which should indicate less oxidative stress in melanocytes. We further showed that H2O2 could permeate by passive diffusion from NHEKs to melanocytes. Nevertheless, despite these previous fi ndings, we now report higher levels of oxidative DNA in mela- nocytes as compared to NHEKs, as well as lower concentrations of GSH, both of which are well-established biomarkers of oxidative status. Because NHEKs and melanocytes had similar UVB-induced 8-oxo-dG potential and because higher levels of 8-oxo-dG were also found in mouse-derived melanocytes, our data suggest that elevated states of oxidation may be characteristic for melanocytes. To account for these differences, we pro- pose that the higher levels of iron that we measured in melanocytes in this study contrib- ute to higher levels of 8-oxo-dG. A possible reaction sequence might involve a Fenton reaction between iron and H2O2 leading to the production of hydroxyl radicals and oxida- tive DNA. Because H2O2 is able to penetrate through membranes and because ferritin has been found in the nucleus (18), the possibility that H2O2 will react with ferrous ions inside the nucleus is suggested as a possible mechanism for increased melanocytic oxida- tive DNA. This reaction sequence might also explain, at least in part, the reduced amount of hydrogen peroxide that we previously determined in melanocytes. In this report, we show that melanocytes are signifi cantly higher in iron, compared not only to NHEKs but also to iron-rich hepatocarcinoma cells. As melanocytes are specialized epidermal cells dedicated to the synthesis and secretion of melanin in order to protect skin against UV-induced photo damage, the increased presence of iron raises the question as to why a melanocyte would tolerate such a high risk/benefi t iron ratio. Although ferritin seques- ters approximately 4500 ferric ions per molecule, it also releases iron as reactive ferrous ions (19). Iron ions have been shown to induce DNA base modifi cations in cellular DNA (20), and understanding this reaction dynamic in melanocytes may be an important factor in under- standing the melanogenic process. Intriguingly, 8-oxoguanine has also recently been de- scribed as a signaling molecule (21), and perhaps future research will link it to melanocyte gene activation, as well. Further, recent work by Gruber and Holtz (22) demonstrated increased levels of ferritin gene expression after treatment with conventional skin lighteners and their data also support the concept of iron fl ux in relation to melanocyte function. As postmenopausal women appear to have higher levels of cutaneous iron (11), our data suggest a possible correlation between this group and the development of age-related melanopathologies, such as lentigines. If future clinical studies support this hypothesis, then research should be targeted toward producing iron-reduction treatments (23). More- over, increased levels of melanocytic oxidative DNA due to increased iron may be a com- mon denominator that may help to explain the occurrence of melanomagenesis even in sun-protected areas of skin. In conclusion, higher levels of oxidative DNA appear to be present in normal human epidermal melanocytes and correspond to increased amounts of reactive bioavailable iron.

JOURNAL OF COSMETIC SCIENCE 284 REFERENCES (1) Y. Yamaguchi, M. Brenner, and V. J. Hearing, The regulation of skin pigmentation, J. Biol. Chem., 282, 27557–27561 (2007). (2) M. Mastore, L. Kohler, and A. J. Nappi, Production and utilization of hydrogen peroxide associated with melanogenesis and tyrosinase-mediated oxidations of DOPA and dopamine, FEBS J., 272, 2407– 2415 (2005). (3) K. U. Schallreuter, S. Kothari, B. Chavan, and J. D. Spencer, Regulation of melanogenesis: controversies and new concepts, Exp. Dermatol., 17, 395–404 (2008). (4) P. A. Riley, Melanin, Int. J. Biochem. Cell Biol., 29, 1235–1239(1997). (5) K. U. Schallreuter, J. M. Wood, and J. Berger, Low catalase levels in the epidermis of patients with vitiligo, J. Invest. Dermatol., 97, 1081–1085 (1991). (6) E. Pelle, T. Mammone, D. Maes, and K. Frenkel, Keratinocytes act as a source of reactive oxygen species by transferring hydrogen peroxide to melanocytes, J. Invest. Dermatol., 124, 793–797 (2005). (7) H. T. Wang, B. Choi, and M. S. Tang, Melanocytes are defi cient in repair of oxidative DNA damage and UV-induced photoproducts, Proc. Natl. Acad. Sci. U S A, 107, 12180–12185 (2010). (8) K. Frenkel, Carcinogen-mediated oxidant formation and oxidative DNA damage, Pharmacol. Ther., 53, 127–166 (1992). (9) D. B. Yarosh, A. Pena, and D. A. Brown, DNA repair gene polymorphisms affect cytotoxicity in the National Cancer Institute Human Tumour Cell Line Screening Panel, Biomarkers, 10, 188–202 (2005). (10) G. E. Costin and V. J. Hearing, Human skin pigmentation: melanocytes modulate skin color in response to stress, FASEB J., 21, 976–994 (2007). (11) E. Pelle, J. Jian, Q. Zhang, N. Muizzuddin, Q. Yang, J. Dai, D. Maes, N. Pernodet, D. B. Yarosh, K. Frenkel, and X. Huang, Menopause increases the iron storage protein ferritin in skin, J. Cosmet. Sci., 64, 175–179 (2013). (12) A. Palumbo, M. d’Ischia, G. Misuraca, L. Carratu, and G. Prota, Activation of mammalian tyrosinase by ferrous ions, Biochim. Biophys. Acta, 1033, 256–260 (1990). (13) T. Shibata, G. Prota, and Y. Mishima, Non-melanosomal regulatory factors in melanogenesis, J. Invest. Dermatol., 100, 274S–280S (1993). (14) X. Huang, J. Powell, L. A. Mooney, C. Li, and K. Frenkel, Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses, Free Radic. Biol. Med., 31, 1341–1351 (2001). (15) E. Pelle, X. Huang, T. Mammone, K. Marenus, D. Maes, and K. Frenkel, Ultraviolet-B-induced oxida- tive DNA base damage in primary normal human epidermal keratinocytes and inhibition by a hydroxyl radical scavenger, J. Invest. Dermatol., 121, 177–183 (2003). (16) K. Frenkel, Z. J. Zhong, H. C. Wei, J. Karkoszka, U. Patel, K. Rashid, M. Georgescu, and J. J. Solomon, Quantitative high-performance liquid chromatography analysis of DNA oxidized in vitro and in vivo, Anal. Biochem, 196, 126–136 (1991). (17) Q. Zhang and X. Huang, Induction of ferritin and lipid peroxidation by coal samples with different prevalence of coal workers’ pneumoconiosis: role of iron in the coals, Am. J. Ind. Med., 42, 171–179 (2002). (18) K. J. Thompson, M. G. Fried, Z. Ye, P. Boyer, and J. R. Connor, Regulation, mechanisms and proposed function of ferritin translocation to cell nuclei, J. Cell. Sci., 115, 2165–2177 (2002). (19) J. Jian, Q. Yang, J. Dai, J. Eckard, D. Axelrod, J. Smith, and X. Huang, Effects of iron defi ciency and iron overload on angiogenesis and oxidative stress-a potential dual role for iron in breast cancer, Free Radic. Biol. Med., 50, 841–847 (2011). (20) T. H. Zastawny, S. A. Altman, L. Randers-Eichhorn, R. Madurawe, J.A. Lumpkin, M. Dizdaroglu, and G. Rao, DNA base modifi cations and membrane damage in cultured mammalian cells treated with iron ions, Free Radic. Biol. Med., 18, 1013–1022 (1995). (21) I. Boldogh, G. Hajas, L. Aguilera-Aguirre, M. L. Hegde, Z. Radak, A. Bacsi, S. Sur, T. K. Hazra, and S. Mitra, Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine, J. Biol. Chem., 287, 20769–20773 (2012). (22) J. V. Gruber and R. Holtz, Examining the impact of skin lighteners in vitro, Oxid Med Cell Longev., 2013, 702120 (2013). (23) E. Pelle, J. Jian, L. Declercq, K. Dong, Q. Yang, C. Pourzand, D. Maes, N. Pernodet, D. B. Yarosh, and X. Huang, Protection against ultraviolet A-induced oxidative damage in normal human epidermal ke- ratinocytes under post-menopausal conditions by an ultraviolet A-activated caged-iron chelator: A pilot study, Photodermatol. Photoimmunol. Photomed., 27, 231–235 (2011).