J. Cosmet. Sci., 65, 277–284 (September/October 2014) 277 Increased endogenous DNA oxidation correlates to increased iron levels in melanocytes relative to keratinocytes EDWARD PELLE, XI HUANG, QI ZHANG, NADINE PERNODET, DANIEL B. YAROSH, and KRYSTYNA FRENKEL, Estee Lauder Research Laboratories, Melville, NY (E.P., N.P., D.B.Y.), Environmental Medicine, New York University School of Medicine, New York, NY (E.P., X.H., K.F.), ImClone Systems/ Eli Lilly, Branchburg, NJ (Q.Z.). Accepted for publication May 28, 2014. Synopsis The endogenous oxidative state of normal human epidermal melanocytes was investigated and compared to normal human epidermal keratinocytes (NHEKs) in order to gain new insight into melanocyte biology. Previously, we showed that NHEKs contain higher levels of hydrogen peroxide (H2O2) than melanocytes and that it can migrate from NHEKs to melanocytes by passive permeation. Nevertheless, despite lower concen- trations of H2O2, we now report higher levels of oxidative DNA in melanocytes as indicated by increased levels of 8-oxo-2’-deoxyguanosine (8-oxo-dG): 4.49 (±0.55 SEM) 8-oxo-dG/106 dG compared to 1.49 (±0.11 SEM) 8-oxo-dG/106 dG for NHEKs. An antioxidant biomarker, glutathione (GSH), was also lower in mela- nocytes (3.14 nmoles (±0.15 SEM)/cell) in comparison to NHEKs (5.98 nmoles (±0.33 SEM)/cell). Intrigu- ingly, cellular bioavailable iron as measured in ferritin was found to be nearly fourfold higher in melanocytes than in NHEKs. Further, ferritin levels in melanocytes were also higher than in hepatocarcinoma cells, an iron-rich cell, and it indicates that higher relative iron levels may be characteristic of melanocytes. To account for the increased oxidative DNA and lower GSH and H2O2 levels that we observe, we propose that iron may contribute to higher levels of oxidation by reacting with H2O2 through a Fenton reaction leading to the generation of DNA-reactive hydroxyl radicals. In conclusion, our data support the concept of elevated oxidation and high iron levels as normal parameters of melanocytic activity. We present new evidence that may contrib- ute to our understanding of the melanogenic process and lead to the development of new skin care products. INTRODUCTION Human melanocytes are specialized, neural crest-derived epidermal cells that synthesize melanin and provide coloration to the skin. In order to synthesize melanin, melanocytes engage in a complex, multifactorial polymerization inside subcellular melanosomal organelles. As these melanosomes mature, they travel through the cell’s dendritic projec- tions and, at the completion of melanin synthesis, are transferred to keratinocytes by Address all correspondence to Edward Pelle at epelle@estee.com.

JOURNAL OF COSMETIC SCIENCE 278 attachment to PAR2 receptors whereupon the melanin then migrates to the top of the nucleus to form a protective perinuclear cap (1). Although environmental exposure of human skin to ultraviolet (UV) radiation can in- crease the level of reactive oxygen species (ROS) in melanocytes and initiate melanogen- esis, oxidation/reduction mechanisms are, nevertheless, an integral part of the basal activity of melanocytes (2). For example, a critical fi rst step in melanogenesis occurs when the Cu(II)-centered enzyme, tyrosinase, is reduced to Cu(I) leading to DOPA formation. Additionally, reduction of phenylalanine hydroxylase and tyrosine hydroxylase 1 by the electron donor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) is a specifi c require- ment for tyrosinase activation (3). To date, much biochemical research has been devoted to the etiology of melanin forma- tion and the role that oxidation/reduction mechanisms play in this process (4). However, the endogenous oxidative state of the melanocyte has received less attention, yet it may yield clues to understanding some of the paradoxes encountered in melanocyte biology. In support of this concept, Schallreuter et al. (5) reported higher concentrations of hydro- gen peroxide in melanocytes of vitiligo patients and, in normal epidermal cells, we ob- served the transfer of hydrogen peroxide from keratinocytes to melanocytes (6). Further, Wang et al. (7) found that reduced levels of oxidative DNA damage repair in melanocytes may also contribute to higher levels of oxidation. Because oxidative stress contributes to mutations, infl ammatory processes, and aging (8,9), understanding how oxidative differences in melanocytes affect their normal activity may lead to new insights regarding melano- genesis, melanoma, and solar lentigenes (10). Moreover, a recent study on postmeno- pausal skin (11) showed higher levels of ferritin and may indicate that postmenopausal women may be more susceptible to UV-induced oxidative damage than other groups due to the presence of more cutaneous bioavailable iron and, thus, iron may also play a critical role in melanocytic function in this group. Although the role of iron in melanocytes has been explored by others (12,13), much of their work was focused on tyrosinase activity. Here, we compare the difference in oxidative state between melanocytes and keratinocytes, two contiguous epidermal cells that cross talk and interact intimately with each other in human skin. We compare oxidative lesions in DNA in the form of 8-oxo-dG, which is a well- characterized indicator of oxidation, ferritin as a biomarker for bioavailable iron, and glutathione (GSH) as a measure of cellular antioxidant status. We also discuss a possible mode of action involving iron in melanocytes in order to account for these differences. MATERIALS AND METHODS CELLS Primary normal human epidermal keratinocytes (NHEKs), melanocytes, and growth me- dia were obtained from Life Technologies (Carlsbad, CA) as primary culture cells isolated from fetal foreskin. NHEKs were cultured in EpiLife (calcium-free) medium containing 1% human keratinocyte growth factors. Eumelanin-producing melanocytes were cul- tured in Medium 254 and also supplemented to 1% with human melanocyte growth factors. At approximately 50% confl uency of the third passage, there were suffi cient numbers of cells to perform experiments. Melan a and melan c cells were kind gifts from Drs. S. Orlow and S. Pifko-Hirst (New York University School of Medicine, New York,