ENDOGENOUS DNA OXIDATION CORRELATES TO INCREASED IRON LEVELS IN MELANOCYTES 281 although melanocytes have higher endogenous levels of oxidative DNA, they have the same relative susceptibility as NHEKs to UVB-induced oxidative stress (Figure 1B). One possible explanation for the higher 8-oxo-dG values in melanocytes may have been the difference in growth media. To test this, melanocyte medium was incubated with NHEKs overnight to determine the contribution of medium components to oxidative DNA damage. The next day, cells were harvested and 8-oxo-dG analyzed. In these ex- periments, the average control value for NHEKs was 1.96 (±0.5 SEM)/106 dG while the melanocyte-medium-treated NHEK value was 1.65 (±0.57 SEM)/106 dG. Thus, the higher oxidative levels observed in the DNA of melanocytes were not due to differences in medium components. Additionally, NHEK and melanocyte cell cultures were syn- chronized by serum starvation and their 8-oxo-dG levels assessed for any possible cell cycle effects. The results from this experiment were consistent with our other data, which indicated higher oxidative DNA levels in melanocytes (data not shown). To further understand the nature of the relative differences in 8-oxo-dG between NHEKs and melanocytes, two murine melanocytic cell lines referred to as melan a and melan c were also evaluated. Although both types of cells are melanocytic in origin, the melan c line has a defective tyrosinase gene and is devoid of melanin. If melanogenesis increases the level of oxyradicals in the cell, the level of 8-oxo-dG should be lower in the melan c cells. However, as shown in Figure 2, only a nonsignifi cant decrease in 8-oxo-dG was observed in melan c cells as compared to melan a cells. Nevertheless, both melan a and melan c cells were higher in oxidative DNA than in NHEKs and show a greater similar- ity to human melanocytes, which might be characteristic of these cells. Glutathione. As a gauge on the overall oxidative state of the cells, total GSH levels were also measured and found to be lower in melanocytes than in NHEKs. GSH in NHEKs was calculated to contain 5.98 nmoles (±0.33 SEM)/cell while in eumelanin-producing melanocytes this level was 3.14 nmoles (±0.15 SEM)/cell and would appear to be consis- tent with an oxidatively challenged cellular environment. However, the oxidized dimer of GSH, GSSG, was 12.2% for both cell types and these results are summarized in Figure 3. Although lower total GSH levels in melanocytes should foster a more oxidatively Figure 2. DNA extracted from NHEKs and melanocytes were digested to the nucleoside level and analyzed by HPLC/EC for 8-oxo-dG. Oxidative DNA levels in two melanocyte-derived cell lines: melan a and melan c were more similar to melanocytes than to NHEKs. Data are expressed as mean ± SEM.

JOURNAL OF COSMETIC SCIENCE 282 challenged cellular environment, the fi nding that GSSG is the same in both cell types raises the possibility that eumelanin-producing melanocytes may normally utilize less GSH for their cellular functions. Ferritin. As iron plays a signifi cant role in the generation of ROS, we next measured the levels of ferritin, a storage protein of bioavailable iron, in both types of cells. Because serum is an exogenous source of iron in cell culture, we were careful to standardize both cell lines in 1% serum. As shown in Figure 4, ferritin levels in melanocytes were nearly fourfold higher than in NHEKs. Thus, NHEKs contained 23.3 ng (±5 SEM) ferritin/mg protein, whereas melanocytes contained 97.1 ng (±6 SEM) ferritin/mg protein. To further understand this difference in the context of other cells, we compared ferritin levels in other cell types. MCF-10a cells, immortalized mammary epithelial cells, had 13.0 ng Figure 3. GSH and GSSG were measured in NHEKs and melanocytes using a chemiluminescence-based assay. GSH levels were lower in melanocytes than in NHEKs but GSSG levels were both 12.2% of the total GSH. A linear regression curve was also developed with GSH standards to determine cell molar concentra- tions and then normalized to cell number as reported in the text. Data are expressed as mean ± SEM. Figure 4. Ferritin concentrations were assayed using an ELISA technique and showed increased levels of ferritin in melanocytes in comparison to NHEKs, MCF-10, as well as in HepG2, an iron-rich hepatocarci- noma cell. Data are expressed as mean ± SEM.