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Keratinocytes Act as a Source of Reactive Oxygen Species by Transferring Hydrogen Peroxide to Melanocytes

2005; Elsevier BV; Volume: 124; Issue: 4 Linguagem: Inglês

10.1111/j.0022-202x.2005.23661.x

1523-1747

Edward Pelle, Thomas Mammone, Daniel Maes, Krystyna Frenkel,

Phytochemicals and Antioxidant Activities

Basal hydrogen peroxide (H2O2) levels in normal human epidermal keratinocytes (NHEK) and melanocytes (mel) were compared on a per cell basis and found to be significantly higher in keratinocytes. Since H2O2 is a neutral molecule capable of permeating through cellular membranes, we then investigated the possibility that H2O2 transfer might occur between these two types of cells. Because the ratio of keratinocytes to mel in skin is 36:1, keratinocytes may act as a source of reactive oxygen species (ROS) even by passive diffusion and, thus, affect melanocytic functions. In order to measure H2O2 transfer, a fluorescence-based co-culture system was developed in which mel were first pre-labeled with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (DCFdA). When mel were co-cultured with keratinocytes, fluorescence increased as a function of keratinocyte cell number. Thus, for mel incubated with 1-, 1.5-, and 2-fold the number of keratinocytes, fluorescence increased by 22.6% (±2.8%), 25.6% (±4.8%), and 39.9% (±4.1%), respectively. Separating the cells with a transwell membrane did not prevent the transfer, whereas the addition of catalase to media significantly reduced the transfer of H2O2 to mel. In conclusion, keratinocytes appear to be a previously unexamined source of ROS that may affect neighboring skin cells, such as mel, and, as a result, may influence the process of melanogenesis or contribute to the progression of vitiliginous lesions. Basal hydrogen peroxide (H2O2) levels in normal human epidermal keratinocytes (NHEK) and melanocytes (mel) were compared on a per cell basis and found to be significantly higher in keratinocytes. Since H2O2 is a neutral molecule capable of permeating through cellular membranes, we then investigated the possibility that H2O2 transfer might occur between these two types of cells. Because the ratio of keratinocytes to mel in skin is 36:1, keratinocytes may act as a source of reactive oxygen species (ROS) even by passive diffusion and, thus, affect melanocytic functions. In order to measure H2O2 transfer, a fluorescence-based co-culture system was developed in which mel were first pre-labeled with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (DCFdA). When mel were co-cultured with keratinocytes, fluorescence increased as a function of keratinocyte cell number. Thus, for mel incubated with 1-, 1.5-, and 2-fold the number of keratinocytes, fluorescence increased by 22.6% (±2.8%), 25.6% (±4.8%), and 39.9% (±4.1%), respectively. Separating the cells with a transwell membrane did not prevent the transfer, whereas the addition of catalase to media significantly reduced the transfer of H2O2 to mel. In conclusion, keratinocytes appear to be a previously unexamined source of ROS that may affect neighboring skin cells, such as mel, and, as a result, may influence the process of melanogenesis or contribute to the progression of vitiliginous lesions. hydrogen peroxide melanocytes normal human epidermal keratinocytes reactive oxygen species Increased levels of reactive oxygen species (ROS) have been associated with many pathological processes (Sies, 1985Sies H. Oxidative Stress. Academic Press, London1985Crossref Google Scholar; Frenkel, 1992Frenkel K. Carcinogen-mediated oxidant formation and oxidative DNA damage.Pharm Ther. 1992; 53: 127-166Crossref PubMed Scopus (335) Google Scholar; Packer and Fuchs, 1993Packer L. Fuchs J. Oxidative Stress in Dermatology. Marcel Dekker, New York1993Google Scholar; Halliwell and Gutteridge, 1999Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 3rd edn. Oxford University Press, Oxford1999Google Scholar) and the effects of these reactive intermediates on epidermal cells in skin have been extensively studied. Beginning withPathak and Stratton, 1968Pathak M.A. Stratton K. Free radicals in human skin before and after exposure to light.Arch Biochem Biophys. 1968; 123: 463-476Crossref Scopus (155) Google Scholar, the potential for free radical-mediated damage was established by the observation that melanin radicals were generated in the epidermis after ultraviolet (UV) radiation. Later work byDixit et al., 1983Dixit R. Mukhtar H. Bickers D.R. Studies on the role of reactive oxygen species in mediating lipid peroxide formation in epidermal microsomes of rat skin.J Invest Dermatol. 1983; 81: 369-375Crossref PubMed Scopus (62) Google Scholar demonstrated an increase in lipid peroxides in UV-irradiated epidermal homogenates, whereas subcutaneous injection of superoxide dismutase (SOD), a superoxide anion radical scavenger, byDanno et al., 1984Danno K. Horio T. Takigawa M. Imamura S. Role of oxygen intermediates in UV-induced epidermal cell injury.J Invest Dermatol. 1984; 83: 166-168Crossref PubMed Scopus (124) Google Scholar, reduced epidermal sunburn cell formation. In addition to ROS production as a result of photodynamic action, influx of ROS into skin also occurs during inflammatory episodes due to the action of phagocytic NADPH oxidase (Babior, 1984Babior B.M. Oxidants from phagocytes: Agents of defense and destruction.Blood. 1984; 64: 959-966Crossref PubMed Google Scholar). Ischemia/reperfusion during dermatological surgical procedures is yet another situation in which ROS ingression has also been observed (Knight, 1994Knight K.R. Review of postoperative pharmacological infusions in ischemic skin flaps.Microsurgery. 1994; 15: 675-684Crossref PubMed Scopus (38) Google Scholar). The subject of free radicals in skin biology has been reviewed byDarr and Fridovich, 1994Darr D. Fridovich I. Free radicals in cutaneous biology.J Invest Dermatol. 1994; 102: 671-675Abstract Full Text PDF PubMed Google Scholar and more recently an entire volume has been devoted to this topic (Thiele and Elsner, 2001Thiele J. Elsner P. Oxidants and Antioxidants in Cutaneous Biology. Current Problems in Dermatology. Vol. 29. Karger, Basel2001Google Scholar). During oxidative stress, molecular oxygen is reduced to form superoxide anion radicals, thereby eliminating the electron spin restriction imposed by the paramagnetic nature of oxygen and, thus, leads to the increased reactivity of oxygen. Further, superoxide anion radicals dismutate to hydrogen peroxide (H2O2) either spontaneously (k=1 × 105 per M per s) or by the action of SOD (k=1 × 109 per M per s) and there is much evidence that H2O2 is a mediator of oxidative damage in cells (Davies, 1998Davies K.J. Oxidative stress: The paradox of aerobic life.Biochem Soc Symp. 1998; 61: 1-31Crossref Scopus (872) Google Scholar). For example, 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative lesion in DNA, was decreased in the presence of catalase (Zhang et al., 1997Zhang X. Rosenstein B.S. Wang Y. Lebwohl M. Wei H. Identification of possible reactive oxygen species involved in ultraviolet radiation-induced oxidative DNA damage.Free Radical Biol Med. 1997; 23: 980-985Crossref PubMed Scopus (175) Google Scholar) and membrane damage has been associated with increased levels of hydrogen peroxide (Chatterjee and Agarwal, 1988Chatterjee S.N. Agarwal S. Liposomes as membrane model for study of lipid peroxidation.Free Radical Biol Med. 1988; 4: 51-72Crossref PubMed Scopus (236) Google Scholar).Frenkel and Chrzan, 1987aFrenkel K. Chrzan K. Hydrogen peroxide formation and DNA base modification by tumor promoter-activated polymorphonuclear leukocytes.Carcinogenesis. 1987; 8: 455-460Crossref PubMed Scopus (92) Google Scholar also demonstrated an increase in H2O2 when HeLa cells were treated with a tumor promoter and additional studies showed that leukemic cells could be protected by chemopreventive agents from the effects of tumor promoter-induced H2O2 (Bhimani et al., 1995Bhimani R.S. Zhong Z. Schleifer E. Troll W. Frenkel K. Human promyelocyte leukemia cells (HL-60): A new model to study the effects of chemopreventive agents on H2O2 production.Cancer Detect Prev. 1995; 19: 292-298PubMed Google Scholar). In epidermal cells, H2O2 levels increase in response to oxidative stress from environmental trauma, such as ultraviolet radiation B (UVB, 290–320 nm) (Peus et al., 1998Peus D. Vasa R.A. Meves A. Pott M. Beyerle A. Squillace K. Pittelkow M.R. H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes.J Invest Dermatol. 1998; 110: 966-971Crossref PubMed Scopus (169) Google Scholar), causing damage to biomolecules (Peus and Pittelkow, 2001Peus D. Pittelkow M.R. Reactive oxygen species as mediators of UVB-induced mitogen-activated protein kinase activation in keratinocytes.in: Thiele J. Elsner P. Current Problems in Dermatology: Oxidants and Antioxidants in Cutaneous Biology. Vol. 29. Karger, Basel2001: 114-127Google Scholar; Pelle et al., 2003Pelle E. Huang X. Mammone Marenus K. Maes D. Frenkel K. UVB-induced oxidative DNA base damage in primary normal human epidermal keratinocytes.J Invest Dermatol. 2003; 121: 177-183Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Moreover, there is a basal level of oxidants in cells that is a by-product of normal endogenous processes. Although a system of enzymatic and non-enzymatic antioxidants provides cells with protection against ROS (Lopez-Torres et al., 1998Lopez-Torres M. Thiele J.J. Shindo Y. Han D. Packer L. Topical application of alpha-tocopherol modulates the antioxidant network and diminishes ultraviolet-induced oxidative damage in murine skin.Br J Dermatol. 1998; 138: 207-215Crossref PubMed Scopus (178) Google Scholar), these antioxidant defenses can be overwhelmed during times of oxidative stress and can lead to a disruption of cellular function (Aruoma, 1998Aruoma O.I. Free radicals, oxidants and antioxidants: trends towards the year 2000 and beyond.in: Aruoma O.I. Halliwell B. Molecular Biology of Free Radicals in Human Diseases. OICA International, St. Lucia, WI1998: 13Google Scholar). Yohn et al., 1991Yohn J.J. Norris D.A. Yrastorza D.G. Buno I.J. Leff J.A. Hake S.S. Repine J.E. Disparate antioxidant enzyme activities in cultured human cutaneous fibroblasts, keratinocytes, and melanocytes.J Invest Dermatol. 1991; 97: 405-409Abstract Full Text PDF PubMed Google Scholar evaluated enzymatic antioxidant levels in epidermal cells and found higher levels of catalase and glutathione peroxidase in keratinocytes as compared with melanocytes. These findings indicated that either catalase was higher in keratinocytes in response to H2O2 or, alternatively, may have been lower in mel due to the lower steady-state levels of H2O2. Modulation of H2O2 in mel appears to be crucial for the process of melanogenesis (Nappi and Vass, 1996Nappi A.J. Vass E. Hydrogen peroxide generation associated with the oxidations of the eumelanin precursors 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid.Melanoma Res. 1996; 6: 341-349Crossref PubMed Scopus (66) Google Scholar), which seems to require a precise redox balance. This latter point is supported by the work ofJimbow et al., 2001Jimbow K. Chen H. Park J.-S. Thomas P.D. Increased sensitivity of melanocytes to oxidative stress and abnormal expression of tyrosinase-related protein in vitiligo.Br J Dermatol. 2001; 144: 55-65Crossref PubMed Scopus (188) Google Scholar, who demonstrated that, despite the utilization of ROS in melanogenesis, mel are particularly sensitive to oxidative stress. In patients with vitiligo, a disease characterized by the destruction of mel,Schallreuter et al., 1991Schallreuter K.U. Wood J.M. Berger J. Low catalase levels in the epidermis of patients with vitiligo.J Invest Dermatol. 1991; 97: 1081-1085Abstract Full Text PDF PubMed Google Scholar detected low levels of epidermal catalase in affected areas. Successful repigmentation of vitiliginous patches was then achieved by these workers using topical application of a psuedocatalase (Schallreuter, 1999Schallreuter K.U. Successful treatment of oxidative stress in vitiligo.Skin Pharmacol Appl Skin Physiol. 1999; 12: 132-138Crossref PubMed Scopus (60) Google Scholar). Since H2O2 is diamagnetic (Halliwell and Gutteridge, 1999Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 3rd edn. Oxford University Press, Oxford1999Google Scholar), it has no unpaired electrons and, as a result, is a neutral species with the ability to permeate membranes (Frenkel and Chrzan, 1987bFrenkel K. Chrzan K. Radiation-like modification of DNA and H2O2 formation by activated polymorphonuclear leukocytes (PMNs).in: Cerutti P. Nygaard O.F. Simic M. Anticarcinogenesis and Radiation Protection. Plenum Publishing, New York1987: 97-102Crossref Google Scholar) and, thus, has the potential to migrate into neighboring cells. In human epidermis, the ratio of keratinocytes to mel is 36:1 (Jakubovic and Ackerman, 1992Jakubovic H.R. Ackerman A.B. Chapter 1. Structure and function of skin: development, morphology, and physiology;a.in: Moschella S.L. Hurley H.J. 3rd edn. Dermatology. Vol. 1. WB Saunders, Philadelphia1992: 3-87Google Scholar) and, as a consequence, the potential for transfer of H2O2 from keratinocytes to mel, even by a passive diffusion mechanism, appears to be quite high. In this study, our first objective was to compare the relative levels of H2O2 in keratinocytes and mel, whereas our second objective was to measure the transfer of H2O2 from keratinocytes to mel. Herein, we present evidence demonstrating that human keratinocytes transfer H2O2 to mel and, therefore, can act as a source of oxidative stress in mel. Due to its lipophilic nature, DCFdA readily permeates through cell membranes, whereupon non-specific esterases in the cytosol remove the acetyl moieties and produce non-fluorescent 2′,7′-dichlorodihydrofluorescein (DCFH), which is trapped inside the cell. In order to measure the ability of H2O2 to permeate into mel, cells were first incubated in the presence of glucose oxidase in Dulbecco's phosphate-buffered saline (D-PBS) over a range of concentrations (5–50 mU per mL) and as a function of time. Table I shows a linear (p<0.001) increase in fluorescence in mel caused by increasing doses of glucose oxidase, which indicates that the product of glucose oxidase, H2O2 (Rubin and Farber, 1984Rubin R. Farber J.L. Mechanisms of the killing of cultured hepatocytes by hydrogen peroxide.Arch Biochem Biophys. 1984; 228: 4450-4459Crossref Scopus (148) Google Scholar), was generated in situ followed by its migration into mel.Table IH2O2 measurement in melGlucose oxidase (mU per mL)Time (h)05102550089 (±0.8)124 (±8.1)103 (±5.8)109 (±9.8)104 (±2.2)1216 (±1.8)1159 (±53.2)1316 (±64.9)1606 (±66.6)1964 (±54.1)2365 (±2.7)3555 (±121.2)4118 (±90.3)4911 (±101.1)5903 (±144.0)3558 (±3.6)7110 (±200.4)8171 (±238.4)9593 (±135.5)10,814 (±193.7)Human mel were labeled with 10 μM DCFdA, at 37°C for 15 min, followed by addition of NaN3 (catalase inhibitor) to a final concentration of 12.5 mM in D-PBS. Glucose oxidase (0–50 mU per mL) was then added and increases in fluorescence observed as a function of time of the exposure to glucose oxidase. Data are expressed as means±SE (n=3, ANOVA: p<0.001). Open table in a new tab Human mel were labeled with 10 μM DCFdA, at 37°C for 15 min, followed by addition of NaN3 (catalase inhibitor) to a final concentration of 12.5 mM in D-PBS. Glucose oxidase (0–50 mU per mL) was then added and increases in fluorescence observed as a function of time of the exposure to glucose oxidase. Data are expressed as means±SE (n=3, ANOVA: p<0.001). In Figure 1, the basal levels of hydrogen peroxide in NHEK are compared with those in mel. NHEK had a significantly (p<0.001) higher titer of oxidants than mel on a per cell basis at each time point, which indicates the potential for passive transfer of H2O2 from NHEK to mel due to the imbalance of the basal levels of H2O2. To test the hypothesis that NHEK contribute to the oxidative load in mel, mel were co-cultured with increasing numbers of NHEK. Mel were first labeled with 10 μM DCFdA at 37°C and then washed five times with D-PBS in order to remove any unincorporated DCFdA. Increasing numbers of NHEK were then added to the mel, co-incubated overnight, and their fluorescence was measured. The results of these experiments are shown in Figure 2 and demonstrate increasing fluorescence in mel as a function of the incubation with increased numbers of NHEK. Thus, as the number of NHEK increased to 1-, 1.5-, and 2-times the number of mel, the fluorescence intensity in the mel increased 22.6% (±SE 2.8%), 25.6% (±SE 4.8%), and 39.9% (±SE 4.1%), respectively. To further illustrate that fluorescence was specific only to mel in co-cultures with NHEK, phase contrast microscopy was used to visualize a co-culture of both cell types followed by fluorescence microscopy of the same field. As shown in Figure 3a, both mel and NHEK are visible as morphologically distinct entities by phase contrast, whereas in Figure 3b, only those cells with dendritic processes, which are characteristic of mel, are fluorescent.Figure 3Detection of hydrogen peroxide in melanocytes (mel) grown in co-cultures with normal human epidermal keratinocytes (NHEK) by fluorescence microscopy. 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (DCFdA)-labeled mel were co-cultured with NHEK at 37°C for 16 h, and then visualized by fluorescence microscopy (× 100, 1 cm=10 μm). (a) Phase contrast of mel and NHEK showed the presence of both types of cells; (b) fluorescence of the same field illuminated only DCFdA-labeled mel.View Large Image Figure ViewerDownload (PPT) Since mel transfer melanosome-containing melanin to NHEK by direct contact through its dendrites, it was possible that H2O2 could have been transferred to mel in the reverse direction along its dendritic processes. In order to determine whether direct contact was a prerequisite for H2O2 transfer, mel were again co-cultured with NHEK but physically separated by a transwell membrane. In this experiment, when mel and NHEK were co-cultured together, there was a 28% (±SE 3.9%) increase in fluorescence and when co-cultured but separated by a transwell membrane there was an increase of 38% (±SE 5.5%), which is a non-significant difference between both sets. Thus, these results indicate that NHEK did not need to be in direct contact with mel in order to transfer H2O2. To further prove that it was H2O2 that was transferred from NHEK to mel, an experiment was performed in which catalase was present in the cell culture medium during co-culture incubation. The results from this experiment show a reduced H2O2 transfer in the presence of catalase (Figure 4). Although other oxidants are capable of DCFH oxidation, the inhibitory effects of catalase prove that in these experiments it was H2O2 that was transferred from NHEK to mel. In this study, we compared oxidant levels of human keratinocytes with those in mel and found that keratinocytes had significantly higher basal levels of H2O2 on a per cell basis by approximately 4-fold under conditions that inhibited catalase. Based on this finding, and the fact that keratinocytes far outnumber mel in the skin (36:1), we assessed the potential of H2O2 to pass from keratinocytes to mel. Since only mel were labeled with DCFdA in the co-culture system with keratinocytes, we were able to develop a fluorescence-based assay that detected the transfer of H2O2 in a concentration-dependent manner from keratinocytes to mel. As a result, these data show the potential of keratinocytes to act as a source of ROS in mel. These data also demonstrate a sensitive way to measure the effect that one type of cell has on the oxidative state of a different type of cell since, even at a cell-to-cell ratio of 1:1, a significant increase in ROS (22.6%) was detected in mel. This increase was also visualized by fluorescence microscopy of our co-cultured cells, which clearly depicted the localization of DCFdA only in the mel. Because of the proximity of keratinocytes to mel in human epidermis, the transfer of H2O2 may have wide-ranging effects in vivo on the biology of the melanocyte. One putative effect of H2O2 transfer may involve the destruction or impairment of mel at the periphery of vitiliginous lesions leading to the characteristic discoloration of skin observed in these lesions. Thus, as H2O2 passes into the melanocyte, oxidative damage may occur, perhaps due to an increased level of exported H2O2 from the keratinocyte. This is supported bySchallreuter et al., 2001Schallreuter K.U. Moore J. Wood J.M. et al.Epidermal H2O2 accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: Identification of a general mechanism in regulation of all 6BH4-dependent processes.J Invest Dermatol. 2001; 116: 167-174Crossref PubMed Scopus (178) Google Scholar, who showed that catalase and tetrahydrobiopterin dehydratase activities are inhibited by H2O2 and that these enzymes are also lower in patients with vitiligo. Alternatively, vitiligo-involved mel may have a higher sensitivity to normal levels of H2O2. Since tyrosinase-related protein (TRP-1) has been shown to have peroxidase activity (Schallreuter, 1999aSchallreuter K.U. A review of recent advances on the regulation of pigmentation in the human epidermis.Cell Mol Biol. 1999; 45: 943-949PubMed Google Scholar), and autoantibodies to TRP-1 have also been found in the sera of vitiligo patients (Kemp et al., 1998Kemp E.H. Waterman E.A. Gawkrodger D.J. Watson P.F. Weetman A.P. Autoantibodies to tyrosinase-related protein-1 detected in the sera of vitiligo patients using a quantitative radiobinding assay.Br J Dermatol. 1998; 139: 798-805Crossref PubMed Scopus (84) Google Scholar), it is possible that an influx of H2O2 from keratinocytes may render mel vulnerable to oxidative attack due to a defective or incapacitated TRP-1, which is unable to effectively remove H2O2. Although other molecules secreted from keratinocytes, such as cytokines, may affect H2O2 levels in mel, our data show that H2O2 is directly involved in the increase in oxidation because the presence of catalase in the co-culture medium negated the increase in fluorescence. Moreover, H2O2 transfer between cells was not contingent upon direct cell-to-cell contact. When keratinocytes were separated from mel by a transwell membrane in our co-culture system, we still observed an increase in fluorescence in mel. A future area of investigation will be to study the transfer of H2O2 in co-cultures that contain ratios that reflect the 36:1 ratio and our preliminary data suggest a linear correlation to H2O2 levels as the keratinocyte to melanocyte ratio increases. Also, since catalase and glutathione peroxidase affect H2O2 levels, our next steps include determining the relative expression of these two enzymes by RT-PCR in both cell types. Another area of investigation will assess the downstream effects of H2O2 on cell signaling. Previously, we reported that UVB increased H2O2 levels in keratinocytes (Pelle et al., 2003Pelle E. Huang X. Mammone Marenus K. Maes D. Frenkel K. UVB-induced oxidative DNA base damage in primary normal human epidermal keratinocytes.J Invest Dermatol. 2003; 121: 177-183Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and, in a parallel set of experiments, we also observed higher levels of H2O2 and Activator Protein-1 (AP-1) in mel following UVB irradiation (unpublished observations). Delineating transduction pathways in response to increased levels of H2O2 in normal as well as abnormal mel in the future may provide insights into the mechanisms of melanogenesis. In summary, this study proves that H2O2 can be transferred from keratinocytes to mel and demonstrates that keratinocytes can act as a source of ROS, which may contribute to oxidant-related modulation of biochemical processes in the melanocyte. DCFdA was obtained from Molecular Probes (Eugene, Oregon). Transwell membranes (3472, clear) from Corning (Corning, New York) were used for the separation of keratinocytes and mel in co-culture experiments. Glucose oxidase, Grade 1 from Aspergillus niger, was obtained from Roche (Indianopolis, Indiana), whereas catalase was purchased from Sigma (St Louis, Missouri). Primary NHEK were obtained from Cascade (Portland, Oregon) as a primary culture of cells isolated from fetal foreskin. Cells were cultured in 10 ml EpiLife (calcium-free) medium containing growth factors and serum (Cascade) using T75 Falcon culture flasks with screw-capped HEPA filters; cells were subcultured by trypsinization. At approximately 50% confluency of the third passage, there were sufficient numbers of cells to perform experiments. Normal human epidermal neonatal mel were obtained from Cascade as tertiary proliferating cultures and grown in Medium 154 supplemented with 1% melanocyte growth serum using T75 Falcon culture flasks with screw-capped HEPA filters. Cells were subcultured by trypsinization and used for experimentation when they were in the log-phase of growth. H2O2 was determined by adding DCFdA to cells, which was a modification of the technique used byBhimani et al., 1995Bhimani R.S. Zhong Z. Schleifer E. Troll W. Frenkel K. Human promyelocyte leukemia cells (HL-60): A new model to study the effects of chemopreventive agents on H2O2 production.Cancer Detect Prev. 1995; 19: 292-298PubMed Google Scholar andHuang et al., 1994Huang X. Zhuang Z. Frenkel K. Klein C.B. Costa M. The role of nickel and nickel-mediated reactive oxygen species in the mechanism of nickel carcinogenesis.Environ Health Perspect. 1994; 102: 281-284Crossref PubMed Scopus (71) Google Scholar. For the analysis, 2 × 104 cells were plated into the wells of a 96-well microtiter plate because it was determined that this number of cells attained approximately 50% of confluence after overnight incubation. The next day, the medium was aspirated, 100 μL of a 10 μM DCFdA solution in D-PBS was added to the cells, and incubated at 37°C for 15 min. The DCFdA solution was prepared by adding 100 μL ethanol to a vial containing 50 μg DCFdA and then transferring 50 μL to 5 mL D-PBS to a final concentration of 10 μM. After incubation with DCFdA, 100 μL 25 mM sodium azide (NaN3, a catalase inhibitor) was added and incubated with the cells at 37°C for 2 h. But NaN3 was not added to cells for overnight incubations due to its cytotoxicity. Fluorescence measurements were made in a fluorescence plate reader (Cytofluor, PerSeptive Systems, Framingham, Massachusetts) with a 485/20 nm excitation filter and a 530/25 nm emission filter set at a gain of 75. Although NaN3 is used to inhibit catalase in order to obtain a stronger fluorescent signal, an additional control was also carried out to demonstrate the validity of our technique. This was achieved by substituting NaN3 with 3-amino-1,2,4-triazole, which inhibits catalase more specifically than NaN3. Cells treated with 12.5 mM NaN3 increased fluorescence over untreated cells by 165% (±2.4%, SE, n=3), whereas those treated with the same concentration of 3-amino-1,2,4-triazole increased fluorescence by 352% (±19.1%, SE, n=3). Thus, both treatments increased fluorescence and demonstrate the validity of this technique for H2O2 measurements. For microscopic visualization of melanocyte-labeled co-cultures, mel were plated in 60 mm culture dishes at 3 × 105 cells per dish, treated with DCFdA, washed five times with D-PBS, and then incubated overnight with an equal number of keratinocytes. Cell cultures were then viewed at × 100 magnification by phase contrast and fluorescence microscopy with an Olympus, BX60 microscope (Olympus, Melville, New York). Microscope fluorescence was generated with a mercury arc lamp and observed through blue light filters. Probability (p) values were calculated using a Student's two-tailed t test assuming unequal variances or one-way analysis of variance (ANOVA) where indicated.