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Carcinogenesis vol.20 no.11 pp.2137–2142, 1999

Inhibitory effects of deferoxamine on UVB-induced AP-1 transactivation

Kim Kramer-Stickland, Andrew Edmonds, Warner B.Bair III and G.Tim Bowden1 Department of Radiation Oncology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA 1To

whom correspondence should be addressed Email: [email protected]

Production of reactive oxygen species (ROS) by iron can contribute directly to DNA and protein damage and may contribute to cell signaling and proliferation. We have examined the effects of the iron(III) chelator deferroxamine (DFO) and iron (FeCl3) on UVB (290–320 nm)-induced activator protein 1 (AP-1) signaling. The ability of DFO to inhibit UVB-induced AP-1 transactivation was tested in a human keratinocyte cell line stably transfected with a luciferase reporter driven by a single AP-1 element. DFO treatment 24 h prior to UVB irradiation reduced UVBinduced AP-1 transactivation by ~80%, with the effect of DFO diminishing as pre-treatment time was shortened. Treatment with FeCl3 a minimum of 6 h prior to UVB potentiated the UVB induction of AP-1 transactivation by 2–3-fold. DFO was able to ablate both the UVB induction of AP-1 transactivation as well as the potentiation by FeCl3. The antioxidants Trolox and N-acetyl cysteine were both able to inhibit UVB-induced AP-1 transactivation and Trolox was able to inhibit the potentiation of UVB-induced AP-1 by FeCl3. These results indicate that UVB-induced AP-1 activation may be in part due to oxidant effects of UVB and intercellular iron. Introduction Diseases of iron overload result in significant tissue damage to the liver and other organs if not managed with iron chelators (1). Elevated body iron is also associated with an increased risk of cancer in humans, and in rodents iron overload has been shown to enhance colon and mammary carcinogenesis (2–12). The ability of iron to generate reactive oxygen species (ROS) via the Fenton reaction and cause oxidative damage is thought to be one of the primary mechanisms of iron-induced tissue damage (13–15). Oxidative stress is thought to cause release of iron from its storage proteins as well, leading to further oxidative damage (12,16,17). Many diseases are initiated or exacerbated by oxidative damage, and may thus be sensitive to changes in iron in the cell. Inhibition of UVBinduced tumors in mice with antioxidant treatments (18–27) suggests the importance of oxidative damage in the induction of skin cancer by UVB. Oxidative damage to lipids, proteins and DNA by UVB have all been implicated in skin tumor Abbreviations: AP-1, activator protein 1; DFO, deferoxamine; DMEM, Dulbecco’s modified Eagle’s medium; EGTA, ethylene glycol tetraacetic acid; HCL14, HaCaT cells containing AP-1 regulated luciferase reporter gene; NAC, N-acetyl cysteine; RLU, relative light units; ROS, reactive oxygen species; Trolox-Me, Trolox methyl ether. © Oxford University Press

formation. Chronic UV has also been shown to cause iron and ferritin accumulation in human skin (28). ROS resulting from acute UVB exposures may release iron from ferritin in the cell, causing a temporary increase in free iron which may participate in oxidative chemistry in the cell. Thus, iron may contribute to the ROS formed by UVB irradiation and be involved in the pathogenesis of UVB-induced skin cancer. Promotion of UVB-induced epidermal tumors occurs over a long period of time, via outgrowth of initiated cells. UVB light can cause growth-stimulatory effects via induction of cell signaling pathways, or secondary to inflammation. UVB causes activation of both the NFκB and activator protein 1 (AP-1) transcription factor complexes (29–33), and antioxidants inhibit UVB-induced AP-1 expression (34). AP-1 is a transcription factor complex that is involved in the activation of multiple genes by UVB (the ‘UV response’). The transient induction of AP-1 has been shown to be involved in the promotion of epidermal tumors, and AP-1 activity has been implicated in neoplastic transformation in vitro and in epidermal tumors (35–39). Constitutive AP-1 activity has been associated with the malignant conversion of papillomas to carcinomas (40) as well. Chemopreventive agents or modifications of AP-1 proteins that inhibit AP-1 activation are effective in preventing cell transformation or tumorigenesis due to chemicals or UV light (30,36,37,41,42). Inhibition of UVB-induced AP-1 signaling by antioxidants or by iron chelators would lend validity to the idea that UVB- and iron-induced ROS are important in UVB-induced cell signaling. Deferoxamine (DFO) is an iron chelator that has long been used in patients with acute iron poisoning (43) and iron overload resulting from multiple transfusions in patients with β-thallassaemia major, or in patients with primary hemochromatosis (44–47). More recently, DFO has been tested in animal models of diseases known to be influenced by oxidative damage, such as cardiovascular disease, neurodegenerative disease, hepatocarcinogenesis and ischemia-reperfusion injury (48–52). DFO has been shown to inhibit phorbol ester-induced hyperplasia in mouse ear skin (53), UVB-induced oxidative stress in corneal epithelial cells and murine fibroblasts (54,55) and the formation of an oxidative DNA lesion, 8-OHdG in neutrophils (56), suggesting inhibition of oxidative damage, presumably via iron chelation and inhibition of iron-induced Fenton chemistry. DFO itself is a reductant, and may affect the redox status of the cell in that manner as well. In human dermal fibroblasts DFO inhibits UVB-induced lipid peroxidation, UVB-induced collagenase and c-jun mRNA levels and UVB-induced JNK2 activity (57), suggesting a potential role for iron in UVB-induced cell signaling, which may be linked to the formation of ROS in the cell. We have hypothesized that sustained induction of the transcription factor complex, AP-1, plays a functional role in UVB-induced skin tumor promotion and the development of non-melanoma skin cancers (NMSC). In addition we are testing the hypothesis that inhibition of UVB-induced AP-1 will 2137

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contribute to chemoprevention of NMSC. We have examined the effects of UVB, iron, an iron chelator and antioxidants on AP-1 transactivation in a human keratinocyte cell line (HaCaT). UVB-induced ROS may be involved in induction of AP-1 transactivation in human keratinocytes, and iron-driven generation of ROS may increase UVB-induced AP-1 transactivation. Materials and methods Chemicals, reagents and equipment DFO, FeCl3, N-acetyl cysteine (NAC) and Trolox were purchased from Sigma (St Louis, MO). Trolox methyl ether (Trolox-Me) was synthesized from Trolox by Dr Bhasker Reddy Avula at the Southwest Environmental Health Sciences Center Synthetic Core Laboratory at The University of Arizona. Trolox-Me was prepared by NaH-catalyzed alkylation of Trolox by methyl iodide in dimethylformamide. The final product was obtained by alkaline hydrolysis of the Trolox methyl ester. R- and S-Trolox-Me are also available from Fluka (Milwaukee,WI). D-luciferin was purchased from Promega (Madison, WI). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco BRL (Grand Island, NY). Westinghouse FS-20 UVB lamps were purchased from National Biological (Twinsburg, OH) and a UVX digital radiometer with a UVX-31 sensor was purchased from Ultraviolet Products (San Gabriel, CA). Approximately 80% of the lamp output was in the UVB spectrum (290–320), ,1% in the UVC spectrum (,290 nm), 4% in the UVA spectrum (320–400 nm), and the remainder was in the visible spectrum according to manufacturer’s specifications. Other reagents were obtained commercially from standard sources and used without purification. Cells and UV irradiation HaCat cells were stably transfected with a luciferase reporter construct (containing a single cis AP-1 response element) driven by the human collagenase type I promoter as described previously (30). The cell line HCL14 was isolated by ring cloning, selected with G418, and screened for 12-Otetradecanoyl phorbol-13-acetate-induced luciferase activity. Logarithmically growing HCL14 cells were plated at a density of 30 000 cells per 35 mm dish in DMEM (10% fetal bovine serum, 1% penicillin–streptomycin) and allowed to adhere for 24 h. Cells were then serum starved in serum-free DMEM for 24 h. Immediately prior to UVB irradiation, the cells were washed twice in PBS [3 M NaCl, 14 mM KH2PO4, 85 mM K2HPO4 (pH 7.4)] and UVB irradiated without media to a dose of 250 J/m2 under a bank of two Westinghouse FS20 lamps. Immediately after irradiation, serum-free DMEM was added to the cells. At various times post-irradiation (up to 12 h) the medium was aspirated, the cells were washed twice with PBS, and lysis buffer [1% (v/v) Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA (pH 8.0) and 1 mM DTT] was added. Cells were scraped off the plate and cell lysates were frozen at –80°C. DFO and FeCl3 treatments HCL14 cells were treated with the iron chelator DFO or with FeCl3 to determine the effects on UVB-induced AP-1. DFO (0.025, 0.1, 0.4, 0.8 mM, final concentration) or FeCl3 (0.3, 0.6, 1.2 mM, final concentration) in ddH2O were added to cells in serum-free DMEM at various times prior to UVB irradiation, and replaced in serum-free DMEM post-irradiation. Alternately, cells were treated at the time of initial serum starvation with DFO (0.4 mM) for 18 h followed by removal of DFO-containing serum-free media and addition of FeCl3 (0.6 mM)-containing serum-free media for the remaining 6 h prior to UVB irradiation. Cells were then treated with DFO and FeCl3 post-UVB irradiation. Cells were irradiated with UVB as indicated above and were harvested 12 h post-UVB treatment. Antioxidant treatments HCL14 cells were treated with the antioxidants Trolox and NAC to determine the effect on UVB- and FeCl3-induced AP-1. HCL14 cells were treated with the water-soluble vitamin E derivative Trolox (1 and 3 mM) or Trolox-Me (1 and 3 mM) for up to 24 h prior to UVB treatment and for 12 h post-UVB. Trolox and Trolox-Me were dissolved directly in serum-free DMEM and the pH was adjusted back to 7.2 with a small volume of 10 M NaOH. Alternately, cells were treated with NAC (1, 15, 30 mM) at various times following UVB treatment. NAC was dissolved directly in serum-free DMEM, and the pH was adjusted back to 7.2 with a small volume of 10 M NaOH. Cells were irradiated with UVB as indicated above and were harvested 12 h post-UVB treatment. In vitro luciferase assays Frozen cell extracts were thawed on ice and centrifuged at 14 000 r.p.m. for 5 min. Supernatants were collected and protein content was measured using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Luciferase assay buffer [180 ml; 25 mM glycylglycine (pH 7.8), 15 mM K3PO4 (pH 7.8),

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Fig. 1. (A) DFO inhibits UVB-induced AP-1 in a dose-dependent manner. HCL14 cells were treated with DFO (0.025, 0.1, 0.4, 0.8 mM) in serum-free DMEM 18 h prior to UVB (250 J/m2) treatment, and for 12 h following UVB treatment. (B) Time dependence of the effect of DFO on UVBinduced AP-1. HCL14 cells were treated with DFO (0.4 mM) at various times before UVB treatment (250 J/m2) and for 12 h following UVB treatment. Cells were harvested 12 h after UVB treatment in both experiments. Luciferase protein levels are expressed as RLU/mg protein. Data are the averages 6 SD of three measurements; *P , 0.05 as compared with 1UV control.

15 mM MgSO4, 4 mM EGTA (pH 8.0), 2 mM ATP and 1 mM DTT] was added to 100 mg of extract protein. Activity of luciferase protein in lysates was measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratories, Sparks, MD) with the injection of 100 µl of 0.2 mM D-luciferin [in 25 mM glycylglycine (pH 70.8), 15 mM MgSO4, 4 mM EGTA (pH 8.0) and 2 mM DTT]. After 2 s, emission was integrated over a 10 s interval and expressed as relative light units (RLU). A background reading of lysis buffer plus assay buffer was subtracted from all values before further calculations. Statistics Results were analyzed for statistical significance by ANOVA. Treatment groups were compared with control (UVB-irradiated) values. Experimental data shown in the figures are representative of at least three repeat experiments, with three samples per treatment group.

Results DFO inhibits UVB- and iron-induced AP-1 UVB-irradiation (250 J/m2) increases AP-1 transactivation in HCL-14 cells ~6–10-fold over unirradiated controls (Figures 1, 3, 4 and 5). DFO treatment inhibits UVB-induced AP-1 in both a time- and dose-dependent manner, with ~80% inhibition occurring with 12–24 h of pre-treatment and doses of 0.4 and 0.8 mM DFO (Figure 1A and B). Inhibition of UVB-induced AP-1 decreases with decreasing DFO pretreatment time. This suggests that endogenous iron is involved in UVB-induced AP-1 signaling, and that DFO accumulation is necessary for chelation of free iron in the cell. To determine if excess iron would exacerbate the UVBinduction of AP-1 transactivation, HCL14 cells were treated with FeCl3 for up to 18 h prior to UVB treatment. UVB-induced AP-1 activity was potentiated 2–3-fold by iron treatment at doses of 0.6 and 1.2 mM FeCl3 (Figure 2A), whereas basal

DFO- and UVB-induced AP-1

Fig. 2. (A) FeCl3 potentiates the induction of AP-1 transactivation by UVB. HCL14 cells were treated with FeCl3 (0.3, 0.6, 1.2 mM) 18 h prior to UVB treatment (250 J/m2) and for 12 h following UVB treatment. (B) Time dependence of the effect of FeCl3 on UVB-induced AP-1. HCL14 cells were treated with FeCl3 (0.6 mM) at various times before UVB treatment (250 J/m2) and for 12 h following UVB treatment. Cells were harvested 12 h after UVB treatment in both experiments. Luciferase protein levels are expressed as RLU/mg protein. Data are the averages 6 SD of three measurements; *P , 0.05 as compared with 1UV control.

Fig. 3. DFO inhibits UVB and FeCl3-induced AP-1. HCL14 cells were treated with DFO alone (0.4 mM) for 18 h, followed by a 6 h treatment with FeCl3 alone (0.6 mM) prior to UVB treatment (250 J/m2). DFO and FeCl3 were replaced following UVB treatment. Cells were harvested 12 h after UVB treatment in both experiments. Luciferase protein levels are expressed as RLU/mg protein. Data are the averages 6 SD of three measurements; *P , 0.05 as compared with 1UV control.

AP-1 activity was not affected by iron in unirradiated cells (data not shown). A minimum of 6 h of FeCl3 treatment prior to UVB was necessary to cause a significant increase in UVBinduced AP-1 (Figure 2B), suggesting that the cells capacity to store free iron has been exceeded by this extended iron treatment. DFO (0.4 mM) was able to inhibit both UVB-induced AP-1 and the potentiation of UVB-induced AP-1 by FeCl3 (0.6 mM) (Figure 3), suggesting that the iron-chelating properties of DFO account for the inhibition of UVB-induced AP-1, and not a non-specific effect of DFO on cell signaling. Due to the fact that both UVB and iron induce an oxidative insult in cells and the fact that UVB treatment can release FeCl3 from ferritin in the cell, causing further oxidative insult via Fenton chemistry,

Fig. 4. (A) NAC Inhibits UVB-induced AP-1 in a dose-dependent manner. HCL14 cells were treated with NAC (1, 15, 30 mM) for 6 h immediately following UVB treatment (250 J/m2). (B) Early oxidative events are important in UVB-induced AP-1 transactivation. HCL14 cells were treated with NAC (15 mM) at various times post-UVB treatment (250 J/m2). Cells were harvested 12 h after UVB treatment in both experiments. Luciferase protein levels are expressed as RLU/mg protein. Data are the averages 6 SD of three measurements; *P , 0.05 as compared with 1UV control.

we postulated that ROS may be affecting the AP-1 signaling pathway. Cells were then treated with antioxidants to determine their effects on UVB- or iron-induced AP-1 activity. Antioxidants inhibit UVB- and FeCl3-induced AP-1 NAC, a thiol reductant, antioxidant and glutathione precursor, and Trolox, a water-soluble vitamin E derivative, were both effective in inhibiting UVB-induced AP-1 transactivation, and Trolox was effective in inhibiting the potentiation of UVBinduced AP-1 by FeCl3 (Figures 4 and 5). In Figure 4, cells were treated with NAC immediately following UVB, and NAC containing media was removed from the cells 6 h after UVB treatment due to toxicity associated with longer NAC treatments. NAC doses of 15 and 30 mM caused approximately 75% inhibition of UVB-induced AP-1 transactivation (Figure 4A). If initiation of NAC treatment was delayed to .3 h after UVB, NAC treatment was ineffective in inhibiting UVBinduced AP-1 activity (Figure 4B), suggesting that early oxidative events are important in the induction of AP-1 transactivation. We attempted to ascertain the effect of NAC on the potentiation of UVB-induced AP-1 by iron as well. However, the combination of iron and NAC was toxic to the cells, and no reliable estimate of AP-1 activity could be ascertained (data not shown). In Figure 5A, Trolox was added to the cells 24 h prior to UVB treatment, and Trolox treatment was continued following UVB irradiation. Shorter Trolox treatment times prior to UVB treatment were ineffective in inhibiting UVB-induced AP-1 (data not shown). Treatments of 1 and 3 mM Trolox inhibited UVB-induced AP-1 transactivation by ~30 and 75% respectively. A complication introduced into the experiment by adding Trolox to the cells prior to UVB involves the UVB-absorbing characteristics of Trolox. One 2139

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Fig. 5. (A) Trolox inhibits UVB-induced AP-1. HCL14 cells were treated with Trolox (1, 3 mM) 24 h prior to UVB treatment (250 J/m2) and for 12 h following UVB treatment. (B) Trolox-Me does not inhibit UVBinduced AP-1. HCL14 cells were treated with Trolox-Me (1 and 3 mM) 24 h prior to UVB treatment (250 J/m2), and for 12 h following UVB treatment. (C) Trolox inhibits FeCl3 potentiation of UVB-induced AP-1. HCL14 cells were treated with FeCl3 (0.6 mM) 6 h prior to UVB treatment (250 J/m2) or with FeCl3 in the presence of Trolox (1 and 3 mM). Trolox treatment was initiated 24 h prior to UVB treatment. Both Trolox and FeCl3 were replaced following UVB treatment. Cells were harvested 12 h after UVB treatment in all three experiments. Luciferase protein levels are expressed as RLU/mg protein. Data are the averages 6 SD of three measurements. (A and B) *P , 0.05 as compared with 1UV control; (C) *P , 0.05 as compared with UV1FeCl3.

cannot distinguish between sunscreening versus antioxidant effects of Trolox on UVB-induced AP-1 if Trolox is added to the cells before UVB irradiation. To address this point, cells were also treated with a Trolox derivative, containing a methyl ether at the 6-OH position of the Trolox chromanol ring. This non-hydrolyzable modification blocks the antioxidant capabilities of the chromanol hydroxyl group, but maintains the UVB-absorbing characteristics of Trolox. Trolox-Me did not significantly inhibit UVB-induced AP-1 at 1 and 3 mM doses (Figure 5B), suggesting that the actions of Trolox on UVB-induced AP-1 are due primarily to its antioxidant actions rather than its sunscreening actions. Trolox also inhibits the potentiation of UVB-induced AP-1 by FeCl3 (Figure 5C), suggesting that iron induction of UVB-induced AP-1 is dependent on the generation of ROS by iron. Discussion NMSC, which includes both basal and squamous cell carcinomas, is a very common malignancy in modern sunbathing societies. Many recent chemopreventive strategies for NMSC have centered on prevention of UVB-induced skin damage and tumorigenesis, with the inhibition of UVB-induced oxidative damage at the forefront. UVB-induced ROS in the 2140

cell may cause lipid, protein and DNA damage, and proliferation via cell signaling pathways. Much is known about the effects of UVC-induced ROS on cell signaling pathways in a variety of cell types but, in contrast, very little is known about cell signaling pathways affected by the more biologically relevant UVB. In the present study, we have demonstrated the ability of the iron chelator DFO and the antioxidants Trolox and NAC to inhibit UVB-induced AP-1 signaling. In addition, the contribution of free cellular iron to UVB-induced AP-1 signaling has been shown. These observations suggest a role for iron chelation in the prevention of tumor promotion by UVBinduced oxidants. The majority of iron in the cell is tightly bound to ferritin, the major iron-storage protein in the cell. Iron is also associated with other low affinity ligands such as proteins, ATP and cellular lipids (58–62). This loosely bound iron and a cellular fraction of ‘free iron’ also known as the labile iron pool, or LIP, is available for oxidative chemistry via the Fenton reaction. Free iron levels are closely regulated by ferritin synthesis and binding. If there is an increase in iron or oxidative damage in the cell, iron regulatory proteins initiate the synthesis of ferritin in the cell, to decrease levels of free iron (14,63). In the presence of UVB-induced oxidants, a transient increase in free iron can occur via release of iron from ferritin (12,17,64). The changes in cellular iron localization in the presence of UVB light suggests a role for iron in the exacerbation of ROS formation by UVB, which can contribute to UVB-induced proliferative signaling, lipid, DNA and protein damage, and tumorigenesis. The iron chelator DFO is able to inhibit UVB-induced AP-1 signaling in human keratinocytes. This effect of DFO is lessened with decreasing pre-treatment time before UVB, suggesting that accumulation of DFO is necessary to chelate all potential free iron in the cell prior to UVB irradiation. DFO does not absorb UVB radiation and, thus, is not acting as a sunscreen. The fact that DFO inhibits UVB-induced AP-1 strongly indicates that endogenous free iron is important in the induction of AP-1 signaling by UVB. These data suggest that indeed ‘free iron’ is increased with UVB treatment, and the ROS cycle established with UVB and iron may contribute to the UVB-induced AP-1 signaling in the cell. If this is true, then additional increases in free iron should further increase UVB-induced AP-1. We have modeled a significant increase of ‘free iron’ in the cell via the addition of exogenous iron in the form of FeCl3. FeCl3 causes a potentiation of UVB-induced AP-1 signaling in HCL14 cells, and DFO is able to inhibit the effects of exogenously added iron on UVB-induced AP-1 signaling. DFO is loaded into the cell prior to the exposure of the cells to exogenous iron; therefore, the effect of DFO is not to inhibit the entrance of iron into the cell, it is to chelate the free iron in the cell. The fact that a 6 h treatment with iron is necessary to elicit a potentiating response on AP-1 signaling suggests that the cellular capacity of ferritin for the incoming iron has been exceeded, and ferritin is not being synthesized fast enough to bind the incoming iron. Interestingly, iron treatment alone is not sufficient to increase AP-1 signaling in the cell. There is no increase in basal AP-1 signaling in control unirradiated cells treated with iron. The combination of UVB and free iron is necessary to initiate the cycle of Fenton chemistry. UVB causes increases in lipid peroxide, superoxide and other oxidants in the cell which can lead to oxidative damage if they are not first scavenged by cellular antioxidants. Hydrogen peroxide is produced secondary to

DFO- and UVB-induced AP-1

superoxide production via superoxide dismutase. Iron is able to potentiate UVB-induced oxidative damage via its interaction with the superoxide radical. Superoxide reduces free ferrous iron to ferric iron, which then interacts with peroxides in the cell to form the highly reactive hydroxyl radical. The reactivity of hydroxyl radical is much greater than other UVB-induced oxidants, thus making hydroxyl radical less likely to be scavenged by endogenous antioxidants, and more likely to cause oxidative damage (61). The ability of antioxidants to inhibit UVB-induced AP-1 signaling lends further evidence to support this hypothesis of oxidant signaling via the AP-1 pathway. NAC is able to inhibit UVB-induced AP-1 signaling, and NAC must be present early after UVB-irradiation to have an effect. This suggests that early oxidative events are important in AP-1 signaling. This also supports the idea that the increase in free iron and ROS occurs soon after UVB-irradiation. Trolox also inhibits UVBinduced AP-1; however, the duration of pre-treatment time with Trolox is important, suggesting that it takes time for Trolox to accumulate in the cell to a large enough degree to have an effect on UVB-induced ROS. Trolox is also able to inhibit the potentiating effect of iron on UVB-induced AP-1, and perhaps this is the most convincing data that iron affects UVB-induced AP-1 signaling via generation of oxidant species. However, exactly where oxidants are acting in the UVBinduced signaling cascade is unknown. AP-1 DNA-binding activity has been shown to be redox regulated (65), and ROS have been shown to induce expression of the AP-1 protein c-fos (66). Activity of MAP kinases upstream of AP-1 has been shown to be redox sensitive as well (67–70). UVBirradiation is known to activate the JNK, ERK and p38 MAP kinases, and preliminary studies in our laboratory show that DFO inhibits MAP kinase activation in UVB-irradiated HCL14 cells; therefore, oxidants most likely affect UVB-induced signaling at or above the level of MAP kinases (K.KramerStickland and M.Gonzales, unpublished observation) in these cells. In conclusion, given the knowledge that cellular free iron may be an important factor in UVB-induced oxidative damage and UVB-induced signaling in the cell, further investigation of iron chelators such as DFO may be warranted as potential chemopreventive agents for NMSC. To the authors’ knowledge no work has been done to estimate the topical absorption of DFO or other iron chelators. However, preliminary data in AP-1 transgenic mice indicate that topical DFO is able to inhibit UVB-induced AP-1 signaling in mouse skin (Z.Dong and C.Shu-Huang, unpublished observation). Acknowledgements We thank Dr Bhasker Reddy Avula for the synthesis and purification of Trolox-Me. This work was supported in part by NIH grant CA-27502 and The Ladies’ Auxiliary to the Veterans of Foreign Wars.

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