Photoprotective Effects of Bucillamine Against UV-Induced Damage in an SKH-1 Hairless Mouse Model[Dagger]

By Anwar, Adil Gu, Mallikarjuna; Brady, Sara; Qamar, Lubna; Behbakht, Kian; Shellman, Yiqun G; Agarwal, Rajesh; Norris, David A; Horwitz, Lawrence D; Fujita, Mayumi

ABSTRACT UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non- DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2- methylpropionyl)-L-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm^sup -2^) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg^sup -1^ of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.

INTRODUCTION

Epidemiologic, clinical and biologic studies have implicated repeated exposures of human skin to solar UV irradiation, especially the UVB (290-320 nm) wavelength, as a cause of both melanoma and nonmelanoma skin cancers (1-4). There is considerable evidence that UVB-induced skin cancer is associated with DNA damage (5). DNA damage induces activation of p53 protein and upregulation of p53 transcription.

UVB also causes the generation of reactive oxygen species (ROS) that damage both DNA and non-DNA targets (6-8). Singlet oxygen can be generated as a result of UV absorption by endogenous photosensitive chromophores, such as porphyrins and cytochromes (9- 11). Newly formed ROS including hydroxyl radical and superoxide anion can activate genes (such as cyclooxygenase [COX]-1 which is linked to the inflammatory response), damage DNA or oxidize cell lipids and proteins (7,12), and thereby modify cellular function (6,13). Signals transduced from the cell surface to the nucleus through protein phosphorylation at serine/threonine are altered by ROS and redox reactions (14).

Similarly, UVB induces inflammatory responses including edema, dermal infiltration of leukocytes and production of cytokines and growth factors (15,16). There is mounting evidence that inflammation plays a pivotal role in tumor initiation and promotion (16,17). Therefore, inhibiting UVB-induced ROS generation and inflammation may be expected to prevent or mitigate photodamage and photocarcinogenesis in the skin.

Several studies have reported that both natural and synthetic antioxidants may have beneficial effects against UV-mediated damage to the intracellular redox state (14). Studies have also shown the beneficial effects of anti-inflammatory drugs against cancer and UV- mediated damage (13,18). Long-term use of anti-inflammatory drugs, such as aspirin and selective COX-2 inhibitors, significantly reduces cancer risk (18). For example, sulindac, a nonsteroidal anti- inflammatory drug, attenuated UVB-induced inflammatory responses and reduced UVB-induced events relevant to carcinogenesis (13).

Bucillamine [N-(2-mercapto-2-methylpropionyl)-L-cysteine] (Fig. 1) is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups (19). Bucillamine is a potent antioxidant and reduces inflammation through this mechanism.

Figure 1. Chemical structure of bucillamine showing two donatable thiol groups.

Here, we investigated the effects of bucillamine on UVB-induced photodamage in the SKH-1 hairless mouse.

MATERIALS AND METHODS

Chemicals and reagents. Powdered bucillamine (> 99% purity) was obtained from Keystone Biomedical, Inc. (Los Angeles, CA). Stock solutions of bucillamine (10 mg mL^sup -1^) were made in normal saline, pH adjusted to approximately 7.4 with equimolar NaOH, and filtered sterilized before injecting into the animals. Anti-actin (mouse monoclonal) was purchased from Sigma (St. Louis, MO). Anti- p53 (rabbit polyclonal) was obtained from Novocastra (UK). Phospho- p53 (ser15) and phospho-p53 (ser20) antibodies were from Cell Signaling Technology (Danvers, MA). Anti-ubiquitin antibody was a rabbit polyclonal from Sigma, while anti-p53-upregulated modulator of apoptosis (PUMA) rabbit polyclonal was purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA). Protease inhibitor cocktail tablets (cat. no. 11 836 153 001) were obtained from Roche Diagnostics (Mannheim, Germany) and added to lysis buffer for skin lysate preparation. All chemicals and reagents used in this study were of highest purity grade available commercially.

Animals and treatment. Female SKH-1 hairless mice (6 weeks old) were purchased from Charles River laboratories (Wilmington, MA). After their arrival, animals were housed at the University of Colorado at Denver and Health Sciences Center vivarium under specific pathogen-free conditions, following National Institutes of Health Animal Care Guidelines under an institutional protocol reviewed and approved by the Institutional Animal Care and Use Committee. Animals were allowed to acclimatize for a week prior to the experiment. Animals were fed Purina chow diet and water ad libitum. Throughout the experimental protocol, the mice were maintained at standard conditions-temperature 24 +- 2[degrees]C, relative humidity 50 +- 10% and 12 h room light/12 h dark cycle. Mice were divided into four groups. The first group of six mice did not receive any exposure or treatment and served as control (group 1). The remaining animals were divided into UV exposure alone (group 2), UV + saline treatment (group 3) and UV + bucillamine treatment (group 4). Mice in groups 2-4 were exposed to two doses of 230 mJ cm^sup -2^ UVB, 24 h apart. Two hours prior to each UVB exposure, mice in groups 3 and 4 were treated with normal saline (group 3) or bucillamine at a dose of 20 mg kg^sup -1^ of body weight (group 4) subcutaneously. Animals were killed after the last UVB exposure at various time points, and the dorsal skin was surgically removed and used for further analysis.

UVB source. We employed four FS-40-T-12-UVB sunlamps with UVB spectra 305 Dosimeter (Daavlin, Bryan, OH), which emitted about 80% radiation in the range of 280-340 nm with a peak emission at 314 nm as monitored with a SEL 240 photodetector, 103 filter and 1008 diffuser attached to an IL1400A NIST Traceable Radiometer/ Photometer from International Light (Newburyport, MA). The UVB irradiation doses were also calibrated using an IL1400A radiometer.

Histopathology. Skin samples were fixed in 10% formalin and embedded in paraffin. Vertical sections (4 [mu]m thickness) were cut and mounted on a glass slide, and stained with hematoxylin and eosin followed by microscopic evaluation of the slides.

Immunohistochemical analysis. Formalin-fixed and paraffin- embedded samples were sectioned by microtome, heat immobilized and deparaffinized using xylene, and rehydrated in a graded series of ethanol with a final wash in distilled water. Antigen retrieval was achieved by boiling the sections in citric acid buffer (pH 6.0) in a microwave oven (at 650 W) until the solution boiled. At this time, the power of the microwave was lowered to 100 W for 15 min. The samples were allowed to cool at room temperature followed by rinsing with PBS. The tissue sections were then subjected to incubation with 3% H2O2 in methanol for quenching the endogenous peroxidase activity. Nonspecific binding sites were blocked by incubating with PBS containing 1% bovine serum albumin (BSA) and 0.1% Tween 20 for 10 min. Sections were incubated with the appropriate primary antibody for 1 h at 25[degrees]C, followed by appropriate biotinylated secondary antibody for 1 h at room temperature and conjugated horseradish peroxidase streptavidin for 45 min in a humid chamber. Color development was achieved by incubation with 3,3′- diaminobenzidine for 10 min at room temperature. The sections were counterstained with Harris hematoxylin, dehydrated and mounted for microscopic observation. Immunohistochemical analyses were performed using a Zeiss Axioscop 2 microscope (Carl Zeiss, Inc., Jena, Germany). All samples were coded and evaluated by at least two investigators in a blinded manner. Pictures were taken using a Kodak DC 290 camera and processed using Kodak Microscopy Documentation System 290 (Eastman Kodak Company, Rochester, NY).

Preparation of epidermal skin lysate. Total tissue lysates were prepared as described previously (20). Briefly, mouse skin samples were homogenized in ice-cold lysis buffer (50 mM Tris-Hcl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na^sub 3^VO^sub 4^, 0.5% NP-40, 1% Triton X-100 and 1 mM PMSF, pH 7.4) with a freshly added protease inhibitor cocktail (Roche Diagnostics, Mannhein, Germany). The homogenate was centrifuged at 13 200 g for 15 min at 4[degrees]C, and the supernatant (total cell lysate) was collected, aliquoted and stored at -80[degrees]C. Protein analysis was carried out using the Bio-Rad (Bio-Rad Laboratories, Hercules, CA) assay kit as per the manufacturer’s protocol. BSA was used as a standard for the protein measurements. SDS-PAGE and immunoblot analysis. Proteins were analyzed by SDS-PAGE (12% gels) followed by transfer to a polyvinylidene difluoride membrane (0.4 [mu]m) in 25 mM Tris, 192 mM glycine and 20% methanol at 110 V for 1 h. Membranes were blocked overnight in 10 mM Tris-HCl, pH 8.0, 125 mM NaCl and 0.2% (vol/vol) Tween 20 containing 5% (wt/vol) nonfat dry milk (TBST-M5). Immunoblot analysis of the total protein bands were visualized using enhanced chemiluminescence as described by the manufacturer (Pierce Biotechnology, Inc., Rockford, IL). Immunoblots were subsequently stripped of antibodies by incubating for 15-20 min at 37[degrees]C with Restore Western Stripping Buffer (Pierce Biotechnology, Inc.) and re-probed with beta-actin antibody to confirm protein loading. Densitometric analysis of the membranes was performed using GelDoc 200 (Bio-Rad Laboratories).

RESULTS

In order to assess any adverse effects of bucillamine and UV exposure separately, a preliminary experiment was carried out in which SKH-1 mice were either treated with bucillamine (20 mg kg^sup – 1^ s.c.) or UVB (230 mJ cm^sup -2^) irradiation. Bucillamine can be administered intravenously, subcutaneously, intraperitoneally or orally. We used subcutaneous administration because it is a relatively easy route and the least likely to injure the animal during administration. The selection of bucillamine and UVB doses was based on previously published studies (13,19,21,22). Doses of bucillamine between 10 and 20 mg kg^sup -1^ have offered a high, probably maximal, level of efficacy without toxicity in a previous work. We elected to use the upper end of this range as the administration was only two doses and we wished to be certain whether the drug would have therapeutic efficacy in this model. A high dose of UVB (230 mJ cm^sup -2^) was used as the current study was intended to investigate the effect of bucillamine on acute photodamage. Each group of animals received two doses of bucillamine or UVB, 24 h apart. UVB exposure caused edema, erythema and thickening of the exposed skin (data not shown). Bucillamine had no adverse effects. Therefore this dose of bucillamine was found to be safe and tolerable to SKH-1 hairless mice.

Figure 2. Histopathology of the skin from SKH-1 hairless mice unexposed (A) or irradiated with UVB (230 mJ cm^sup -2^) twice, 24 h apart (B-E). Mice irradiated with UVB were untreated (B, D) or pretreated with bucillamine (C, E). Skin samples were collected at 6 h (B, C) and 24 h (D, E) after the last UVB exposure, and processed for histopathologic analysis as described in Materials and Methods. Arrows indicate dilated blood vessels. Asterisk indicates dermal edema. Open arrows indicate leukocyte infiltration. Bar = 100[mu]m.

Effects of bucillamine on histology in UV-exposed skin

UVB exposure induced mild edema, erythema and thickening of the dorsal skin in untreated SKH-1 mice, while bucillamine pretreatment attenuated the erythema (data not shown). UV-exposed skin in untreated mice showed scattered necrotic epidermal keratinocytes, papillary dermal edema and dermal infiltration of leukocytes at 6 h after the last UVB exposure (Fig. 2B). In bucillamine pretreated mice there were similar abnormalities at 6 h after the last UVB exposure, but the epidermal necrosis was less prominent (Fig. 2C). At 24 h after the last UVB exposure, UV-exposed skin in untreated mice showed hyperkeratosis and acanthosis (thickening of the epidermis) in the epidermis and papillary dermal edema, infiltration of leukocytes and dilated blood vessels in the dermis (Fig. 2D). In contrast, bucillamine pretreatment attenuated the effects of UV on inflammation (dermal edema, leukocyte infiltration and dilatation of blood vessels) at 24 h after the last UVB exposure (Fig. 2E).

Effects of bucillamine on UVB-mediated p53 activation

p53 plays a pivotal role in cellular stress responses. It governs the adaptive and protective responses to multiple stresses. When normal cells are subjected to stress signals, such as DNA damage or oxidative stress, p53 is activated, resulting in transcription of downstream targets that coordinate cellular growth arrest or apoptosis (23).

UVB exposure resulted in a strong induction of total p53 at 6, 12 and 24 h after the last UVB exposure. Bucillamine pretreatment attenuated this effect at 6 h after the last UVB exposure (Fig. 3A). The p53 level was further decreased at 12 and 24 h after the last UVB exposure in bucillamine pretreated mice. Consistent with these findings, immunohistochemical analysis showed increased nuclear p53 staining in the epidermal keratinocytes from UV-exposed skin, whereas bucillamine pretreatment diminished this effect at 24 h after the last UVB exposure (Fig. 3B-F).

Stabilization and upregulation of p53 by UVB is mediated by phosphorylation but not inhibition of degradation

p53 levels are regulated by protein modifications and proteolytic degradation. In particular, phosphorylation at serine (Ser) 15 and serine (Ser) 20 residues of p53 are known to be essential for stabilization and activation of p53. It is also well documented that p53 is predominantly targeted for destruction by the ubiquitin proteasomal pathway (UPP), and inhibition of UPP results in the upregulation of the p53 protein (24). Hence we assessed the effect of UVB and bucillamine on the phosphorylation of p53 and the UPP pathway. UVB exposure at 230 mJ cm^sup -2^ resulted in a strong induction of phosphorylation at both Ser-15 and Ser-20 at 6, 12 and 24 h after the last UVB exposure. Bucillamine pretreatment attenuated this effect at 6 h after the last UVB exposure and further diminished the phosphorylation at 12 and 24 h after the last UVB exposure (Fig. 4A).

UVB exposure did not result in the formation of high molecular weight ubiquitin-positive material, a characteristic feature of inhibition of the UPP pathway (25) (data not shown). As a positive control of UPP inhibition, we used 8B20 mouse melanoma cells treated with UPP inhibitors MG 132 (10 [mu]M) and lactacystin (10 [mu]M), and as a negative control we used 8B20 cells treated with etoposide (50 [mu]M), a DNA-damaging agent. Positive controls showed an increase in the ubiquitin reactive high molecular weight bands between 50 and 250 kDa, while a negative control showed no polyubiquitinated species (data not shown).

Effect of bucillamine on PUMA, a downstream target of p53

In an attempt to investigate the effect of p53 activation by UVB, we analyzed its downstream target PUMA, a p53-upregulated modulator of apoptosis. The activation of PUMA coincided with the activation of p53 at 6, 12 and 24 h after the last UVB exposure in UV-exposed skin samples (Fig. 4B). However, pretreatment with bucillamine attenuated the activation of PUMA at 12 and 24 h after the last UVB exposure. Thus, while UVB exposure induced p53 and its downstream target, PUMA, bucillamine treatment inhibited activation of both p53 and PUMA at 12 and 24 h after the last UVB exposure.

Figure 3. Effect of UVB irradiation and bucillamine treatment on p53 from the SKH-1 hairless mouse skin. SKH-1 hairless mice were irradiated as described previously. They were untreated (UV only), or pretreated with saline (UV+S) or bucillamine (UV+BUC). Skin samples were collected at 6, 12 and 24 h after the last UVB exposure, and processed for immunoblotting analysis and immunohistochemical analysis as described in Materials and Methods. (A) Immunoblot was probed with the anti-p53 antibody. Protein loading was confirmed by stripping the blots and re-probing for beta- actin. C = control with no UVB exposure and no treatment; UV + S = UV exposure and saline pretreatment; UV + BUC = UV exposure and bucillamine pretreatment. Blot shown is a representative of three independent experiments. (B-F) Immunohistochemical staining of p53. Mice were unexposed and untreated (B), exposed with UV and untreated (C, E), or exposed with UV and pretreated with bucillamine (D, F). Samples were collected at 6 h (C, D) and 24 h (E, F) after the last UVB exposure. Tissue sections were stained with the anti-p53 antibody. Bar = 50[mu]m.

DISCUSSION

Solar UV radiation is the most prominent and ubiquitous carcinogen in our environment and the skin is its major target. Several animal studies have shown that UV radiation can act both as a tumor initiator and as a tumor promoter (26-28). UVB exposure of skin results in various adverse biologic responses either through direct (5) or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, ROS and inflammation (29-31). Therefore, protecting the skin against UVB-induced biologic responses would be expected to inhibit the development of photodamage and photocarcinogenesis.

Figure 4. (A) Effect of UVB irradiation and bucillamine treatment on p53-Ser-15 and p53-Ser-20 from SKH-1 hairless mouse skin. The same skin lysates shown in Fig. 3 were used. Mice were untreated (UV only), or pretreated with saline (UV+S) or bucillamine (UV+BUC). Immunoblot was probed with anti-p-Ser-15-p53 and anti-p-Ser-20-p53 antibodies. Protein loading was confirmed by stripping the blots and re-probing for beta-actin. C = control with no UVB exposure and no treatment; UV + S = UV exposure and saline pretreatment; UV + BUC = UV exposure and bucillamine pretreatment. Blot shown is a representative of three independent experiments. (B) Effect of UVB irradiation and bucillamine treatment on PUMA levels from the SKH-1 hairless mouse skin. The same skin lysates shown in Fig. 3 were used. Mice were untreated (UV only), or pretreated with saline (UV+S) or bucillamine (UV+BUC). The immunoblot was probed with the anti-PUMA antibody. Actin levels are shown as loading controls. The blot shown is representative of three independent observations. One of the major aims of the current study was to evaluate the effect of bucillamine, an antioxidant agent, against acute UVB-induced photodamage and to identify the molecular mechanisms for the photoprotection. We have demonstrated that (1) UV exposure of the dorsal skin of SKH-1 mice induced inflammatory responses and p53 activation as early as 6 h after the last UVB exposure and (2) bucillamine pretreatment attenuated UVB-mediated inflammatory responses and p53 activation at 6 h after the last UVB exposure and further diminished these effects at 12 and 24 h after the last UVB exposure.

Bucillamine is a cysteine derivative and functions as an antioxidant by transferring thiol groups to the endogenous glutathione or thioredoxin systems and maintaining them in a reduced state (32,33). Animal studies have shown that bucillamine can attenuate tissue damage during myocardial infarction, cardiac surgery and oxidative injury in reperfusion during organ transplantation (19,21,22). Bucillamine can also inhibit diesel exhaust particle-enhanced allergic sensitization in mice and blood- retinal barrier permeability in streptozotocin-induced diabetic rats by reducing ROS accumulation (34-37). Two closely related compounds, N-acetylcysteine and N-(2-mercaptopropionyl)-glycine, have been shown to work through an antioxidant mechanism (32,38,39). All three compounds contain the basic cysteine molecule, but bucillamine has two donatable thiol groups while the other two have only one, which explains the greater potency of bucillamine as an antioxidant. Previously, we have demonstrated the antioxidant mechanism of action of bucillamine in various experimental settings (19,21,22). Therefore, in this study, we did not attempt to further demonstrate the antioxidant mechanism of bucillamine.

Bucillamine may also function as an anti-inflammatory drug through effects of an oxidized metabolite. It has been used as an effective oral medication in several Asian countries for the treatment of rheumatoid arthritis (RA), a chronic multisystem inflammatory disease (40). Bucillamine inhibits T cell proliferation and production of pro-inflammatory cytokines (41). UV exposure to keratinocytes induces the release of pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, IL-10 and tumor necrosis factor- alpha (TNF-alpha) (42). In particular, IL-1 is a pleiotropic pro- inflammatory cytokine and plays a critical role in cell growth and differentiation, tissue repair, and regulation of immune response by inducing other cytokines (IL-6, IL-18 and TNF-alpha), growth factors (granulocyte macrophage colony-stimulating factor, vascular endothelial growth factor), proteases and inflammatory mediators (COX-2 and inducible nitric oxide synthase) (43). Inflammatory responses induce ROS at the sites of inflammation, which can exacerbate tissue damage. We have observed that UVB irradiation induced inflammation and that pretreatment with bucillamine before UVB irradiation attenuated the inflammation at 12 and 24 h after the last UVB exposure.

p53 is regulated by proteolytic degradation and activation of p53. We have demonstrated that the UVB-mediated increase in p53 is not due to the inhibition of UPP proteolytic degradation. Activation of p53 in response to DNA damage involves an increase in p53 protein levels, through stabilization of the p53 protein (31,44,45). The stabilization of p53 in response to DNA damage has been attributed biochemically to the phosphorylation of p53 serine residues, including Ser-15 and Ser-20 (46,47). DNA-damaging agents activate phosphorylation of p53 at Ser-15 by a family of protein kinases, including ATM and ATR, and Ser-20 by the Chk2 kinase. These phosphorylations prevent the binding of Mdm2, a negative regulator of p53. Consistent with these studies, we have observed that UVB irradiation induced the phosphorylation of p53-Ser-15 and p53-Ser- 20 at 6, 12 and 24 h after the last UVB exposure. However, pretreatment with bucillamine before UV irradiation resulted in a decrease in the activation of p53-Ser-15 and p53-Ser-20 at 12 and 24 h after the last UVB exposure. Therefore, bucillamine appeared to protect against UVB-mediated p53 activation and UVB-mediated p53 phosphorylation.

In this study, we have also shown that bucillamine had an inhibitory effect on the activation of PUMA, a downstream target of p53. It is well known that DNA damage, such as occurs with UV exposure, can cause p53-mediated cell cycle arrest and/or apoptosis. PUMA is a BH3-only protein which is an essential trigger for the induction of apoptosis by binding to the anti-apoptotic protein Bcl- 2 (48). Further investigations are warranted to study the molecular mechanisms of photoprotection by bucillamine.

Bucillamine has been widely used as an oral medication in long- term treatment of RA in Asia (40,49,50). Further studies to optimize dosage and timing of administration (before or after UVB exposure) as well as development of a suitable topical application would be instrumental for the development of future use of bucillamine as an effective agent against UV-induced skin damage.

In summary, the findings of the present study demonstrate that pretreatment of SKH-1 mice with bucillamine before UVB irradiation has a protective effect to quickly attenuate the inflammatory responses and p53 activation via phosphorylation of Ser-15 and Ser- 20 residues of the p53 protein. Bucillamine also had an inhibitory effect on the activation of a downstream target of p53, PUMA, a key pro-apoptotic molecule. Results presented in this paper warrant further investigations for the use of bucillamine as a photoprotective agent against UV-mediated skin damage.

Acknowledgements-The authors thank Dr. Yvonne K. Hodges for her help with bucillamine injections. Financial support in part by the “Skin Cancer Foundation” to A.A. and M.F. is gratefully acknowledged.

[dagger] This paper is part of a special issue dedicated to Professor Hasan Mukhtar on the occasion of his 60th birthday.

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Adil Anwar1, Mallikarjuna Gu2, Sara Brady1, Lubna Qamar3, Klan Behbakht3, Yiqun G. Shell man1, Rajesh Agarwal2, David A. Norris1, Lawrence D. Horwitz4 and Mayumi Fujlta*1

1 Department of Dermatology, School of Medicine, University of Colorado Health Sciences Center, Anschutz Medical Campus, Aurora, CO

2 Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, School of Pharmacy, Denver, CO

3 Department of Obstetrics and Gynecology, School of Medicine, University of Colorado Health Sciences Center, Anschutz Medical Campus, Aurora, CO

4 Division of Cardiology, Department of Medicine, University of Colorado Health Sciences Center, Anschutz Medical Campus, Aurora, CO

Received 31 October 2007, accepted 28 November 2007, DOI: 10.1111/ j.1751-1097.2007.00288.x

* Corresponding author email: mayumi.fujita@uchsc.edu (Mayumi Fujita)

(c) 2008 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/08

Copyright American Society for Photobiology Mar/Apr 2008

(c) 2008 Photochemistry and Photobiology. Provided by ProQuest Information and Learning. All rights Reserved.