14 - Current Topics in Photobiology, I
Florida 3 14:00 - 16:00
|Chair(s): Josh Williams, Rebecca Justiniano|
14:00 Molecular Engineering of Photosensitizers for Enhanced Photodynamic Therapy Against Pigmented Melanoma JM Dabrowski*, Faculty of Chemistry, Jagiellonian University, Krakow, Poland
; B Pucelik, Faculty of Chemistry, Jagiellonian University, Krakow, Poland; G Stochel, Faculty of Chemistry, Jagiellonian University, Krakow, Poland; LG Arnaut, Chemistry Departament, University of Coimbra, Coimbra, Portugal
Abstract: Photodynamic therapy (PDT) has emerged as a promising strategy in oncology. Its multiple approaches to optimize therapeutic regimens and the targeting of tumor vasculature may be especially useful in the treatment of melanoma. However, PDT of pigmented melanoma has generally been unsuccessful because insufficient light penetration through tissue and high concentration of melanin that acts as an optical shield and as antioxidants. PDT photosensitizers and protocols need to be carefully optimized in order to overcome these challenges. The treatment of melanoma requires highly active photosensitizers that absorb at long wavelengths (700"800 nm) where melanin does not absorb and the energy is still sufficient to generate reactive oxygen species (ROS). In this work the relevant photosensitizer properties (electronic absorption, photostability, nature of generated ROS, polarity and delivery) and treatment parameters (drug and light doses, drug-to-light intervals, radiant exposure and tumor margin) to optimize PDT for melanoma are discussed. Comprehensive in vitro studies against B16F10 melanoma cells with photostable NIR absorbing bacteriochlorin (redaporfin) showed that redaporfin-P123 micelles led to higher cellular uptake and increased oxidative stress compared with photosensitizer alone after short incubation times. Neither redaporfin encapsulated in Pluronics nor P123 micelles alone exhibited cytotoxicity in a broad concentration range. On the other hand, they cause strong light dose depended apoptosis and necrosis. Vascular-targeted PDT using redaporfin in P123 against B16F10 tumors in C57BL/6J mice with light doses of 74 J/cm2 led to 100% complete cures (no tumor regrowth ca. one year post-treatment). This remarkable result reveals that redaporfin has nearly ideal properties and its modification with Pluronic block copolymers helps to overcome the resistance of melanoma cells via increased tumor selectivity and enhanced ROS generation in Pluronic micelles.
14:15 Wavelengths from 385 " 405nm cause photodamage to skin cells KP Lawrence*, King's College London
; RPE Sarkany, King's College London; S Acker, BASF Grenzach GmbH; B Herzog, BASF Grenzach GmbH; AR Young, King's College London
Abstract: The adverse effects of solar UVR (~295 " 400nm) on the skin are well documented, especially in the UVB region (~295-315/320nm), and sunscreens have been shown to be beneficial in inhibiting a wide range of photodamage. The effects of long-wave UVA (>380nm) and visible radiation on the skin are much less well known, but increasingly studied. Most sunscreen formulations provide very little protection in the long wave UVA region (380-400nm) and almost none from shortwave visible wavelengths (400-420nm). We demonstrate photodamage in this region using high irradiance, narrowband LED arrays at 385nm and 405nm, using environmentally relevant doses. The endpoints include cell viability, DNA damage (cyclobutane pyrimidine dimers " CPD), differential gene expression and oxidative stress in vitro in HaCat keratinocytes, and pigmentation and erythema in vivo in human volunteers. For most endpoints we found a clear dose-response relationship for both sources. There was a highly significant reduction in cell viability (385nm p<0.0001; 405nm p<0.0001), increase in reactive oxygen species (385nm p<0.0001; 405nm p=0.0001), and several genes associated with adverse effects were significantly upregulated including HMOX-1, MMP-1, IL-1a, IL-6 and IL-8 (385nm p<0.02; 405nm p<0.003). PON-2 was upregulated by 385nm but not 405nm (385nm p=0.0002; 405nm p=0.1122). At high doses of 385nm radiation there was an increase in CPD production, but no effect at 405nm. In addition we demonstrate that these sources can induce skin-type dependent changes in erythema, IPD and delayed tanning in vivo. This work provides new insight into photodamage and may lead to new strategies to provide improved photoprotection in this poorly protected region.
14:30 Potential Hot Spots for Solar-Induced DNA Lesions RR Ramazanov*, St.Petersburg State University
; DA Maksimov, St.Petersburg State University; AI Kononov, St.Petersburg State University
Abstract: UV-induced DNA lesions are not randomly distributed among the bases. Local DNA structure can affect both photophysical and photochemical aspects of DNA photo-damage. We report on the electronic spectra of DNA stacked bases calculated for a wide range of base stacking conformations taken from PDB data bank and molecular dynamics simulations. Quantum mechanical calculations on stacked thymine, cytosine, adenine (R. R. Ramazanov, D. A. Maksimov, A. I. Kononov, J. Am. Chem. Soc. 2015, 137, 11656), and guanine dimers reveal low-lying excited excitonic states in the stacked bases with reduced inter-base distance. Absorption of terrestrial solar radiation at 300 nm by such structures increases dramatically with respect to canonical forms of stacking. The low-lying excited states at about 300 nm in the dimers may also serve as a trap for the excitation energy transfer from the neighboring bases in the helix. We thus conclude that such sites with non-canonical stacked geometries can be hot spots for sunlight-induced DNA damage.
14:45 Long-term Skin Pigmentation after a Single Sunburn Reveals Altered Hemidesmosome Plasticity SG Coelho*, Center for Drug Evaluation and Research, FDA
; SA Miller, Center for Devices and Radiological Health, FDA; TM Michele, Center for Drug Evaluation and Research, FDA
Abstract: Despite educational efforts to reduce the risk for skin cancers from preventable UV overexposure, sunburns occur quite frequently, especially in vulnerable populations such as children. Severe sunburn events can occur in the absence of using protective measures, but may also occur during careless use of sunscreens and/or other protective measures to prolong exposure times in the sun. Until now, no long-term evaluation of the consequences from a single sunburn had ever been done. Based on our work, approximately 32-61% of individuals with moderate sunburn from a single exposure (2-4x their minimal erythema dose) developed a long-lasting pigmentation (LLP) effect that persisted for greater than 9 months. By studying 6 individuals (3 LLP+ and 3 LLP-) in detail, the goal was to investigate whether this long-term effect was signaling overt changes to melanin production/distribution, cellular proliferation and/or skin morphology. Based on immunohistochemistry, the increased visual pigmentation was corroborated with increased melanin staining in the basal layer of the epidermis in LLP+ individuals. This increase in pigmentation was not due to any increased melanogenesis, but rather retention of melanin in basal keratinocytes. There was a measureable increase in basement membrane interdigitation at the epidermal-dermal junction. This basement membrane plasticity was characterized by decreased hemidesmosome density by electron microscopy, attenuation of hemidesmosomal partners (integrin alpha 6 beta 4 and plectin) by immunofluorescence and spatial regulation of SoxF family transcription factor (Sox7) by proximity ligation assays. The detected histopathological features resemble characteristics of solar lentigos, which are known risk factors for precancerous lesions. Our results further underscore advice to encourage individuals to use various sun protective measures.
15:00 Rapid deamination of cyclobutane pyrmidine dimers at TCG sites in the FOS nucleosome in vivo VJ Cannistraro, Washington University
; S Pondugula, Washington University; Q Song, Washington University; JS Taylor*, Washington University
Abstract: C to T UV signature mutation hotspots in the p53 tumor suppressor gene of skin cancers occur primarily at methylated PyCpG sites that have been correlated with UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations can be explained by the rapid deamination of the C or 5-methyl-C in the CPD's to U or T followed by insertion of A by the DNA damage bypass polymerase Î·. As a consequence, the relative mutagenicity of C-containing CPDs is expected to depend on the relative deamination rates. In vivo, DNA is largely bound to nucleosomes which we have shown in vitro modulate the deamination rates of TmCG CPD's by 12-fold over a complete turn of the DNA helix. To determine the extent to which this occurs in vivo, we determined the deamination rates of CPD's at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells by LMPCR. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared to two other sites located at outside positions. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Most importantly, deamination of CPDs at TCG sites were faster than at all other C-containing dipyrimidine sites.
15:15 Towards Label-Free Evaluation of Oxidative Stress in Human Skin Exposed to Sun Filters S Osseiran*, Massachusetts General Hospital
; H Wang, Massachusetts General Hospital; Y Suita, Massachusetts General Hospital; E Roider, Massachusetts General Hospital; DE Fisher, Massachusetts General Hospital; CL Evans, Massachusetts General Hospital
Abstract: Skin cancer is the most common form of cancer in North America, and its incidence remains on the rise despite growing use of sunscreen. Given the regulatory history of cosmetics in the US, there is a concern regarding the safety of sun filters that remains to be tested. Indeed, previous experiments have shown that they induce oxidative stress in in vitro cultures, warranting the study of these compounds in intact human skin. Label-free optical methods for evaluating oxidative stress rely on fluorescence of NADH and FAD, two key metabolic co-enzymes. However, sun filters exhibit fluorescence that interferes with that of NADH. Here, we present a method based on fluorescence lifetime imaging microscopy to decompose the fluorescence of NADH and sun filters to (1) specifically localize the compounds spatiotemporally as they penetrate through skin, and (2) to exclude the sun filter signal from that of NADH to measure oxidative stress.
15:30 Ultraviolet A - Induced Oxidation in Cornea : Characterization Of The Early Oxidation-Related Events C Zinflou*, University Laval and Centre Hospitalier Universitaire de Quebec Research Center, Canada
; PJ Rochette, University Laval and Centre Hospitalier Universitaire de Quebec Research Center, Canada
Abstract: Molecular and epidemiological data show that chronic exposure to sunlight ultraviolet (UV) rays, especially to UVA wavelengths (315 " 400 nm), is toxic for eyes and might be linked to many ocular diseases. UVA are the main component of solar UV reaching the eyes. Their absorption by cellular photosensitizers efficiently enhances oxygen reactive species production, leading to an oxidative stress induction. It has been described that UVA-induced oxidative stress plays a role in UVA toxicity. However, events triggered by oxidation and involved in the toxic effects of UVA radiations remain unknown. We have thus investigated the early cellular and molecular changes linked to oxidation in the cornea, the most UV-exposed ocular structure, following an ocular exposure to UVA rays. Three markers were used to assess changes throughout the three corneal layers (epithelium, stroma and endothelium) of UVA-irradiated (6000 kJ/m^2) rabbit eyes: (1) oxidized mitochondrial flavins/reduced NAD(P) (ox-Fvm/NAD(P)H) ratio as a sensor of mitochondrial activity and redox state; (2) 8-oxo-7,8-dihydroguanine (8-oxodG) formation in nuclei and mitochondria as a marker of photo-oxidative DNA damage and (3) levels of S-glutathionylated proteins (SG-Prot) as a marker of transiently impaired redox signaling. In response to UVA, we found significantly higher ox-Fvm/NAD(P)H ratio, reflective of a disrupted mitochondrial redox balance, in the most posterior parts of the cornea. Besides, UVA-induced 8-oxodG are concentrated in nuclear DNA of the epithelium, while they are found in both nuclear and mitochondrial DNA in stromal and endothelial layers. Finally, altered patterns of SG-Prot appear in the three layers immediately after UVA exposure and seem to correlate with UVA penetrance. Our data indicate that mitochondrial anomalies and prolonged disruption of proteins normal functions, under stress favored by a chronic UVA exposure, are events potentially critical for the development of UVA toxicity.