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Photobiology
PHOTOBIOLOGY
Photobiology
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315
Photocytotoxicity and Photochemistry of the Selective Non-Steroidal Androgen Receptor Modulator TDPQ Piotr Bilski1, Boris Risek2, Colin F Chignell1, and William T Schrader2 1 2 Laboratory of Pharmacology and Chemistry, Laboratory of Reproductive and Developmental Toxicology, NIEHS/NIH, RTP, NC
Photosensitized Oxidation of Hypoxanthine and Xanthine by AlPcS4. Role of the Alkylating Quinone 2,5-Dicloro-Diaziridinyl-1,4-Benzoquinone Antonio E. Alegria1, and Yaritza Inostroza1 1 University of Puerto Rico-Humacao Photoirradiation of nitrogen-saturated aqueous solutions containing aluminum phthalocyanine tetrasulfonate (AlPcS4) at 675 nm in the presence of 2,5-dicloro-diaziridinyl-1,4benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX derivatives, xanthine (X) and uric acid (UA). Concentrations of the AZDClQ semiquinone, X and UA increase at the expense of HX with an increase in irradiation time. Almost negligible decomposition of HX, as well as, very low amounts of X are detected if photolysis occurs under identical conditions but in the absence of AZDClQ. Addition of calf-thymus DNA produces quinone-DNA covalent adducts after photolysis of anaerobic samples containing quinone, DNA and AlPcS4, in the presence or absence of HX and at pH 5.5. However, larger amounts of quinone-DNA adducts are detected if HX is present. The results presented here could have applications in the photodynamic treatment of hypoxic tissues such as solid tumors, under conditions of high HX concentration, where type I pathways could be more important than singlet oxygen generation.
316 Mechanisms of Phototoxicity of Peroxidized Docosahexaenoate and Protection by Antioxidants Linda Bakker1, Michael E Boulton2, and Malgorzata Rozanowska1 1 2 Cardiff University, UK, University of Texas Medical Branch, USA Docosahexaenoate (DHA) is found in abundance in neurons and is essential for their function. Many beneficial products are formed by enzymatic peroxidation of DHA, but in the retina, due to high metabolic rates and availability of oxygen, presence of photosensitizers and high light fluxes, DHA may also undergo non-enzymatic peroxidation. This results in production of potentially toxic products able to photosensitize the production of singlet oxygen and free radicals. The aim of this study was to investigate mechanisms of peroxidized DHA (pDHA) toxicity using antioxidants with different modes of action. Exposure of retinal 2 pigment epithelium (RPE) cells in vitro to pDHA and 15 mW/cm white light led to a dose-dependent loss of mitochondrial activity, morphological changes and cell detachment. The free radical scavenger α-tocopherol reduced toxicity, while the singlet oxygen quencher zeaxanthin did not. To investigate the role of reactive aldehydes in toxicity, phosphatidyl-ethanolamine (PE) was used. PE reduced the quantum yield of singlet oxygen from 0.14 to 0.08, but led to an increased absorption of blue light so the net yields of singlet oxygen were similar with and without PE. Despite this, PE increased cell survival from 18 to 79%. To determine if reactive aldehydes can be detoxified by glutathione S-transferase (GST), cells were treated with GST, glutathione and N-acetyl-Lcysteine, which increased cell survival by ~60%. In conclusion, we have shown that pDHA is phototoxic with reactive aldehydes playing a key role, and have identified 3 detoxifying pathways. Our results suggest pDHA may play a role in age-related macular degeneration, the leading cause of blindness in the developed world, and provide a basis for potential therapeutic strategies.
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Non-steroidal androgen receptor (AR) ligand 1,2,3,4-tetrahydro2,2-dimethyl-6-(trifluoromethyl)-8-pyridono [5,6-g]quinoline (TDPQ) is a fluorescent compound that we found to be photochemically active and photocytotoxic. We have characterized its spectral properties in comparison to its structural precursor carbostyril 151 (C151). The absorption maximum of TDPQ is approximately 400 nm in CH3CN, whereas the absorption maximum of C151 is at 350 nm. The fluorescence quantum yields in CH3CN measured against a quinine sulfate standard are 0.8 for TDPQ and 0.02 for C151, while the corresponding dominant fluorescence lifetimes are: 4.2ns and 0.2ns, respectively. Both fluorescences were little affected by different solvents. TDPQ was photobleached upon irradiation by 405 nm light typically used for excitation in biological studies. 1 Singlet oxygen ( O2) could be involved in this bleaching process 1 because we found TDPQ to be a moderate O2 photosensitizer. In 1 contrast, the more photostable C151 did not produce O2 and was unsuitable for experiments in cells due to its ultra-violet light 1 absorption. The quantum yield of O2 photosensitization by TDPQ was about 7% in CH3CN, which is usually sufficient to induce phototoxicity. We confirmed that TDPQ was indeed photocytotoxic to human prostate cancer cells in culture. Combination of the well-defined TDPQ’s AR modulating activity with the compound’s photocytotoxic potential makes it a promising photosensitizer that has the attractive potential to selectively target AR-positive malignant cells during photodynamic therapy.
318 Singlet Oxygen-Mediated Photo-Oxidation of Tryptophan Residues: Characterization of Intermediate Peroxides and Stable Products Michelle Gracanin1, David I. Pattison1, Clare L. Hawkins1, and Michael J. Davies1 1 The Heart Research Institute, Sydney, Australia Proteins are major biological targets for oxidative damage within cells due to their high abundance and their rapid rates of reaction with free radicals and excited state species such as singlet oxygen. Exposure of some free amino acids and proteins to singlet oxygen generates peroxides in high yield, with these formed predominately on Tyr, Trp, and His residues. Peroxides have also been detected on proteins within intact cells on exposure to a photosensitizer and light. The structures of some of these materials have been elucidated for free amino acids, but less is known about the species formed on peptides and proteins. In the current study we have characterized Trp-derived peroxides and breakdown products generated on free Trp, and Trp residues in peptides and proteins, using LC-MS/MS. Exposure of free Trp to visible light in the presence of the sensitizer Rose Bengal gave 7 major products that have been characterized in detail. These include two isomeric hydroperoxides, two alcohols, two diols and N-formylkynurenine, with these materials consistent with photooxidation occurring via the formation and subsequent reactions of singlet oxygen. The hydroperoxides are readily decomposed at elevated temperatures and in the presence of reductants, to give increased yields of the alcohols. These materials are acidsensitive. In order to examine whether these species can be used as markers of damage induced by singlet oxygen we have employed pronase digestion to release these products from oxidized proteins. Under the conditions employed, some of these
SFRBM 2007
materials can be quantified post-hydrolysis by LC-MS/MS, providing direct evidence of peroxide formation on proteins and the involvement of singlet oxygen. This approach may allow the quantification of protein modification in intact cells arising from singlet oxygen formation.
319 Photo Oxidative Stress in Human Epidermal Melanocytes: How to Cope with Photoreactivity of Melanogenesis Related Compounds? Laurent Marrot1, Jean-Philippe Belaidi1, Christophe Jones1, Philippe Perez1, and Jean-Roch Meunier1 1 L'Oreal Life Sciences Research Melanin has been described as a double edge sword because of its ability to both generate and scavenge reactive species. The melanocyte faces thus a very peculiar situation towards oxidative stress, especially when exposed to sunlight. Here, we report data which exemplify this paradox using normal human epidermal melanocytes (NHEM) in culture. First, using the comet assay, we have shown that endogenous melanin (and/or its chemical intermediates) could be involved in UVA-induced lesions in genomic DNA: the more pigmented the cells, the more intense the DNA damage and stimulation of melanogenesis further increased this photogenotoxic stress. Moreover, even in the dark, oxidative DNA damage were higher in cells prepared from highly pigmented dysplastic nevi than in less pigmented melanocytes from a normal skin area from the same donor. In order to get some more information at the molecumar level, the antioxidant status of melanocytes was questioned. We first noticed that the Heme Oxygenase 1 gene (HO1) was over-expressed when NHEM in culture were exposed to UV radiation from a solar simulator (300400 nm) or to UVA radiation only (320-400 nm). Stimulation of melanogenesis prior to irradiation further increased HO1 induction, consistently with our previous results on UVA-induced DNA damage. HO1 being a member of phase 2 antioxidant genes controlled by the transcription factor Nrf2, we finally compared the status of this metabolic pathway in NHM and NHK (normal human keratinocytes) in response to either UVA or to the chemical sulforaphane (Nrf2-inducer). From the differences we observed, we could confirm that HO1 plays a peculiar role in NHEM. Altogether, our results strongly suggest that melanocytes have got specific defense mechanisms to cope with endogenous and exogenous oxidative stress.
320 Determination of Phototoxicity, Crystalline Form and Light-Induced Free Radical Formation for Tattoo Inks Containing TiO2 Wayne Wamer1, and Jun Jie Yin1 1 U.S. Food and Drug Administration TiO2 is a pigment widely used in tattoo inks and has been anecdotally associated with increased sensitivity of tattooed skin to sunlight. Because of the potential importance of light-induced effects on tattooed skin, we investigated the photoactivity of several tattoo inks containing TiO2. The crystalline form of TiO2 in 10 inks was determined by X-ray diffraction. The phototoxicity of the inks was determined using human skin fibroblasts. Skin fibroblasts, exposed for 18 hr to suspensions of an ink in culture media, were washed twice with phosphate-buffered saline and 2 2 irradiated with 10 J/cm UVA light (320 - 400 nm) and 45 J/cm visible light (400 – 800 nm). The fibroblasts were then re-plated and incubated for 10 to 14 days for colony formation. Phototoxicity was expressed as inhibition of colony formation for fibroblasts treated with an ink and light compared to fibroblasts treated with TiO2 or light alone. Light-induced generation of free radicals was studied using ESR. Each ink was suspended in a
solution containing the spin-trap, DMPO, in deionized water. The suspension was irradiated at 320 nm and the ESR spectrum was recorded. We determined that 7 of the 10 inks were phototoxic. Xray diffraction studies revealed that these 7 inks contained anatase, the more photoactive form of TiO2. These inks also photosensitized the formation of free radicals. The remaining 3 inks were not phototoxic, contained the rutile form of TiO2 and did not photosensitize the formation of free radicals. These results suggest that determination of the crystalline form and lightinduced free radical formation are useful tools for identifying phototoxic inks containing TiO2.
321 Identification of Small Molecule Nrf2-Activators Targeting Skin Cell Photo-Oxidative Stress Georg Thomas Wondrak1, Shirley Zhang1, Christopher Michael Cabello1, Nicole Frances Villeneuve1, Stephanie Ley1, Zheng Sun1, and Donna Daoji Zhang1 1 College of Pharmacy & Arizona Cancer Center, University of Arizona, Tucson, AZ, USA The rising incidence of nonmelanoma and melanoma skin cancers and the established role of solar UV-radiation as a major environmental carcinogen create an urgent need for better agents for skin photoprotection. Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2 activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that exhibit activity as modulators of the Nrf2-Keap1 signaling pathway. A series of cinnamon-derived electrophilic Michael acceptors has been identified as potent Nrf2-activators active in the low micromolar range. Hit confirmation was performed in a secondary screen based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts and HaCaT keratinocytes exposed to test compounds that passed primary screening. Subsequent bioefficacy profiling of positive test compounds was performed measuring compound-induced upregulation of hemeoxygenase I (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), two well studied Nrf2 target genes involved in the cellular antioxidant response. Induction of target gene expression was detected using real time RT-PCR analysis and confirmed by determination of NQO1 specific enzymatic activity and HO-1 immunoblotting, respectively. Finally, test compounds were assessed for their photoprotective efficacy in human skin cells. Suppression of intracellular oxidative stress and photo-oxidative induction of apoptosis by pretreatment with cinnamon-derived Nrf2-activators was achieved in skin cells exposed to high doses of singlet oxygen generated by dye-sensitization. Our pilot studies suggest feasibility of developing small molecule Nrf2-activators into novel photo-chemopreventive agents for topical suppression of skin cell photo-oxidative stress. Supported in part by National Institutes of Health grants (SWEHSC pilot research grant [ES06694]).
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322 Photo-Induced Reactive Oxygen Species Generation and Cytotoxicity of Different Water-Soluble Fullerenes in Human HaCaT Keratinocytes Baozhong Zhao1, Piotr J. Bilski1, Yu-Ying He1, Li Feng1, and Colin F. Chignell1 1 Laboratory of Pharmacology and Chemistry, NIEHS/NIH, Research Triangle Park, NC 27709, USA Nanoparticles are currently an area of intense scientific interest, due to a wide variety of potential applications in biomedical, optical, and electronic fields. Nanoparticles encompass a wide variety of chemical structures including fullerenes, nanotubes, dendrimers and quantum dots. The increasing use of nanomaterials has prompted widespread concern over their safety. We have therefore studied the (photo)toxicity of the fullerene C60 employing four different preparations: (γ-CyD)2/C60 (γ-cyclodextrin complexed C60); THF/nC60 (prepared by THF solvent exchange); Son/nC60 (sonication of a toluene solution of C60 mixed with water); and γ-CyD/nC60 (by heating (γ-CyD)2/C60 water solution). The absorption spectra indicated that (γCyD)2/C60 in water was present in monomeric state whereas in 1 the other preparations C60 was aggregated. The O2 quantum yield of (γ-CyD)2/C60 in D2O was ~1.0, the same as C60 dissolved in 1 toluene; emission maximum was 1273 nm. In contrast O2 phosphorescence generated from the aggregated state of C60 was red-shifted by 10 nm with a very short lifetime in D2O (2.9 µs). Also it was very sensitive to oxygen but was not quenched by NaN3. Irradiation (λ>300 nm) of an aqueous solution of (γCyD)2/C60 in the presence of NADH generated the anion radical – – (C60)• . When DMPO was present the DMPO/O2• adduct was observed, confirming the generation of superoxide as a result of – the reaction of (C60)• with oxygen. Superoxide was also generated during the irradiation of THF/nC60 and Son/nC60 but only in the presence of NADH. The phototoxicity of the C60 preparations was tested against HaCaT keratinocytes, an immortal human cell line. (γ-CyD)2/C60 showed high phototoxicity and no dark toxicity. The aggregated C60 preparations showed no evidence of (photo)cytotoxicity, with the exception of THF/nC60 which exhibited dark toxicity.
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Photobiology
317
315
Photocytotoxicity and Photochemistry of the Selective Non-Steroidal Androgen Receptor Modulator TDPQ Piotr Bilski1, Boris Risek2, Colin F Chignell1, and William T Schrader2 1 2 Laboratory of Pharmacology and Chemistry, Laboratory of Reproductive and Developmental Toxicology, NIEHS/NIH, RTP, NC
Photosensitized Oxidation of Hypoxanthine and Xanthine by AlPcS4. Role of the Alkylating Quinone 2,5-Dicloro-Diaziridinyl-1,4-Benzoquinone Antonio E. Alegria1, and Yaritza Inostroza1 1 University of Puerto Rico-Humacao Photoirradiation of nitrogen-saturated aqueous solutions containing aluminum phthalocyanine tetrasulfonate (AlPcS4) at 675 nm in the presence of 2,5-dicloro-diaziridinyl-1,4benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX derivatives, xanthine (X) and uric acid (UA). Concentrations of the AZDClQ semiquinone, X and UA increase at the expense of HX with an increase in irradiation time. Almost negligible decomposition of HX, as well as, very low amounts of X are detected if photolysis occurs under identical conditions but in the absence of AZDClQ. Addition of calf-thymus DNA produces quinone-DNA covalent adducts after photolysis of anaerobic samples containing quinone, DNA and AlPcS4, in the presence or absence of HX and at pH 5.5. However, larger amounts of quinone-DNA adducts are detected if HX is present. The results presented here could have applications in the photodynamic treatment of hypoxic tissues such as solid tumors, under conditions of high HX concentration, where type I pathways could be more important than singlet oxygen generation.
316 Mechanisms of Phototoxicity of Peroxidized Docosahexaenoate and Protection by Antioxidants Linda Bakker1, Michael E Boulton2, and Malgorzata Rozanowska1 1 2 Cardiff University, UK, University of Texas Medical Branch, USA Docosahexaenoate (DHA) is found in abundance in neurons and is essential for their function. Many beneficial products are formed by enzymatic peroxidation of DHA, but in the retina, due to high metabolic rates and availability of oxygen, presence of photosensitizers and high light fluxes, DHA may also undergo non-enzymatic peroxidation. This results in production of potentially toxic products able to photosensitize the production of singlet oxygen and free radicals. The aim of this study was to investigate mechanisms of peroxidized DHA (pDHA) toxicity using antioxidants with different modes of action. Exposure of retinal 2 pigment epithelium (RPE) cells in vitro to pDHA and 15 mW/cm white light led to a dose-dependent loss of mitochondrial activity, morphological changes and cell detachment. The free radical scavenger α-tocopherol reduced toxicity, while the singlet oxygen quencher zeaxanthin did not. To investigate the role of reactive aldehydes in toxicity, phosphatidyl-ethanolamine (PE) was used. PE reduced the quantum yield of singlet oxygen from 0.14 to 0.08, but led to an increased absorption of blue light so the net yields of singlet oxygen were similar with and without PE. Despite this, PE increased cell survival from 18 to 79%. To determine if reactive aldehydes can be detoxified by glutathione S-transferase (GST), cells were treated with GST, glutathione and N-acetyl-Lcysteine, which increased cell survival by ~60%. In conclusion, we have shown that pDHA is phototoxic with reactive aldehydes playing a key role, and have identified 3 detoxifying pathways. Our results suggest pDHA may play a role in age-related macular degeneration, the leading cause of blindness in the developed world, and provide a basis for potential therapeutic strategies.
S126
Non-steroidal androgen receptor (AR) ligand 1,2,3,4-tetrahydro2,2-dimethyl-6-(trifluoromethyl)-8-pyridono [5,6-g]quinoline (TDPQ) is a fluorescent compound that we found to be photochemically active and photocytotoxic. We have characterized its spectral properties in comparison to its structural precursor carbostyril 151 (C151). The absorption maximum of TDPQ is approximately 400 nm in CH3CN, whereas the absorption maximum of C151 is at 350 nm. The fluorescence quantum yields in CH3CN measured against a quinine sulfate standard are 0.8 for TDPQ and 0.02 for C151, while the corresponding dominant fluorescence lifetimes are: 4.2ns and 0.2ns, respectively. Both fluorescences were little affected by different solvents. TDPQ was photobleached upon irradiation by 405 nm light typically used for excitation in biological studies. 1 Singlet oxygen ( O2) could be involved in this bleaching process 1 because we found TDPQ to be a moderate O2 photosensitizer. In 1 contrast, the more photostable C151 did not produce O2 and was unsuitable for experiments in cells due to its ultra-violet light 1 absorption. The quantum yield of O2 photosensitization by TDPQ was about 7% in CH3CN, which is usually sufficient to induce phototoxicity. We confirmed that TDPQ was indeed photocytotoxic to human prostate cancer cells in culture. Combination of the well-defined TDPQ’s AR modulating activity with the compound’s photocytotoxic potential makes it a promising photosensitizer that has the attractive potential to selectively target AR-positive malignant cells during photodynamic therapy.
318 Singlet Oxygen-Mediated Photo-Oxidation of Tryptophan Residues: Characterization of Intermediate Peroxides and Stable Products Michelle Gracanin1, David I. Pattison1, Clare L. Hawkins1, and Michael J. Davies1 1 The Heart Research Institute, Sydney, Australia Proteins are major biological targets for oxidative damage within cells due to their high abundance and their rapid rates of reaction with free radicals and excited state species such as singlet oxygen. Exposure of some free amino acids and proteins to singlet oxygen generates peroxides in high yield, with these formed predominately on Tyr, Trp, and His residues. Peroxides have also been detected on proteins within intact cells on exposure to a photosensitizer and light. The structures of some of these materials have been elucidated for free amino acids, but less is known about the species formed on peptides and proteins. In the current study we have characterized Trp-derived peroxides and breakdown products generated on free Trp, and Trp residues in peptides and proteins, using LC-MS/MS. Exposure of free Trp to visible light in the presence of the sensitizer Rose Bengal gave 7 major products that have been characterized in detail. These include two isomeric hydroperoxides, two alcohols, two diols and N-formylkynurenine, with these materials consistent with photooxidation occurring via the formation and subsequent reactions of singlet oxygen. The hydroperoxides are readily decomposed at elevated temperatures and in the presence of reductants, to give increased yields of the alcohols. These materials are acidsensitive. In order to examine whether these species can be used as markers of damage induced by singlet oxygen we have employed pronase digestion to release these products from oxidized proteins. Under the conditions employed, some of these
SFRBM 2007
materials can be quantified post-hydrolysis by LC-MS/MS, providing direct evidence of peroxide formation on proteins and the involvement of singlet oxygen. This approach may allow the quantification of protein modification in intact cells arising from singlet oxygen formation.
319 Photo Oxidative Stress in Human Epidermal Melanocytes: How to Cope with Photoreactivity of Melanogenesis Related Compounds? Laurent Marrot1, Jean-Philippe Belaidi1, Christophe Jones1, Philippe Perez1, and Jean-Roch Meunier1 1 L'Oreal Life Sciences Research Melanin has been described as a double edge sword because of its ability to both generate and scavenge reactive species. The melanocyte faces thus a very peculiar situation towards oxidative stress, especially when exposed to sunlight. Here, we report data which exemplify this paradox using normal human epidermal melanocytes (NHEM) in culture. First, using the comet assay, we have shown that endogenous melanin (and/or its chemical intermediates) could be involved in UVA-induced lesions in genomic DNA: the more pigmented the cells, the more intense the DNA damage and stimulation of melanogenesis further increased this photogenotoxic stress. Moreover, even in the dark, oxidative DNA damage were higher in cells prepared from highly pigmented dysplastic nevi than in less pigmented melanocytes from a normal skin area from the same donor. In order to get some more information at the molecumar level, the antioxidant status of melanocytes was questioned. We first noticed that the Heme Oxygenase 1 gene (HO1) was over-expressed when NHEM in culture were exposed to UV radiation from a solar simulator (300400 nm) or to UVA radiation only (320-400 nm). Stimulation of melanogenesis prior to irradiation further increased HO1 induction, consistently with our previous results on UVA-induced DNA damage. HO1 being a member of phase 2 antioxidant genes controlled by the transcription factor Nrf2, we finally compared the status of this metabolic pathway in NHM and NHK (normal human keratinocytes) in response to either UVA or to the chemical sulforaphane (Nrf2-inducer). From the differences we observed, we could confirm that HO1 plays a peculiar role in NHEM. Altogether, our results strongly suggest that melanocytes have got specific defense mechanisms to cope with endogenous and exogenous oxidative stress.
320 Determination of Phototoxicity, Crystalline Form and Light-Induced Free Radical Formation for Tattoo Inks Containing TiO2 Wayne Wamer1, and Jun Jie Yin1 1 U.S. Food and Drug Administration TiO2 is a pigment widely used in tattoo inks and has been anecdotally associated with increased sensitivity of tattooed skin to sunlight. Because of the potential importance of light-induced effects on tattooed skin, we investigated the photoactivity of several tattoo inks containing TiO2. The crystalline form of TiO2 in 10 inks was determined by X-ray diffraction. The phototoxicity of the inks was determined using human skin fibroblasts. Skin fibroblasts, exposed for 18 hr to suspensions of an ink in culture media, were washed twice with phosphate-buffered saline and 2 2 irradiated with 10 J/cm UVA light (320 - 400 nm) and 45 J/cm visible light (400 – 800 nm). The fibroblasts were then re-plated and incubated for 10 to 14 days for colony formation. Phototoxicity was expressed as inhibition of colony formation for fibroblasts treated with an ink and light compared to fibroblasts treated with TiO2 or light alone. Light-induced generation of free radicals was studied using ESR. Each ink was suspended in a
solution containing the spin-trap, DMPO, in deionized water. The suspension was irradiated at 320 nm and the ESR spectrum was recorded. We determined that 7 of the 10 inks were phototoxic. Xray diffraction studies revealed that these 7 inks contained anatase, the more photoactive form of TiO2. These inks also photosensitized the formation of free radicals. The remaining 3 inks were not phototoxic, contained the rutile form of TiO2 and did not photosensitize the formation of free radicals. These results suggest that determination of the crystalline form and lightinduced free radical formation are useful tools for identifying phototoxic inks containing TiO2.
321 Identification of Small Molecule Nrf2-Activators Targeting Skin Cell Photo-Oxidative Stress Georg Thomas Wondrak1, Shirley Zhang1, Christopher Michael Cabello1, Nicole Frances Villeneuve1, Stephanie Ley1, Zheng Sun1, and Donna Daoji Zhang1 1 College of Pharmacy & Arizona Cancer Center, University of Arizona, Tucson, AZ, USA The rising incidence of nonmelanoma and melanoma skin cancers and the established role of solar UV-radiation as a major environmental carcinogen create an urgent need for better agents for skin photoprotection. Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2 activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that exhibit activity as modulators of the Nrf2-Keap1 signaling pathway. A series of cinnamon-derived electrophilic Michael acceptors has been identified as potent Nrf2-activators active in the low micromolar range. Hit confirmation was performed in a secondary screen based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts and HaCaT keratinocytes exposed to test compounds that passed primary screening. Subsequent bioefficacy profiling of positive test compounds was performed measuring compound-induced upregulation of hemeoxygenase I (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), two well studied Nrf2 target genes involved in the cellular antioxidant response. Induction of target gene expression was detected using real time RT-PCR analysis and confirmed by determination of NQO1 specific enzymatic activity and HO-1 immunoblotting, respectively. Finally, test compounds were assessed for their photoprotective efficacy in human skin cells. Suppression of intracellular oxidative stress and photo-oxidative induction of apoptosis by pretreatment with cinnamon-derived Nrf2-activators was achieved in skin cells exposed to high doses of singlet oxygen generated by dye-sensitization. Our pilot studies suggest feasibility of developing small molecule Nrf2-activators into novel photo-chemopreventive agents for topical suppression of skin cell photo-oxidative stress. Supported in part by National Institutes of Health grants (SWEHSC pilot research grant [ES06694]).
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322 Photo-Induced Reactive Oxygen Species Generation and Cytotoxicity of Different Water-Soluble Fullerenes in Human HaCaT Keratinocytes Baozhong Zhao1, Piotr J. Bilski1, Yu-Ying He1, Li Feng1, and Colin F. Chignell1 1 Laboratory of Pharmacology and Chemistry, NIEHS/NIH, Research Triangle Park, NC 27709, USA Nanoparticles are currently an area of intense scientific interest, due to a wide variety of potential applications in biomedical, optical, and electronic fields. Nanoparticles encompass a wide variety of chemical structures including fullerenes, nanotubes, dendrimers and quantum dots. The increasing use of nanomaterials has prompted widespread concern over their safety. We have therefore studied the (photo)toxicity of the fullerene C60 employing four different preparations: (γ-CyD)2/C60 (γ-cyclodextrin complexed C60); THF/nC60 (prepared by THF solvent exchange); Son/nC60 (sonication of a toluene solution of C60 mixed with water); and γ-CyD/nC60 (by heating (γ-CyD)2/C60 water solution). The absorption spectra indicated that (γCyD)2/C60 in water was present in monomeric state whereas in 1 the other preparations C60 was aggregated. The O2 quantum yield of (γ-CyD)2/C60 in D2O was ~1.0, the same as C60 dissolved in 1 toluene; emission maximum was 1273 nm. In contrast O2 phosphorescence generated from the aggregated state of C60 was red-shifted by 10 nm with a very short lifetime in D2O (2.9 µs). Also it was very sensitive to oxygen but was not quenched by NaN3. Irradiation (λ>300 nm) of an aqueous solution of (γCyD)2/C60 in the presence of NADH generated the anion radical – – (C60)• . When DMPO was present the DMPO/O2• adduct was observed, confirming the generation of superoxide as a result of – the reaction of (C60)• with oxygen. Superoxide was also generated during the irradiation of THF/nC60 and Son/nC60 but only in the presence of NADH. The phototoxicity of the C60 preparations was tested against HaCaT keratinocytes, an immortal human cell line. (γ-CyD)2/C60 showed high phototoxicity and no dark toxicity. The aggregated C60 preparations showed no evidence of (photo)cytotoxicity, with the exception of THF/nC60 which exhibited dark toxicity.
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SFRBM 2007