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Hydroxyl Radical Production and DNA Damage via Photolysis by Sunlight of the Genotoxic Hydroxamic Acid Intermediate of Polyaromatic Amine Carcinogen
forward electron transport (FET) and (4) reverse electron transport (RET). S1QELs and rotenone suppressed IQr, with relative potency of S1QEL1 > rotenone > S1QEL2. None of the compounds suppressed IQf at any tested concentration. S1QELs were functionally different from rotenone: S1QELs and rotenone suppressed IQr at submicromolar concentrations, but the inhibition of FET and RET by S1QELs was far less potent than that of rotenone. Rotenone also inhibited FET more strongly than RET, where the opposite was true for S1QEL1. S1QEL1 was also found to be different from S1QEL2: S1QEL1 inhibited FET and RET but S1QEL2 did not, which suggests they have different mechanisms.
doi: 10.1016/j.freeradbiomed.2016.10.080 40 Novel Selective Fluorescent Probes for Imaging Superoxide, Peroxynitrite, Hypochlorous Acid, Hydrogen Peroxide and Hydroxyl Radical Naikei Wong1, Jun Jacob Hu1, Tao Peng1, Xiaoyu Bai1, Mingyang Lu1, Sen Ye1, and Dan Yang1 1 The University of Hong Kong, People's Republic of China Remarkable advances of biological understanding have been made on the complex roles of reactive oxygen/nitrogen species (ROS/RNS) both as signaling molecules and pathogenic contributors in health and disease contexts. However, due to a persistent lack of reliable molecular tools, particularly smallmolecule fluorogenic probes with decisively robust properties such as selectivity, sensitivity, chemo-/photo-stability, organellespecificity, and rapid turn-on, researchers continue to face challenges of how to establish a strong mechanistic base for ever more intricate phenomena. Here we report the design, synthesis and application of several efficient fluorescent probes (HKSOX, HKGreen/Yellow, HKOCl, HKPerox, and HKOH series) for the selective detection and molecular imaging of endogenous superoxide, peroxynitrite, hypochlorous acid, hydrogen peroxide and hydroxyl radical, respectively, with cytosol, mitochondria and lysosome targeting capacity. Those probes open up exciting opportunities for establishing new experimental paradigms and accelerating mechanistically focused biological discoveries.
doi: 10.1016/j.freeradbiomed.2016.10.081 41 Nitrosodisulfide [S2NO] − (Perthionitrite) is a True Intermediate during the "Cross-Talk"; of Nitrosyl and Sulfide Ari Zeida1, Juan P. Marcolongo1, Uriel N. Morzan1, Damián A. Scherlis1, and José A. Olabe1 1 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina NO and H2S are endogenously produced in humans and show multiple actions relevant to animal and plant physiology. Both gases often exert similar and, in part, interdependent biological actions within the same model system resulting in either mutual attenuation or potentiation responses, the so-called NO/H2S “cross-talk”. New biological mediators might be involved, and attention is being directed to some early-described N-S hybrid
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species such as thionitrous acid (HSNO) and thionitrites NOS−, as well as to nitrosodisulfide S2NO− (perthionitrite), the sulfur analog of peroxynitrite. The reactions of H2S with low-molecular weight and/or protein Snitrosothiols might afford a new scenario for the modulation of the S-nitrosothiol profile in the cells. It is currently accepted that HSNO can be initially formed via the transnitrosation of S-nitrosothiols RSNO with H2S. This mechanism shows an intermediate (Iyellow) with a growing UV-vis absorption band centered at 412 nm.1 Iyellow attained its maximum absorbance in ~1 min, and was moderately stable for ~1 h, revealing a slow decay. This initial UV-vis display does not match with HSNO, given that HSNO absorb at ~330 nm. 1,2 A high subsequent reactivity of HSNO leading to I yellow has been invoked for explaining this fact, and two species have been proposed for identifying Iyellow, namely polysulfides HSn− (n = 2-7)1 or perthionitrite, S2NO−,2,3 via the transnitrosation process as follows: HSNO + HS2− S2NO− + HS− + H+ (eq. 1) In this contribution, we performed QM-MM molecular dynamics free energy profile determinations, combined with a TD-DFT analysis, in order to contribute to a clear identification of S 2NO– in different solvents, accounting for the UV-Vis signatures and broadening the mechanistic picture of N/S signaling in biochemistry. Our results strongly support the early formation and survival of the S 2NO– intermediate in the aqueous solutions, arising from the reaction between HSNO and HS2–. Filipovic MR, et al., J Am Chem Soc 2012,134 (29). Cortese-Krott MM, et al., Redox Biol 2014, 2 (234). Cortese-Krott MM, et al., Proc Natl Acad Sci USA 2015, 112 (4651).
doi: 10.1016/j.freeradbiomed.2016.10.082 42 Hydroxyl Radical Production and DNA Damage via Photolysis by Sunlight of the Genotoxic Hydroxamic Acid Intermediate of Polyaromatic Amine Carcinogen Ben-Zhan Zhu1, Dan Xu1, and Chun-Hua Huang1 1 Research Centre for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing, People's Republic of China The hydroxyl radical (●OH) has been considered to be one of the most reactive oxygen species produced in biological systems, which can cause DNA, protein, and lipid oxidation. N-hydroxy-2acetamidofluorene (N-OH-AAF) has been identified as a major genotoxic metabolite of 2-nitroflurene, a polyaromatic amine carcinogen commonly found in diesel exhaust. The carcinogenicity of N-OH-AAF has been mainly attributed to the formation of DNA adducts via aryl nitrenium ion. Recently, we found, unexpectedly, that exposure of N-OH-AAF to sunlight can induce the formation of not only DNA single and double strand breaks, but also 8-OHdG, both of which were inhibited by the typical ●OH scavengers (DMSO and ethanol), but not by the classic iron and copper chelating agents. ESR secondary radical spin-trapping with DMPO and fluorescent studies with terephthalic acid confirmed that ●OH was indeed generated from N-OH-AAF photolysis, with the formation of the characteristic DMPO/•OH and the fluorescent 2hydroxyterephthalic acid, respectively. This is the first report that the highly reactive ●OH can be readily produced via homolysis of N-OH-AAF by sunlight, which represents a new mechanism of ● OH production that does not require redox-active metal ions, and provide a new perspective to understand the molecular mechanism for the carcinogenicity of polycyclic aromatic amines.
doi: 10.1016/j.freeradbiomed.2016.10.083
SfRBM / SFRRI 2016
doi: 10.1016/j.freeradbiomed.2016.10.080 40 Novel Selective Fluorescent Probes for Imaging Superoxide, Peroxynitrite, Hypochlorous Acid, Hydrogen Peroxide and Hydroxyl Radical Naikei Wong1, Jun Jacob Hu1, Tao Peng1, Xiaoyu Bai1, Mingyang Lu1, Sen Ye1, and Dan Yang1 1 The University of Hong Kong, People's Republic of China Remarkable advances of biological understanding have been made on the complex roles of reactive oxygen/nitrogen species (ROS/RNS) both as signaling molecules and pathogenic contributors in health and disease contexts. However, due to a persistent lack of reliable molecular tools, particularly smallmolecule fluorogenic probes with decisively robust properties such as selectivity, sensitivity, chemo-/photo-stability, organellespecificity, and rapid turn-on, researchers continue to face challenges of how to establish a strong mechanistic base for ever more intricate phenomena. Here we report the design, synthesis and application of several efficient fluorescent probes (HKSOX, HKGreen/Yellow, HKOCl, HKPerox, and HKOH series) for the selective detection and molecular imaging of endogenous superoxide, peroxynitrite, hypochlorous acid, hydrogen peroxide and hydroxyl radical, respectively, with cytosol, mitochondria and lysosome targeting capacity. Those probes open up exciting opportunities for establishing new experimental paradigms and accelerating mechanistically focused biological discoveries.
doi: 10.1016/j.freeradbiomed.2016.10.081 41 Nitrosodisulfide [S2NO] − (Perthionitrite) is a True Intermediate during the "Cross-Talk"; of Nitrosyl and Sulfide Ari Zeida1, Juan P. Marcolongo1, Uriel N. Morzan1, Damián A. Scherlis1, and José A. Olabe1 1 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina NO and H2S are endogenously produced in humans and show multiple actions relevant to animal and plant physiology. Both gases often exert similar and, in part, interdependent biological actions within the same model system resulting in either mutual attenuation or potentiation responses, the so-called NO/H2S “cross-talk”. New biological mediators might be involved, and attention is being directed to some early-described N-S hybrid
S32
species such as thionitrous acid (HSNO) and thionitrites NOS−, as well as to nitrosodisulfide S2NO− (perthionitrite), the sulfur analog of peroxynitrite. The reactions of H2S with low-molecular weight and/or protein Snitrosothiols might afford a new scenario for the modulation of the S-nitrosothiol profile in the cells. It is currently accepted that HSNO can be initially formed via the transnitrosation of S-nitrosothiols RSNO with H2S. This mechanism shows an intermediate (Iyellow) with a growing UV-vis absorption band centered at 412 nm.1 Iyellow attained its maximum absorbance in ~1 min, and was moderately stable for ~1 h, revealing a slow decay. This initial UV-vis display does not match with HSNO, given that HSNO absorb at ~330 nm. 1,2 A high subsequent reactivity of HSNO leading to I yellow has been invoked for explaining this fact, and two species have been proposed for identifying Iyellow, namely polysulfides HSn− (n = 2-7)1 or perthionitrite, S2NO−,2,3 via the transnitrosation process as follows: HSNO + HS2− S2NO− + HS− + H+ (eq. 1) In this contribution, we performed QM-MM molecular dynamics free energy profile determinations, combined with a TD-DFT analysis, in order to contribute to a clear identification of S 2NO– in different solvents, accounting for the UV-Vis signatures and broadening the mechanistic picture of N/S signaling in biochemistry. Our results strongly support the early formation and survival of the S 2NO– intermediate in the aqueous solutions, arising from the reaction between HSNO and HS2–. Filipovic MR, et al., J Am Chem Soc 2012,134 (29). Cortese-Krott MM, et al., Redox Biol 2014, 2 (234). Cortese-Krott MM, et al., Proc Natl Acad Sci USA 2015, 112 (4651).
doi: 10.1016/j.freeradbiomed.2016.10.082 42 Hydroxyl Radical Production and DNA Damage via Photolysis by Sunlight of the Genotoxic Hydroxamic Acid Intermediate of Polyaromatic Amine Carcinogen Ben-Zhan Zhu1, Dan Xu1, and Chun-Hua Huang1 1 Research Centre for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing, People's Republic of China The hydroxyl radical (●OH) has been considered to be one of the most reactive oxygen species produced in biological systems, which can cause DNA, protein, and lipid oxidation. N-hydroxy-2acetamidofluorene (N-OH-AAF) has been identified as a major genotoxic metabolite of 2-nitroflurene, a polyaromatic amine carcinogen commonly found in diesel exhaust. The carcinogenicity of N-OH-AAF has been mainly attributed to the formation of DNA adducts via aryl nitrenium ion. Recently, we found, unexpectedly, that exposure of N-OH-AAF to sunlight can induce the formation of not only DNA single and double strand breaks, but also 8-OHdG, both of which were inhibited by the typical ●OH scavengers (DMSO and ethanol), but not by the classic iron and copper chelating agents. ESR secondary radical spin-trapping with DMPO and fluorescent studies with terephthalic acid confirmed that ●OH was indeed generated from N-OH-AAF photolysis, with the formation of the characteristic DMPO/•OH and the fluorescent 2hydroxyterephthalic acid, respectively. This is the first report that the highly reactive ●OH can be readily produced via homolysis of N-OH-AAF by sunlight, which represents a new mechanism of ● OH production that does not require redox-active metal ions, and provide a new perspective to understand the molecular mechanism for the carcinogenicity of polycyclic aromatic amines.
doi: 10.1016/j.freeradbiomed.2016.10.083
SfRBM / SFRRI 2016