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Cytochrome c as a Source of Superoxide Radical: Role of H2O2 and NADH
observed in the case of rosmarinic acid. The direct participation of the unsaturation is very evident while comparing the result between p-coumaric acid (with one phenolic group and cinnamic acid with devoid of phenolic group. We propose that in the first step of the interaction of HER, an electron transfer from the double bond of the side chain of HCAs, takes place leaving a carbon centered radical. This radical forms the HCA-peroxyl radical in presence of ethanol and oxygen, Supportive experimental data for the proposed mechanism which include product characterization by HPLC and kinetic studies for the reaction of HCA-peroxyl radical with ABTS will also be presented in the paper. doi:10.1016/j.freeradbiomed.2010.10.629
616 The Mechanism of the Oxidative Transformation of Boronate Compounds A Quantum Mechanical Study 1,2
1
doi:10.1016/j.freeradbiomed.2010.10.631
2
Adam Sikora , Agnieszka Dybala-Defratyka , Jacek Zielonka , Andrzej Marcinek1, and Balaraman Kalyanaraman2 1 Institute of Applied Radiation Chemistry, Technical University of 2 Lodz, Poland, Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee Recently, a new class of fluorogenic probes for reactive oxygen species containing a boronate moiety has been developed. These assays are based on the oxidative transformation of non- or weakly fluorescent arylboronates (acids or esters) into strongly fluorescent phenolic products. It was shown that boronate compounds can be oxidized into corresponding phenols by hydrogen peroxide, hypochlorous acid, and peroxynitrite. We have found that at neutral pH the reaction of boronates with – 6 6 -1 -1 peroxynitrite (ONOO ) is about 10 times faster (k ≈ 10 M ⋅s ) -1 -1 than the reaction with hydrogen peroxide (k ≈ 1 M ⋅s ). This reaction is direct and stoichiometric. Thus, boronate based probes open up new possibilities for direct detection of peroxynitrite in biological systems. To better understand the molecular mechanism of oxidation of boronates to phenolic products we applied quantum mechanical calculations. The reaction between arylboronates and peroxynitrite anion, or the anionic form of – hydrogen peroxide (HOO ) was studied in gas phase and in aqueous solution using B3LYP/6-31+G(d,p) and PCM/B3LYP/631+G(d,p) levels of theory, respectively. The initial step of – – proposed mechanisms is the addition of ONOO /HOO to the boron atom with the formation of an anionic intermediate. Further transformations of that anionic adducts with the cleavage of the O-O bond lead to the formation of corresponding phenols. For the peroxynitrite anion adduct of arylboronates, two different pathways of that transformation are observed. The major pathway is the heterolytic cleavage of O-O bond, and the minor pathway is the homolytic cleavage. The latter mechanism results in the •– • formation of a caged radical pair [PhB(OH)2O … NO2] and subsequent formation of phenyl radicals via the protonation of •– PhB(OH)2O radical anion. Evidence for phenyl radical formation was obtained using EPR spin-trapping. doi:10.1016/j.freeradbiomed.2010.10.630
617 Cytochrome c as a Source of Superoxide Radical: Role of H2O2 and NADH Murugesan Velayutham1, Craig Hemann1, and Jay L Zweier1 1 The Ohio State University In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. NADH is a substrate for complex-I in the electron transport chain in
S216
mitochondria. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under ischemia, oxidative or nitrative stress, and apoptosis, the peroxidase activity of cyt c is reported to be increased. The level of NADH is also increased under pathophysiological conditions such as cardiac ischemia and diabetes and a concurrent increase in hydrogen peroxide (H2O2) production occurs. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed to understand the related mechanisms of radical generation and NADH oxidation in the peroxidase activity of cyt c. Our results suggest that cyt c may have a novel role in the deleterious effects of cardiac ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, in cardiac myocytes cytochrome c may play a key role in the mitochondrial “ROS-induced ROS-release (RIRR)” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and degenerative neurological diseases.
618 Molecular Cytotoxic Mechanisms of Estradiol involve Estradiol Quinoid Moieties Luke Wan1, Jeff Bruce1, Di Fan1, and Peter J O'Brien1 1 University of Toronto Background: As a major female sex hormone, estradiol is expected to have beneficial effects in females due to its high antioxidant activity. However, prolonged exposure to estradiol increases the risk of developing cancers in the breast and uterus. The liver is the main site of estradiol metabolism such as catechol estrogen formation and detoxification. Estradiol’s carcinogenic effect has been well characterized, but it has also been found to be cytotoxic. It is important to understand the molecular mechanisms of estradiol cytotoxicity to prevent hidden internal tissue injury. Methods: An accelerated cytotoxicity mechanism screening (ACMS) technique was used with primary rat hepatocytes in order to determine the molecular mechanism of estrogen toxicity and detoxification in the rat. Hepatocytes were isolated by collagenase perfusion and incubated with estradiol and various inhibitors of estradiol metabolizing. The effectiveness of the various metabolic pathways could then be ranked. Cytotoxicity was assessed by trypan blue exclusion and mitochondrial membrane potential while oxidative stress endpoints were measured through hydrogen peroxide and other reactive oxygen species formation. Results: Estradiol was found to cause a concentration and time dependent toxicity. Estradiol cytotoxicity was increased by GSH depletion, UGT inhibition, COMT inhibition, SULT inhibition, NQO inhibition, and CYP3A inhibition. Hepatocytes could be rescued from estradiol using o-quinone trapping agents, increasing glutathione synthesis, inhibiting P450 nonspecifically, and inhibiting CYP1A. When H2O2/peroxidase or tyrosinase was added, estradiol oxidation and toxicity was markedly increased. The peroxidase- and the tyrosinase-enhanced cytotoxicity could also be rescued by addition of o-quinone traps. Conclusions: Estradiol cytotoxicity was found to be caused by the oxidation of the phenolic A ring catalyzed by hepatic P450 or peroxidase/H2O2 to form a catechol that underwent autoxidation to quinoid moieties. Estradiol quinoids can be regarded as an endogenous toxin with many routes of detoxification. Using metabolizing enzyme inhibitors it was found that estradiol or its quinone metabolite was detoxified by NQO, UGT, COMT, SULT, and GSH conjugation in order of decreasing importance. doi:10.1016/j.freeradbiomed.2010.10.632
SFRBM/SFRRI 2010
616 The Mechanism of the Oxidative Transformation of Boronate Compounds A Quantum Mechanical Study 1,2
1
doi:10.1016/j.freeradbiomed.2010.10.631
2
Adam Sikora , Agnieszka Dybala-Defratyka , Jacek Zielonka , Andrzej Marcinek1, and Balaraman Kalyanaraman2 1 Institute of Applied Radiation Chemistry, Technical University of 2 Lodz, Poland, Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee Recently, a new class of fluorogenic probes for reactive oxygen species containing a boronate moiety has been developed. These assays are based on the oxidative transformation of non- or weakly fluorescent arylboronates (acids or esters) into strongly fluorescent phenolic products. It was shown that boronate compounds can be oxidized into corresponding phenols by hydrogen peroxide, hypochlorous acid, and peroxynitrite. We have found that at neutral pH the reaction of boronates with – 6 6 -1 -1 peroxynitrite (ONOO ) is about 10 times faster (k ≈ 10 M ⋅s ) -1 -1 than the reaction with hydrogen peroxide (k ≈ 1 M ⋅s ). This reaction is direct and stoichiometric. Thus, boronate based probes open up new possibilities for direct detection of peroxynitrite in biological systems. To better understand the molecular mechanism of oxidation of boronates to phenolic products we applied quantum mechanical calculations. The reaction between arylboronates and peroxynitrite anion, or the anionic form of – hydrogen peroxide (HOO ) was studied in gas phase and in aqueous solution using B3LYP/6-31+G(d,p) and PCM/B3LYP/631+G(d,p) levels of theory, respectively. The initial step of – – proposed mechanisms is the addition of ONOO /HOO to the boron atom with the formation of an anionic intermediate. Further transformations of that anionic adducts with the cleavage of the O-O bond lead to the formation of corresponding phenols. For the peroxynitrite anion adduct of arylboronates, two different pathways of that transformation are observed. The major pathway is the heterolytic cleavage of O-O bond, and the minor pathway is the homolytic cleavage. The latter mechanism results in the •– • formation of a caged radical pair [PhB(OH)2O … NO2] and subsequent formation of phenyl radicals via the protonation of •– PhB(OH)2O radical anion. Evidence for phenyl radical formation was obtained using EPR spin-trapping. doi:10.1016/j.freeradbiomed.2010.10.630
617 Cytochrome c as a Source of Superoxide Radical: Role of H2O2 and NADH Murugesan Velayutham1, Craig Hemann1, and Jay L Zweier1 1 The Ohio State University In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. NADH is a substrate for complex-I in the electron transport chain in
S216
mitochondria. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under ischemia, oxidative or nitrative stress, and apoptosis, the peroxidase activity of cyt c is reported to be increased. The level of NADH is also increased under pathophysiological conditions such as cardiac ischemia and diabetes and a concurrent increase in hydrogen peroxide (H2O2) production occurs. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed to understand the related mechanisms of radical generation and NADH oxidation in the peroxidase activity of cyt c. Our results suggest that cyt c may have a novel role in the deleterious effects of cardiac ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, in cardiac myocytes cytochrome c may play a key role in the mitochondrial “ROS-induced ROS-release (RIRR)” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and degenerative neurological diseases.
618 Molecular Cytotoxic Mechanisms of Estradiol involve Estradiol Quinoid Moieties Luke Wan1, Jeff Bruce1, Di Fan1, and Peter J O'Brien1 1 University of Toronto Background: As a major female sex hormone, estradiol is expected to have beneficial effects in females due to its high antioxidant activity. However, prolonged exposure to estradiol increases the risk of developing cancers in the breast and uterus. The liver is the main site of estradiol metabolism such as catechol estrogen formation and detoxification. Estradiol’s carcinogenic effect has been well characterized, but it has also been found to be cytotoxic. It is important to understand the molecular mechanisms of estradiol cytotoxicity to prevent hidden internal tissue injury. Methods: An accelerated cytotoxicity mechanism screening (ACMS) technique was used with primary rat hepatocytes in order to determine the molecular mechanism of estrogen toxicity and detoxification in the rat. Hepatocytes were isolated by collagenase perfusion and incubated with estradiol and various inhibitors of estradiol metabolizing. The effectiveness of the various metabolic pathways could then be ranked. Cytotoxicity was assessed by trypan blue exclusion and mitochondrial membrane potential while oxidative stress endpoints were measured through hydrogen peroxide and other reactive oxygen species formation. Results: Estradiol was found to cause a concentration and time dependent toxicity. Estradiol cytotoxicity was increased by GSH depletion, UGT inhibition, COMT inhibition, SULT inhibition, NQO inhibition, and CYP3A inhibition. Hepatocytes could be rescued from estradiol using o-quinone trapping agents, increasing glutathione synthesis, inhibiting P450 nonspecifically, and inhibiting CYP1A. When H2O2/peroxidase or tyrosinase was added, estradiol oxidation and toxicity was markedly increased. The peroxidase- and the tyrosinase-enhanced cytotoxicity could also be rescued by addition of o-quinone traps. Conclusions: Estradiol cytotoxicity was found to be caused by the oxidation of the phenolic A ring catalyzed by hepatic P450 or peroxidase/H2O2 to form a catechol that underwent autoxidation to quinoid moieties. Estradiol quinoids can be regarded as an endogenous toxin with many routes of detoxification. Using metabolizing enzyme inhibitors it was found that estradiol or its quinone metabolite was detoxified by NQO, UGT, COMT, SULT, and GSH conjugation in order of decreasing importance. doi:10.1016/j.freeradbiomed.2010.10.632
SFRBM/SFRRI 2010