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Effect of Chronic Ethanol Consumption on the Increased Oxidative Stress in Rat Corpus Cavernosum Tissue
the vulnerability for ventricular arrhythmias in a murine model of ischemic cardiomyopathy. Whereas no differences were observed in infarct size, characteristics of electrical conduction pointed to aggravated adverse electrical or structural remodeling in WT as compared to Mpo-/- mice. The results underline the important role of inflammatory processes in cardiac remodeling upon myocardial infarction and fibroblast activation and identify MPO as a potential therapeutic target to prevent ventricular arrhythmias in ischemic cardiomyopathy. doi: 10.1016/j.freeradbiomed.2014.10.415
101 Superoxide and Superoxide Dismutase Levels in HighRisk Pregnant Women: A Pilot Feasibility Study Tiffany A Moore1, Iman Ahmad1, Jocelyn Jones1, Adam Case1, and Matthew Zimmerman1 1 University of Nebraska Medical Center, USA Previous studies of the perinatal population have suggested complications, such as pre-eclampsia, are correlated with biomarkers of oxidative stress. While these biomarkers are an indicator of an aberrant redox environment, levels of specific radical species in this high-risk patient population remain unknown. Understanding that superoxide (O2Ɣ-) is a precursor free radical to many other damaging reactive oxygen species, the goals of this pilot feasibility study were to: 1) quantify O2Ɣ- levels and total superoxide dismutase (SOD) activity in the blood of high-risk pregnant women; 2) compare these levels with perinatal outcomes. Whole blood was obtained by venipuncture from 4 pregnant women between 15-19 weeks gestation recruited from a maternal-fetal-medicine clinic at an academic medical center. Thirty minutes after FROOHFWLRQ ȝO of whole blood was incubated with a O2Ɣ--sensitive electron paramagnetic resonance (EPR) spin probe, 1-hydroxy-3-methoxycarbonyl-2,2,5,5tetramethylpyrrolidine (CMH), for 30 minutes at 37.5°C then frozen in liquid nitrogen. Superoxide levels were determined using a Bruker eScan EPR spectrometer and expressed as EPR DUELWUDU\ XQLWV $8 $ VHSDUDWH ȝO of whole blood was centrifuged for 2 minutes at 1.5xg, and protein was obtained from the separated red blood cells (RBC). Total SOD activity was measured from the RBC protein using the Dojindo SOD activity assay. of the four recruited patients, one developed postpartum pre-eclampsia. Interestingly, the CMH oxidation levels (3.9x106AU) and SOD activity levels (129,000 U/ml) for the one patient with postpartum pre-eclampsia were considerably HOHYDWHGFRPSDUHGWRWKHUHPDLQLQJWKUHHSDWLHQWV¶DYHUDJH&0+ oxidation levels (2.5x106 ± 0.30x106 AU) and SOD activity levels (31,000 ± 10,000 U/ml) that did not develop any pre-eclampsia. This is the first-known study to examine circulating redox status in whole blood of high-risk pregnant women using the gold-standard for detecting O2Ɣ- levels, that is, EPR spectroscopy. A larger study designed to examine the redox status in high-risk pregnant women over time, in their newborn infants, and with perinatal outcomes is currently being conducted. doi: 10.1016/j.freeradbiomed.2014.10.416
102 Effect of Chronic Ethanol Consumption on the Increased Oxidative Stress in Rat Corpus Cavernosum Tissue Jaqueline Jóice Muniz1, Leticia Nogueira Leite1, Riccardo Lacchini1, José Eduardo Tanus-Santos1, and Carlos Renato Tirapelli1 1 University of Sao Paulo, Brazil The aim of this study was to assess the effects of chronic ethanol consumption on the endothelinergic system, mitogen-activated protein kinase (MAPK) pathway, and on the oxidative stress in cavernosal tissue as factors involved in erectile dysfunction (ED). All protocols were approved by the local Ethics Committee (12.1.317.53.9). Male Wistar rats were divided in two groups: ethanol group±treated with ethanol (20% vol/vol) for 6 weeks; control group±water for 6 weeks. In cumulative concentrationresponse curves for Endothelin 1 (ET-1), the ET-1-induced contraction was higher in ethanol-treated rats (36.1 ± 2.7% KCl 120mM; n=5) compared to control group (20.7 ± 0.9% KCl 120mM; n=5). In ethanol group, the contraction induced by ET-1 was significantly reduced in the presence of apocynin (100μmol/L), an inhibitor of NAD(P)H oxidase, and SP600125 (100 μmol/L), an inhibitor of SAPK/JNK. Plasma antioxidant activity was higher in ethanol group (4.81± 0.19 mmol/L; n=11) than control group (2.52±0.17 mmol/L; n=10). Catalase levels were higher in ethanol group (0.062±0.005 U/mL; n=10 and 0.090±0.015 U/mg protein; n=9) when compared to control group (0.043±0.004 U/mL; n=10 and 0.037±0.007 U/mg protein; n=9) in plasma and cavernosal tissue, respectively. Superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) levels in the cavernosal tissue were lower in ethanol group (3.13±0.63 pmol/μg protein; n=7 and 3.77±0.32 pmol/μg protein; n=8) when compared to control group (5.12±0.53 pmol/μg protein; n=7 and 6.15±0.76 pmol/μg protein; n=8), respectively. No difference was found in level of glutathione and mRNA of the p38MAPK, SAPK/JNK and ERK1/2 between groups. These results show that the chronic ethanol consumption affects intracellular pathways activated by ET-1 and contributes to increased oxidative stress in cavernosal tissue, which can lead to ED. Financial Support: FAPESP doi: 10.1016/j.freeradbiomed.2014.10.417
103 Downregulation of SIRT3 Exerts Mitochondrial Dysfunction in Cultured Cardiomyocytes Rebecca Maria Parodi-Rullán1, Sergiy Nadtochiy2, Paul Brookes2, and Sabzali Javadov1 1 University of Puerto Rico School of Medicine, Puerto Rico, 2 University of Rochester Medical Center, USA Background: Sirtuins are NAD+dependent deacetylases that regulate protein activity through acetylation/deacetylation. Mitochondria contain three of seven sirtuins, and SIRT3 is a major mitochondrial isoform. Lack of specific pharmacological inhibitors for SIRT3 complicates elucidating the role of the sirtuin in mitochondrial metabolism and function. In this study, we: 1) developed a method for downregulation of SIRT3 expression using the siRNA technique in H9c2 cardiomyocytes, and 2) assessed the effect of SIRT3 genetic ablation on mitochondrial function. Methods: To optimize the conditions for silencing, 1x105, 2x105or 3x105 cells per a 10-cm dish and 2.4x103 or 10x103 cells per well for the 24-well plate were transfected with 100 nm or 200 nm siRNA for 24, 48 or 72h. Protein levels of SIRT3 were analyzed by SDS-PAGE Western blotting using SIRT3 antibodies. Once an optimal condition with maximum SIRT3 silencing was achieved, mitochondrial membrane potential was assessed in 24-
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101 Superoxide and Superoxide Dismutase Levels in HighRisk Pregnant Women: A Pilot Feasibility Study Tiffany A Moore1, Iman Ahmad1, Jocelyn Jones1, Adam Case1, and Matthew Zimmerman1 1 University of Nebraska Medical Center, USA Previous studies of the perinatal population have suggested complications, such as pre-eclampsia, are correlated with biomarkers of oxidative stress. While these biomarkers are an indicator of an aberrant redox environment, levels of specific radical species in this high-risk patient population remain unknown. Understanding that superoxide (O2Ɣ-) is a precursor free radical to many other damaging reactive oxygen species, the goals of this pilot feasibility study were to: 1) quantify O2Ɣ- levels and total superoxide dismutase (SOD) activity in the blood of high-risk pregnant women; 2) compare these levels with perinatal outcomes. Whole blood was obtained by venipuncture from 4 pregnant women between 15-19 weeks gestation recruited from a maternal-fetal-medicine clinic at an academic medical center. Thirty minutes after FROOHFWLRQ ȝO of whole blood was incubated with a O2Ɣ--sensitive electron paramagnetic resonance (EPR) spin probe, 1-hydroxy-3-methoxycarbonyl-2,2,5,5tetramethylpyrrolidine (CMH), for 30 minutes at 37.5°C then frozen in liquid nitrogen. Superoxide levels were determined using a Bruker eScan EPR spectrometer and expressed as EPR DUELWUDU\ XQLWV $8 $ VHSDUDWH ȝO of whole blood was centrifuged for 2 minutes at 1.5xg, and protein was obtained from the separated red blood cells (RBC). Total SOD activity was measured from the RBC protein using the Dojindo SOD activity assay. of the four recruited patients, one developed postpartum pre-eclampsia. Interestingly, the CMH oxidation levels (3.9x106AU) and SOD activity levels (129,000 U/ml) for the one patient with postpartum pre-eclampsia were considerably HOHYDWHGFRPSDUHGWRWKHUHPDLQLQJWKUHHSDWLHQWV¶DYHUDJH&0+ oxidation levels (2.5x106 ± 0.30x106 AU) and SOD activity levels (31,000 ± 10,000 U/ml) that did not develop any pre-eclampsia. This is the first-known study to examine circulating redox status in whole blood of high-risk pregnant women using the gold-standard for detecting O2Ɣ- levels, that is, EPR spectroscopy. A larger study designed to examine the redox status in high-risk pregnant women over time, in their newborn infants, and with perinatal outcomes is currently being conducted. doi: 10.1016/j.freeradbiomed.2014.10.416
102 Effect of Chronic Ethanol Consumption on the Increased Oxidative Stress in Rat Corpus Cavernosum Tissue Jaqueline Jóice Muniz1, Leticia Nogueira Leite1, Riccardo Lacchini1, José Eduardo Tanus-Santos1, and Carlos Renato Tirapelli1 1 University of Sao Paulo, Brazil The aim of this study was to assess the effects of chronic ethanol consumption on the endothelinergic system, mitogen-activated protein kinase (MAPK) pathway, and on the oxidative stress in cavernosal tissue as factors involved in erectile dysfunction (ED). All protocols were approved by the local Ethics Committee (12.1.317.53.9). Male Wistar rats were divided in two groups: ethanol group±treated with ethanol (20% vol/vol) for 6 weeks; control group±water for 6 weeks. In cumulative concentrationresponse curves for Endothelin 1 (ET-1), the ET-1-induced contraction was higher in ethanol-treated rats (36.1 ± 2.7% KCl 120mM; n=5) compared to control group (20.7 ± 0.9% KCl 120mM; n=5). In ethanol group, the contraction induced by ET-1 was significantly reduced in the presence of apocynin (100μmol/L), an inhibitor of NAD(P)H oxidase, and SP600125 (100 μmol/L), an inhibitor of SAPK/JNK. Plasma antioxidant activity was higher in ethanol group (4.81± 0.19 mmol/L; n=11) than control group (2.52±0.17 mmol/L; n=10). Catalase levels were higher in ethanol group (0.062±0.005 U/mL; n=10 and 0.090±0.015 U/mg protein; n=9) when compared to control group (0.043±0.004 U/mL; n=10 and 0.037±0.007 U/mg protein; n=9) in plasma and cavernosal tissue, respectively. Superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) levels in the cavernosal tissue were lower in ethanol group (3.13±0.63 pmol/μg protein; n=7 and 3.77±0.32 pmol/μg protein; n=8) when compared to control group (5.12±0.53 pmol/μg protein; n=7 and 6.15±0.76 pmol/μg protein; n=8), respectively. No difference was found in level of glutathione and mRNA of the p38MAPK, SAPK/JNK and ERK1/2 between groups. These results show that the chronic ethanol consumption affects intracellular pathways activated by ET-1 and contributes to increased oxidative stress in cavernosal tissue, which can lead to ED. Financial Support: FAPESP doi: 10.1016/j.freeradbiomed.2014.10.417
103 Downregulation of SIRT3 Exerts Mitochondrial Dysfunction in Cultured Cardiomyocytes Rebecca Maria Parodi-Rullán1, Sergiy Nadtochiy2, Paul Brookes2, and Sabzali Javadov1 1 University of Puerto Rico School of Medicine, Puerto Rico, 2 University of Rochester Medical Center, USA Background: Sirtuins are NAD+dependent deacetylases that regulate protein activity through acetylation/deacetylation. Mitochondria contain three of seven sirtuins, and SIRT3 is a major mitochondrial isoform. Lack of specific pharmacological inhibitors for SIRT3 complicates elucidating the role of the sirtuin in mitochondrial metabolism and function. In this study, we: 1) developed a method for downregulation of SIRT3 expression using the siRNA technique in H9c2 cardiomyocytes, and 2) assessed the effect of SIRT3 genetic ablation on mitochondrial function. Methods: To optimize the conditions for silencing, 1x105, 2x105or 3x105 cells per a 10-cm dish and 2.4x103 or 10x103 cells per well for the 24-well plate were transfected with 100 nm or 200 nm siRNA for 24, 48 or 72h. Protein levels of SIRT3 were analyzed by SDS-PAGE Western blotting using SIRT3 antibodies. Once an optimal condition with maximum SIRT3 silencing was achieved, mitochondrial membrane potential was assessed in 24-
SFRBM 2014
S51