A Novel Role for Mitochondrial Superoxide in the Development of Erythropoietic Protoporphyria

have been shown to produce reactive oxygen species. Great emphasis has been placed on cytochrome P450 2E1 (CYP2E1) which can be induced by several environmental stressors including ethanol, acetaminophen, and CCl4. During the oxidation of ethanol, CYP2E1, produces superoxide and H2O2, that have been shown to deplete cellular glutathione levels and cause oxidative damage. These findings led us to the hypothesis that overexpression of CYP2E1 would result in altered mitochondrial bioenergetic function. To test this hypothesis, HepG2 cells transfected with human, active microsomal CYP2E1 (E47) and control HepG2 cells transfected with vector alone (C34) were assessed using a Seahorse Bioscience XF24 Extracellular Flux Analyzer. We observed an increase of 40% of the basal oxygen consumption in HepG2-E47 cells with respect to control cells. In addition, specific mitochondrial parameters were evaluated by sequential addition of inhibitors of the respiratory chain. HepG2E47 showed an increase in the maximal and reserve capacity with respect to HepG2-C34 cells, that were abrogated by the use of chlomethiazole (CYP2E1 inhibitor), as well as by the addition of acetaminophen, indicating a loss in maximal respiratory capacity at Complex IV. Western blott analysis showed a decrease in complex IV activity and protein at subunit 4 in HepG2-E47 cells. Moreover, HepG2-E47 showed an increase in citrate synthase activity indicating an increase in the mitochondrial density in this cells. Evaluation of the bioenergetic profile of HepG2 cells will lead us to a better understanding on the mechanisms of ethanolinduced oxidative stress and cell death. doi: 10.1016/j.freeradbiomed.2010.10.233

227 A Novel Role for Mitochondrial Superoxide in the  Development of Erythropoietic Protoporphyria  Adam J Case1, and Frederick E. Domann1 1 The University of Iowa Erythropoietic protoporphyria (EPP) is a disease caused by ineffective erythropoiesis leading to skin irregularities and liver dysfunction. Inactivation of the mitochondrial enzyme ferrochelatase (FECH) leads to improper iron loading into heme during red blood cell (RBC) development, and this defect leads to the overproduction and build-up of porphyrin rings and free iron causing the previously described pathologies. The inactivation of FECH is primarily caused by a somatic mutation, but in a minority of patients the cause remains elusive. Knowing that increased levels of superoxide may inactivate iron-sulfur cluster enzymes like FECH, we hypothesized that a disruption of manganese superoxide dismutase (SOD2) activity may lead to a defect in heme biosynthesis. To examine this, we used a hematopoietic stem cell specific knock-out mouse targeting manganese superoxide dismutase. Superoxide-mediated oxidative stress was increased approximately 3-5 fold in all hematopoietic organs in the knock-out mice (SOD2-/-) as demonstrated by increased dihydroethidium (DHE) staining and decreased aconitase activity. -/SOD2 mice demonstrated significant anemia indicated by decreased hemoglobin and hematocrit (~4 g/dL and ~28% respectively versus ~10 g/dL and ~41% in controls), increased reticulocytes (~22% versus ~3% in controls), and increases in RBC hypochromasia, anisocytosis, and schistocytosis. In addition, the presence of free erythrocyte porphyrin (FEP) was increased approximately 10-fold in the knock-outs. Marrow -/specific SOD2 mice also demonstrated abnormal plasma and organ iron homeostasis which contributed further to the aforementioned oxidative stress as well as to significant liver and spleen pathologies. Studies are currently underway examining the inactivation of FECH as well as the potential rescue of the observed phenotype with small molecule anti-oxidant supplementation. Our study demonstrates the first connection between oxidative stress and EPP, and may lead to the future discovery of new therapeutic molecules for this disease. doi: 10.1016/j.freeradbiomed.2010.10.234

S92

228 Ca2+­insensitive eNOS Activation is Impaired in  Pre­eclamptic Endothelial Cells: a Role for  Mitochondrial ROS?  Sarah Chapple1, David Rowlands1, Richard Siow1, and Giovanni Mann1 1 King's College London, UK Pre-eclampsia (PE) affects ~5% of pregnancies worldwide and is characterized by abnormal placentation, leading to placental ischemia/reperfusion injury and the onset of systemic maternal oxidative stress and endothelial dysfunction which persists post partum. There is also evidence to suggest exposure to PE in utero increases the risk of vascular disease in adulthood (Young 2+ et al., 2010). Enhanced basal and diminished Ca -sensitive eNOS activation has previously been reported in the fetal 2+ endothelium (Joy et al., 2010) and we here investigate Ca insensitive activation by the soy isoflavone equol. Using the mitochondrial ROS indicator MitoSOX red, we report that physiological concentrations of equol (100nM) acutely stimulate mitochondrial ROS (mtROS) generation via orphan estrogen receptor GPR30 linked to transactivation of the EGF receptor. Inhibition of mtROS with the mitochondrial complex I inhibitor rotenone (5µM) or the EGFR kinase inhibitor AG1478 (5µM) abolished Akt/ERK kinase activation and downstream eNOS phosphorylation and NO production. In PE, basal mtROS generation was enhanced and equol-induced mtROS was diminished due to mitochondrial depolarization measured using JC-1, leading to impaired eNOS phosphorylation. To our knowledge, our findings provide the first evidence that PE is associated with enhanced mitochondrial ROS production and 2+ reduced eNOS activation in response to Ca -insensitive agonists. Thus, exposure to PE in utero may lead to permanent phenotypic alterations in fetal endothelial cells, pre-disposing infants to cardiovascular disease in adulthood. Supported by British Heart Foundation Young et al., (2010) Annu. Rev. Pathol. (5)173-192 Joy et al., (2006) J. Biol. Chem. (281) 27335-27345 doi: 10.1016/j.freeradbiomed.2010.10.235

229 Resveratrol Induces Reactive Oxygen Species  Production and Apoptosis in Breast Cancer Cells  Paula Seixas Costa1, Danielly Cristiny Ferraz Costa2, Fabiana Alves Casanova3, Jerson Lima Silva2, and Eliane Fialho Oliveira4 1 2 3 4 UFRJ- Nutrition Institute, UFRJ- IBqM, UFRJ-IBqM, UFRJnutrition Institute It is estimated that in 2020, the worldwide incidence of cancer is on the order of 15 million, of which 60% of new cases will occur in developing countries. The breast cancer is the second most deadly cancer among women. Resveratrol (RV) is a polyphenol found in grapes and red wine with characteristics of promising agent for cancer prevention. In the present study, we investigated the antiproliferative effect of RV in the breast cancer cells (MCF7) and the involvement of reactive oxygen species (ROS). MCF-7 cells was treated with different concentrations of RV (0 – 400 μM) for 24 or 48 hours. RV inhibited the cellular proliferation in time and dose-dependent manner with an IC50 of 238 μM and 151 μM for 24 or 48 hours, respectively. ROS production was increased when MCF-7 was treated with 200 μM of RV for 24 hours. Recently, the effects of several drugs that produce ROS can interfere directly with apoptosis in cancer cells. To evaluate the cell cycle distribution of MCF-7 cells with resveratrol treatment, the DNA content was measured by flow cytometry, and RV arrest the cell cycle in S phase. The western blotting analysis revealed that the RV stimulated PARP cleavage and caspases 7 and 9 activation. Taken together, our results demonstrate that Resveratrol inhibited the growth of MCF-7 cells in a dosedependent manner and the reduction in cells viability resulted

SFRBM/SFRRI 2010