Heme Scavenging Attenuates Bromine Induced Lung Injury

Antioxidants and Novel Therapeutics 168 Heme Scavenging Attenuates Bromine Induced Lung Injury Saurabh Aggarwal1, Adam Lam1, Subhashini Bolisetty1, Amie Traylor1, Matthew A Carlisle1, Anupam Agarwal1, and Sadis Matalon1 1 University of Alabama at Birmingham, USA Bromine (Br2) gas exposure poses an environmental and occupational hazard that frequently results in severe respiratory failure. However, the mechanism(s) by which the inhalation of Br 2 gas induces lung injury and the therapeutic responses required to mitigate lung damage are not clear. Previously, it was demonstrated that exposure to Br2 up-regulates the heme degrading enzyme, heme oxygenase-1 (HO-1). By virtue of converting toxic heme into billiverdin, HO-1 has anti-apoptotic, anti-oxidant, and anti-inflammatory effects. Therefore, we hypothesized that an increase in heme-dependent tissue injury underlies the pathogenesis of Br2 toxicity. We exposed C57/BL6 mice to Br2 gas (600ppm, 30 min) and found that heme levels were significant elevated in plasma, bronchial alveolar lavage fluid (BALF), and whole lung tissue 24 hours post exposure. However, when these mice were given an intraperitoneal injection of the KHPHVFDYHQJLQJSURWHLQKHPRSH[LQȝJJERG\ZHLJKWPLQ post Br2 exposure), heme levels did not increase significantly. Interestingly, hemopexin also reduced the Br2 mediated increase in lung HO-1 protein levels, suggesting that HO-1 induction by Br2 exposure was secondary to an increase in heme. In addition, hemopexin attenuated the Br2 dependent increase in lung protein oxidation, lung inflammation, restored lung function, and improved survival. To further verify that heme scavenging protects against Br2-dependent lung injury, we tested the effects of Br2 using transgenic mice over-expressing the human HO-1 gene -/(hHO-1) and mice with HO-1 gene deletion (HO-1 ). Exposure to Br2 gas (600ppm, 30 min) significantly increased lung heme, lung oxidative stress, and inflammation in the HO-1-/-, but not in hHO-1 mice. In addition, the hHO-1 mice were protected against the Br2 induced increase in lung resistance, lung edema, and lethality. Finally, using human lung epithelial cells (H4441), we found that Br2 (200ppm, 30min) induces mitochondrial dysfunction and cell apoptosis, while hemopexin (20μg/ml, 30min post Br2 exposure) rescued mitochondrial function and cell viability. Together, these data suggest that scavenging heme may prove a useful adjuvant therapy to treat patients with Br2 exposure. doi: 10.1016/j.freeradbiomed.2014.10.274

169 The Melanocortin 1 Receptor (MC1R) Pathway Enhances Expression of MnSOD and Protects against ROS-Induced Oxidative Stress in Human Melanocytes Alexandra Amaro-Ortiz1 and John A D'Orazio1 College of Medicine, University of Kentucky, Lexington KY, USA The generation of free radicals and other reactive oxidative species (ROS) are thought to be a major consequence of UV exposure, leading to cellular damage and mutagenesis. The melanocortin 1 receptor (Mc1r) is a transmembrane Gs-coupled cell surface receptor found on epidermal melanocytes. The Mc1r transmits pro-survival and pro-differentiation signals mediated by the second messenger cAMP. Moreover, MC1R signaling pathway is well known for its role against UV resistance. Therefore, MC1R signaling may be an exploitable target 1

against skin carcinogenesis. We hypothesize that Mc1r signaling protects against carcinogenesis in part through optimized antioxidant cellular defenses and that individuals with defective Mc1r signaling suffer more oxidative damage than those with intact Mc1r signaling. We had demonstrated the topical application of the root extract of forskolin, an adenylate cyclase activator, in extension (Mc1re/e) K14-Scf animals, which model the fair-skinned UV-sensitive human. We showed that twice-daily application of the extract promotes accelerated melanization and that induced epidermal melanin protects against UV-induced sunburn through measurement of ³PLQLPDO HU\WKHPDWRXV GRVH´ (MED). We used human melanoma cell lines, known to harbor a loss-of-function signaling mutation in MC1R, and MC1R wild type to determine effects of cAMP stimulation on manganese superoxide dismutase (MnSOD) expression. We found that MC1R signaling, either through Į06+ RU SKDUPDFRORJLFDOO\ through manipulation of cAMP, resulted in accumulation of MnSOD in the mitochondria. Furthermore, addition of an oxidative agent such as H2O2 enhanced expression of MnSOD at the protein level as early as one hour after MC1R stimulation. Because of this timing, we hypothesize that MC1Rmediated MnSOD increases may not involve transcriptional upregulation but rather MnSOD transport and/or stability. To DGGUHVV WKH PHFKDQLVP ZH ZLOO GHWHUPLQH F$03¶V HIIHFW RQ possible binding partners. We now study the levels of reactive oxygen species in human melanocytes after UV exposure by flow cytometry and Seahorse analysis. Our results suggest that the MC1R signaling has a protective role in the regulation of the oxidative injury by enhancing MnSOD in human melanocytes. Furthermore, the protective effect of MC1R signaling may be dependent on the levels of exogenous ROS in the cells. The MC1R/cAMP signaling pathway holds promise as a novel preventive mechanism against UV-mediated oxidative skin injury and melanoma. doi: 10.1016/j.freeradbiomed.2014.10.275

170 Inactivation of the Ferroptosis Regulator Gpx4 Triggers Acute Renal Failure in a Therapeutically Relevant Mechanism Jose Pedro Friedmann Angeli1, Bettina Proneth1, Victoria J Hammond2, Yulia Y Tyurina3, Vladimir A Tyurin4, Valerie B O`Donnell2, Valerian E Kagan3, and Marcus Conrad5 1 Helmholtz Zentrum München, Germany, 2Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, UK, 3Departments of Environmental and Occupational Health; Departments of Center for Free Radical and Antioxidant Health, United States, 4Departments of Environmental and Occupational Health; Departments of Center for Free Radical and Antioxidant Health, United States, 5IDG, Germany Ferroptosis is a nonǦapoptotic form of cell death induced by small molecules in specific tumor types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in nonǦtransformed cells and tissues is unexplored and remains enigmatic to date. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) leads to cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4 knockout mice, we elucidate an essential role of the glutathione/Gpx4 axis in preventing lipid oxidationǦinduced acute renal failure and associated death. Animals death was preceded by substantial oxidation of phospholipids in kidneys as analysed by global oxilipidomic, therefore providing the first oxilpids signature of tissues undergoing ferroptosis. We furthermore systematically evaluated a library of small molecules for novel

SFRBM 2014

S77