Glutathione / Thioredoxin Nrf2 & Hyperoxia
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1 Glutathione / Thioredoxin Nrf2 & Hyperoxia Trent E. Tipple, MD Principal Investigator, Center for Perinatal Research The Research Institute at Nationwide Children s Hospital Assistant Professor of Pediatrics, The Ohio State University College of Medicine Columbus, OH Trent.Tipple@nationwidechildrens.org....
2 Oxygen is Good GENERATION OF USABLE ENERGY FROM FOOD METABOLISM OF ENDOGENOUS INTERMEDIATES STEROID HORMONES PROSTAGLANDINS GENERATION OF OXIDANTS FOR PROTEIN FOLDING IMMUNE DEFENSE FUNCTIONS, CELL KILLING GENERATION OF NO METABOLISM OF DRUGS AND XENOBIOTICS
3 Bronchopulmonary Dysplasia A disruption of normalvascular and alveolar development O 2 toxicity Infection Barotrauma Volutrauma
4 Birth: A Hyperoxic Challenge The evolutionary adaptation to extrauterine aerobic existence required the development of efficient cellular electron transport systems to produce energy Biochemical defenses including antioxidant enzymes, evolved to protect against oxidation of cellular constituents by ROS There is increased transferof antioxidants during the last days of gestation
5 BREATHING: Fetus transfers from an intrauterine hypoxic environment (PaO 2 of mm Hg) to an extrauterine normoxic (yet relatively hyperoxic) environment (PaO 2 of 100 mm Hg) Immediately prior to birth there is an up ramping of antioxidant enzyme activity Upon exposure to oxygen newborn lungsof many species increase their normal complement of protective antioxidant enzymes
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8 Sources of Radicals UV Light Ionizing Radiation Tissue Smoking Metabolism Injury O2 -. OH- NO. Inflammation O 2 -. H 2 O 2 OH - Air Pollution
9 Oxidative Stress -Simplified
10 Oxidative Stress -Simplified Oxidants Reactive-Oxygen Species Superoxide (O 2 ) H 2 O 2 Peroxynitrite (ONOO ) Hydroxyl Radical (HO ) Antioxidants Superoxide Dismutases Heme Oxygenase Glutathione Thioredoxin
11 Oxidative Stress -Simplified Oxidants Reactive-Oxygen Species Superoxide (O 2 ) H 2 O 2 Peroxynitrite (ONOO ) Hydroxyl Radical (HO ) Antioxidants Superoxide Dismutases Heme Oxygenase Glutathione Thioredoxin
12 Glutathione (GSH) Redox Cycle Transcription Factor & DNA Binding Ribonucleotide Reductase Protein Binding NADP + Trx(SH) 2 O. O - 2 +e - 2 H 2 O 2 SOD GSH NADP + Pentose Shunt TrxR Prx GPx GR Pentose Shunt NADPH Trx-S 2 GSSG NADPH H 2 O Plasma Lymph Bile PSH PSSG PSH Trx(SH) 2 Trx-S 2
13 Glutathione (GSH) & Thioredoxin (Trx) Transcription Factor & DNA Binding Ribonucleotide Reductase Protein Binding Reduction of Protein Disulfides Redox Systems O. O - 2 +e - 2 SOD H 2 O 2 NADP + Trx(SH) 2 GSH NADP + Pentose Shunt TrxR Prx GPx GR Pentose Shunt NADPH Trx-S 2 GSSG NADPH H 2 O Plasma Lymph Bile PSH PSSG PSH Trx(SH) 2 Trx-S 2
14 Neu Mice Glutathione reductase (GR) knock-outs Reduction of GSSG would seem essential to GSH-dependent antioxidant defense mechanisms Not more susceptible to hyperoxia than were wild-type mice Observations suggest compensatory responses Rogers LK. Toxicol Sci Dec;82(2):367-73
15 Trx as an e - shuttle between reduced TrxR and GSSG TrxR NADPH + TrxS 2 + H + NADP + + Trx(SH) 2 Reaction 1 Trx(SH) 2 + GSSG Reaction 2 TrxS 2 + 2GSH NADPH + GSSG + H + TrxR/Trx Reaction 3 NADP + + 2GSH Adapted from: Kanzok et al. J Biol Chem. 22-Dec-2000; 275(51)
16 Aurothioglucose (ATG) & Auranofin (AFN) Anti-inflammatory gold compounds FDA approved to treat rheumatoid arthritis Potent inhibitor of selenium and cysteine-containing enzymes including TrxR MostertV, Hill KE, Burk RF. Loss of Activity of the SelenoenzymeThioredoxin Reductase Causes Induction of Hepatic Heme Oxygenase-1. FEBS Lett. 2003; 541:85-88
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18 Smith AD, Guidry CA, Morris VC, Levander OA. Aurothioglucose Inhibits Murine Thioredoxin Reductase Activities In Vivo. J Nutr. 1999;129(1):194-8
19 Hypothesis: ATG-mediated TrxR inhibition will enhance hyperoxic susceptibility of GR-KO mice ATG? x x
20 Methods 6 wk-old GR-KO (Neu) and C3H/HeN mice Administered 25 mg/kg ATG or saline vehicle control Placed in hyperoxia (>95% O 2 ) or room air for 72 or 96 hours
21 ATG potently inhibits lung TrxR activity in adult mice
22 GR-KO (Neu) Mice: Increased basal Trx levels & TrxR-dependent GSSG Reduction
23 ATG exacerbates hyperoxic lung in GR-KO (Neu) mice and attenuates lung injury in control mice 72 h 96 h
24 ATG exacerbates hyperoxic lung in GR-KO (Neu) mice and attenuates lung injury in control mice
25 The Nrf2 system
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32 Enhanced Nrf2 activation in the airway epithelium of conditional TrxR1-KO mice
33 The Nrf2 system
34 Reflection
35 Hypothesis: TrxR1 inhibition protects against the effects hyperoxia via Nrf2-dependent mechanisms
36 AFN, DNCB, and Sulforaphane Inhibit TrxR & Induce Nrf2 Nuclear Accumulation
37 AFN and DNCB Induce Nrf2-dependent Gene Transcription
38 TrxR1 knockdown induces Nrf2 activation in mtccs
39 AFN enhances GSH levels in mtccs
40 The effects of AFN on mtcc viability are prevented by BSO
41 ATG enhances Nrf2 activation in adult murine lungs
42 Objective: Test the hypothesis that ATG administration would be protective in a murine model of acute respiratory distress syndrome
43 ATG decreases lung injury in a murine model of ARDS A B * BAL Protein Concentration (mg/ml) BAL GSH (nmol/ml) Saline ATG C D Saline ATG * BAL Cell Number (total cells/ml) BAL Cell GSH (nmol/cell) 0 0 Saline ATG Saline ATG
44 ATG enhances lung GSH levels A B Lung GSH (µmol/g) * Saline ATG 5 0 * Lung GCLM Expression Fold Change Ct (Relative to Saline) Saline ATG C D Lung GSSG (nmol/g lung) Lung GSH/GSSG Saline ATG Saline ATG
45 ATG improves survival in a murine model of ARDS 100 % surv viving 50 ATG * $ 0 ATG/BSO $ $ BSO saline Hours
46 At homeostasis: GSH/Trx systems detoxify ROS GSH or Trx disruption welltolerated Nrf2 activation (Keap1) Compensatory upregulation of GSH or Trx system. In hyperoxia: Enhanced free radical production Nrf2 activation Compensatory upregulation of GSH-dependent responses Unable to prevent fatal injury Therapeutic TrxR inhibition Well-tolerated Nrf2 activation (Keap1) Upregulation of GSH-dependent responses Pulmonary cytoprotection Decreased lung injury Summary
47 Hypothesized role of Nrf2 in lung development & BPD Cho et al. Antioxid Redox Signal Oct 15;17(8):
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