1 2 Oxygen Toxicity in the 3 4 Neonate 5 Thinking Beyond the Balance 6 7 8 Trent E. Tipple, MD*, Namasivayam Ambalavanan, MD Q2 Q3 9 Q4 10 11 KEYWORDS 12 Oxygen Prematurity Bronchopulmonary dysplasia Retinopathy of prematurity 13 14 Necrotizing enterocolitis Glutathione Antioxidants Mitochondria 15 16 KEY POINTS 17 18 Oxidative stress has traditionally been presented as an imbalance between oxidants and 19 antioxidants but the situation is far more complex. 20 Neonatal O2 toxicity has been primarily characterized by macromolecular indices of dam- 21 age that are nonspecific and are inadequate to capture dynamic biochemical processes. 22 In premature infants, the fetal to neonatal transition occurs during a period of marked sus- 23 ceptibility to oxidative stressors caused by deficits in antioxidant defenses and impaired endogenous antioxidant response activation. 24 The molecular effects of O on subcellular compartments and developmental pathways 25 2 are poorly understood. 26 State-of-the-art oxidation-reduction biology techniques will enable more robust under- 27 standing of the global impact of O2 toxicity in preterm neonates. 28 29 30 31 INTRODUCTION 32 33 Fetal development occurs normally in a relatively hypoxic (w20–25 Torr) environment 34 in utero, meaning that the transition into room air at birth represents significant oxida- 35 tive stress for prematurely born neonates.1,2 However, the transition from the hypoxic 36 environment of the womb to the relatively hyperoxic extrauterine environment occurs 37 during a period of marked susceptibility to oxidative stressors. Preterm neonates are Q7 38 more susceptible to the effects of O2 toxicity because of developmental deficits in 39 antioxidant defenses and developmental impairments in the ability to mount rapid 3–7 40 antioxidant responses to hyperoxia. In general, the toxicities of O2 during the 41 neonatal period have been characterized by macromolecular indices of oxidative 42 43 44 Disclosure: Dr. Tipple received NIH grant (R01HL119280) and Dr. Ambalavanan recevied NIH 45 grants (U01HL122626, R01HL129907, U01HL133536). 46 Division of Neonatology, Department of Pediatrics, University of Alabama at Birmingham, Q5 47 176 F Suite 9380, 619 19th Street South, Birmingham, AL 35249-7335, USA * Corresponding author. Q6 48 E-mail address: [email protected] Clin Perinatol - (2019) -–- https://doi.org/10.1016/j.clp.2019.05.001 perinatology.theclinics.com 0095-5108/19/ª 2019 Elsevier Inc. All rights reserved. CLP1101_proof ■ 8 June 2019 ■ 12:49 pm 2 Tipple & Ambalavanan 49 protein, lipid, and/or DNA damage. An expanding body of evidence has defined the 50 molecular effects of hyperoxia on developmental pathways that guide organogen- 51 8,9 esis. The sudden and dramatic increase in lung and systemic O2 tension on preterm 52 delivery significantly influence transcription factor activation and related downstream 53 pathways. However, the global impact of O2 toxicity in preterm neonates is incom- 54 pletely characterized because of the lack of sensitive and specific oxidation- 55 reduction (redox) biological techniques that adequately capture these complex 56 biochemical reactions that undoubtedly contribute to the observed morbidity and 57 mortality in this highly vulnerable patient population. 58 59 BASIC TENETS OF OXIDATIVE STRESS 60 Sources of Reactive O2 Species 61 62 A redox reaction refers to a transfer of electrons between molecules. It is essential to 63 remember that matter is neither created nor destroyed in chemical transformations. In 64 the simplified scheme (Fig. 1), molecule A loses an electron and becomes oxidized 65 and molecule B accepts an electron and becomes reduced. Thus, the net reaction 66 is simply the transfer of the electron from molecule A to molecule B. In Fig. 1, “n” 67 and “m” refer to the oxidation state of molecules A and B, respectively. When elec- n11 68 trons are lost, the oxidation number increases (A ). In contrast, when electrons mÀ1 69 are gained, the oxidation number decreases (B ). 70 In order to fully comprehend the effects of O2 tension on neonatal pathophysiology, 71 the complexities of redox biology must be appreciated. Conceptually, this under- 72 standing must extend beyond the oxidant/antioxidant balance concept, which is 73 that oxidative stress represents a deficiency of antioxidants in a setting of enhanced 74 oxidant generation. This overly simplistic model suggests that oxidative stress can 75 be overcome by exogenously administered antioxidants to restore balance. In reality, 76 the complex biochemical reactions responsible for the reduction of O2 are dynamic, 77 highly compartmentalized, sensitive to clinically relevant factors such as pH and tem- 78 perature, and extremely difficult to characterize in vivo with currently available 10 79 techniques. 80 Diatomic O2 is highly reactive because of an unpaired electron in its outer orbital, 81 and it requires 4 electrons for complete reduction (Fig. 2). O2 is also the primary 10 82 cellular metabolic fuel for aerobic metabolism. Under normal conditions, the reactive 83 O2 species (ROS) generated in the process of the 4-electron reduction of O2 to H2O 11 84 are quickly reduced (Fig. 3). ROS generated during cellular metabolism include su- 10,11 85 peroxide (O2 ) and hydrogen peroxide (H2O2). Additional oxidants, including 86 87 88 89 90 91 92 93 94 95 Fig. 1. Basic scheme of redox reactions. Molecule A loses an electron and becomes oxidized 96 and molecule B accepts an electron and becomes reduced. Thus, the net reaction is simply 97 the transfer of the electron from molecule A to molecule B. “n” and “m” refer to the oxida- tion state of molecules A and B, respectively. When electrons are lost, the oxidation number 98 1 increases (An 1). In contrast, when electrons are gained, the oxidation number decreases 99 (BmÀ1). CLP1101_proof ■ 8 June 2019 ■ 12:49 pm Oxygen Toxicity in the Neonate Q1 3 100 101 102 103 104 105 106 107 Fig. 2. Four-electron reduction of O2 to H2O with intermediate generation of reactive O2 - 108 species including superoxide (O2 ), hydrogen peroxide (H2O2), and hydroxyl radical ( OH). 109 110 À - peroxinitrite (ONOO ), generated from the nonenzymatic reaction between O2 and 111 nitric oxide (NO ), and hydroxyl radical ( OH), generated from the reaction between 112 11 1 H O and iron (Fe ) or copper (Cu ), are primarily formed in situations in which 113 2 2 endogenous antioxidant systems are unable to sufficiently provide electrons for 114 reductive processes. Although the primary focus of this article is O toxicity, it is 115 2 important to understand that excessive ROS generation in preterm infants comes 116 from a variety of sources, including ischemia/reperfusion, infection, inflammation, 117 mitochondrial respiratory chain, free iron and Fenton reaction, and hyperoxia.12–14 118 The generation of ROS can lead to the disruption of normal physiologic events.15 119 The extent of the effects of ROS on physiology depends on specific molecular inter- 120 actions, cellular locations, and timing of exposure.15 121 The effects of ROS contribute to quantifiable cellular, tissue, and organ damage that 122 underlies many of the morbidities of prematurity.12 These damaging processes occur 123 in both the placenta and the developing fetus.13 Although premature infants that 124 develop prematurity-related morbidities are usually exposed to only the least required 125 amount of supplemental O postnatally, they show marked evidence of oxidant 126 2 stress.6,12,14 There is evidence that excessive ROS production contributes to retinop- 127 athy of prematurity, bronchopulmonary dysplasia, intraventricular hemorrhage, 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 Fig. 3. Effects of reactions of ROS generated by O2 metabolism in the absence of adequate detoxification. Nitric oxide (NO) can react with O2À to form peroxinitrite (ONOOÀ), which 149 11 1 oxidizes DNA, lipids, and proteins. H2O2 can react with Fe and/or Cu to cause lipid per- 150 oxidation, DNA damage, and protein oxidation. SOD, superoxide dismutase. CLP1101_proof ■ 8 June 2019 ■ 12:49 pm 4 Tipple & Ambalavanan 151 periventricular leukomalacia, necrotizing enterocolitis, kidney damage, and hemoly- 152 sis.13,16,17 Pathophysiologically, many diseases of prematurity likely represent a 153 convergence between injury and ROS-induced alterations in development, probably 154 leading to increases in susceptibility to chronic diseases in adulthood, and perhaps 155 more rapid aging as well.18 156 The appreciation of ROS as something other than a negative entity has grown in the 157 last 20 years. Several cellular processes are actively modulated via ROS production. 158 ROS serve as cell signaling molecules for normal biological processes.15 For example, 159 - nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) produce O2 160 19 and/or H2O2 in tightly regulated and highly specific intracellular events. As such, 161 these processes are governed by transcription factors that are influenced by the redox 162 environment of the tissue, cell, or subcellular compartment in which they are 163 expressed. Changes in electron flux through these pathways, whether it be through 164 reduction of O2 or through NOX influence signaling. NOX-dependent ROS production Q8 165 influences developmental programming by acting on redox-sensitive transcription 166 factors, including hypoxia-inducible factors (HIFs) and nuclear factor kappa-light- 167 chain-enhancer of activated B cells (NF-kB). Dysregulation of HIFs and NF-kB have 168 been linked to one another and to negative outcomes in prematurely born infants.8,20 169 NOX isoforms contribute to signaling during lung development and injury and their 170 function influences pulmonary airway and vascular cell phenotypes, including prolifer- 171 ation, hypertrophy, and apoptosis.19 Oxidative stress is also associated with altered 172 nitric oxide (NO) signaling in which ROS and reactive nitrogen species production 173 are increased and bioavailable NO is decreased.21 174 175 Antioxidant Systems 176 Antioxidants are substances that inhibit or prevent oxidation of a substrate.