<br><h3> Chapter One </h3> <b>Oxidative Stress in Tropical Marine Ecosystems <p> <i>Michael P. Lesser</i></b> Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, USA <p> <p> The accumulation of oxygen in Earth's atmosphere has had profound effects on the geochemistry, physiology, and evolution of life on the planet. However, most organisms must also contend with the negative aspects of living in a world with oxygen. Reactive oxygen species (ROS) production is prevalent in the world's oceans and oxidative stress is an important component of the stress response in marine organisms exposed to a variety of environmental stressors such as thermal stress, which is now becoming more prevalent because of climate change. In tropical environments exposure to high irradiances of visible and ultraviolet radiation (UVR) contributes significantly, through both direct and indirect processes, to ROS production in the water as well as in many tropical marine taxa of plants and animals. The negative effects of ROS must also be balanced by their role in signal transduction that facilitates processes such as apoptosis, autophagy and necrosis. Because of the high irradiances of solar radiation and exposure to high air and seawater temperatures, oxidative stress in tropical marine environments is ubiquitous and is normally kept in check by a suite of antioxidants, both enzymatic and nonenzymatic, in diverse tropical marine taxa in order to survive, grow and reproduce. <p> <p> <b>HISTORY AND CHEMISTRY OF OXYGEN ON EARTH</b> <p> Life on Earth began in the Archean at least 3.5 Gyr, and possibly as far back as 3.8 Gyr (Nisbet and Sleeo 2001). The early atmosphere of the Earth was highly reduced and dominated by microbes (Kasting and Siefert 2002), with additional evidence for the presence of biogenic structures that supported an oxidizing environment as far back as 3.5 Gyr (Nisbet and Sleeo 2001). By the mid- to early–Archean, cyanobacteria had evolved and were carrying out oxygenic photosynthesis (Nisbet and Sleeo 2001; Kasting and Siefert 2002); and with ample amounts of CO<sub>2</sub>, water as a reductant, and solar radiation, oxygenic cyanobacteria flourished and evolved into other taxa by multiple endosymbiotic events (Falkowski et al. 2004). The end result of this was that molecular oxygen, or dioxygen (O<sub>2</sub>), accumulated in significant amounts in the Earth's atmosphere ~2.5 Gyr, and in the upper atmosphere it formed O<sub>3</sub> which filtered out the shortest wavelengths of harmful UVR (<290 mn), and changed the course of biological evolution. <p> The accumulation of oxygen changed terrestrial and shallow oceanic habitats from a reduced state to an oxidized state and provided strongs elective pressures on anaerobic life-forms existing at the end of the Archean. The evolution of aerobic respiration with its greater efficiency and higher yields of energy was critical to the development of complex multicellular eukaryotic organisms but not without having to solve additional problems associated with gas and nutrient transport. The percentage of oxygen in the Earth's atmosphere is now ~21%. This makes oxygen the second most abundant element in the atmosphere, behind nitrogen at ~78%. <p> Oxygen is a stable, odorless, tasteless, and colorless gas at room temperature that was isolated and characterized in the 1770s. While Joseph Priestly (USA) and Antoine Lavoisier (France) are generally given credit for the discovery and naming of oxygen, it is now widely accepted that Carl Scheele (Sweden) discovered it in 1771. Oxygen has a low solubility coefficient in water that decreases with increasing temperature and affects its availability for a wide range of taxa in both aquatic and marine habitats. Normoxic air dissolved in water contains a higher percentage of oxygen (34%) than does ambient air (21%) because, despite its low solubility, it is more soluble in water than nitrogen. These differences in solubility have important implications for availability and transport of oxygen for oxidative metabolism in aquatic and marine organisms. <p> <p> <b>Oxygen Can Be Toxic!</b> <p> In a world where the presence of oxygen in the Earth's atmosphere is taken for granted many biologists still do not comprehend its potential toxicity. Reactive oxygen species are responsible for the toxic effects of oxygen, and this is because in its ground-state oxygen is distinctive among the elements as it is a biradical and has two unpaired electrons in its outer orbit (Asada and Takahashi 1987; Cadenas 1989; Fridovich 1998; Halliwell and Gutteridge 1999). Therefore, oxygen is usually non-reactive with organic molecules that have paired electrons with opposite spins. This spin restriction means that the most common mechanisms of oxygen reduction in biochemical reactions are those involving the transfer of a single, or univalent reduction, electron (Asada and Takahashi 1987; Cadenas 1989; Fridovich 1998; Halliwell and Gutteridge 1999). <p> The univalent reduction of molecular oxygen produces reactive intermediates such as O<sup>•-</sup><sub>2</sub>, <sup>1</sup>O<sub>2</sub>, H<sub>2</sub>O<sub>2</sub>, HO<sup>•</sup>, and finally water (Halliwell and Gutteridge 1999). H<sub>2</sub>O<sub>2</sub> is not technically a free radical because all of its electrons are paired but is usually included in the definition of ROS. All photosynthetic and respiring organisms produce ROS via the univalent pathway, and subsequently H<sub>2</sub>O<sub>2</sub> with the continued reduction of O<sup>•-</sup><sub>2</sub>, and eventually HO<sup>•</sup>, which is then reduced to HO<sup>-</sup> and water as a consequence of exposure to, and use of, molecular oxygen (Halliwell and Gutteridge 1999). <p> The production of ROS is directly, and positively, related to the concentration or [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] of oxygen (Jamieson et al. 1986) and since most photosynthetic organisms are hyperoxic while photosynthesizing, the production of ROS that occurs requires robust antioxidant defenses or oxidative stress will occur (Asada and Takahashi 1987; Cadenas 1989; Fridovich 1998; Halliwell and Gutteridge 1999). Oxidative stress, the production of ROS beyond the capacity of an organism to quench these reactive species, can cause damage to lipids, proteins, and DNA (Halliwell and Gutteridge 1999). The primary purpose of antioxidant defenses in biological systems is to quench <sup>1</sup>O<sub>2</sub> at the site of production, and quench or reduce the flux of other ROS such as O<sup>•-</sup><sub>2</sub> and H<sub>2</sub>O<sub>2</sub> to ultimately prevent the production of HO<sup>•</sup> the most damaging of the ROS (Asada and Takahashi 1987; Cadenas 1989; Fridovich 1998; Halliwell and Gutteridge 1999). <p> H<sub>2</sub>O<sub>2</sub> causes significant damage because it's diffusion within the cell from its point of synthesis is less restrictive than other forms of ROS and it can enter into numerous other reactions. Exposure to H<sub>2</sub>O<sub>2</sub> can damage many cellular constituents directly, such as DNA and enzymes involved in carbon fixation (Asada and Takahashi 1987; Cadenas 1989; Fridovich 1998; Halliwell and Gutteridge 1999), but H<sub>2</sub>O<sub>2</sub> is also involved in pathways such as programmed cell death or apoptosis (Halliwell and Gutteridge 1999). If H<sub>2</sub>O<sub>2</sub> is further reduced, it can produce HO<sup>•</sup>. One source of electrons for that reduction in biological systems is transition metals, such as Fe, in what is known as Fenton chemistry (Halliwell and Gutteridge 1999). <p> Most organisms have developed mechanisms to sequester and transport essential metal ions in order to reduce the occurrence of Fenton chemistry. Proteins such as transferrin and lactoferrin afford significant Fe-binding capacity, and can reduce the availability of free Fe to zero, while intracellular Fe is stored in ferritin (Halliwell and Gutteridge 1999). Many bacteria produce Fe-chelating proteins, known as siderophores, which are an important indicator of pathogenic potential in clinical settings and essential Oxidative Stress In Tropical Marine Ecosystems 11 for the survival of bacteria in natural habitats where free-Fe availability is also low. Exposure of organisms to Fe or Cu beyond their capacity to chelate or bind these metal ions potentially exposes them to the production of HO<sup>•</sup> in the presence of other ROS. <p> While the HaberWeiss and Fenton reactions have dominated the theoretical underpinnings of most studies on oxidative stress, the discovery that many cells produce NO<sup>•</sup>, a molecule initially believed to be primarily involved in signal transduction (e.g., neurotransmission), is now known to be involved in processes involving oxidative stress (Fang 2004). NO<sup>•</sup> is a colorless gas and a weak reducing agent with moderate solubility in water but by itself is relatively nonreactive despite the fact that it contains an unpaired electron making it paramagnetic and a free radical (Halliwell and Gutteridge 1999). It is now known that the microbicidal activity of white blood cells is primarily a function of the inducible enzyme NOS, which produces NO<sup>•</sup> that reacts with O<sup>•-</sup><sub>2</sub>, to form ONOO<sup>-</sup>, a potent oxidant (Fridovich 1986). Because the solubility of NO<sup>•</sup> is similar to water it can readily diffuse across biological membranes, where it reacts at near diffusion-limited rates with free radicals, especially O<sup>•-</sup><sub>2</sub>, to form ONOO<sup>-</sup>, which can diffuse across biological membranes at rates 400 times greater than O<sup>•-</sup><sub>2</sub> (Marla et al. 1997; Denicola et al. 1998). It has been suggested that the high concentrations of NO<sup>•</sup> in a wide range of taxonomically diverse organisms creates significant competition between NO<sup>•</sup> and superoxide dismutase (SOD) for O<sup>•-</sup><sub>2</sub>. This competition for O<sup>•-</sup><sub>2</sub> may be a major determinant of oxidative stress in many organisms. Many investigators are now re-evaluating the role of O<sup>•-</sup><sub>2</sub> in oxidative stress because of these new insights, and because many of the observed <i>in vitro</i> effects ascribed to O<sup>•-</sup><sub>2</sub> may in fact be mediated by ONOO<sup>-</sup> (Halliwell and Gutteridge 1999). <p> <p> <b>Principal Cellular Sites of ROS Production</b> <p> The maximum quantum efficiency of photosynthesis occurs when photosynthesis increases in a linear fashion with the irradiance of visible, or photosynthetically active radiation (PAR: 400–700 nm). Additional increases in PAR cause this relationship to become nonlinear and can be attributed to changes in electron transport and light-harvesting processes (Asada and Takahashi 1987; Asada 1994; Falkowski and Raven 1997). These processes lead to a decrease in photosystem II (PS II) photochemistry, and additional increases in PAR exceed the capacity to protect PS II by dissipating the excess absorbed excitation energy, and will increase the probability of damage to the primary quinone acceptor (e.g., D1 protein) from the production of ROS in the chloroplast (Asada and Takahashi 1987; Richter et al. 1990; Falkowski and Raven 1997; Lupinkov a and Komenda 2004). Oxidative stress in the chloroplasts not only has direct effects on PS II but also inhibits the repair of damage to PS II (Nishiyama et al. 2001). In addition to the production of 1O<sub>2</sub> within PS II (Macpherson et al. 1993) it has been shown that O<sup>•-</sup><sub>2</sub> and HO<sup>•</sup> are also produced in the PS II reaction center (Liu et al. 2004). <p> There are several sites within the chloroplast that can reduce oxygen. Photosensitized chlorophyll is in a singlet-excited state and normally transfers its excitation energy to the photosynthetic reaction centers, but under high irradiance conditions the long-lived chlorophyll triplet state occurs and can interact with oxygen to form <sup>1</sup>O<sub>2</sub>. The reducing side of PS I can reduce oxygen to O<sup>•-</sup><sub>2</sub> by the Mehler reaction and is the most significant site of O<sup>•-</sup><sub>2</sub> production in the chloroplast (Asada and Takahashi 1987; Asada 1994, 1999). The Mehler reaction is often described as an alternative sink for electrons when sink limitation (e.g., carbon or nitrogen limitation) occurs. Under normal circumstances the O<sup>•-</sup><sub>2</sub> produced is rapidly dismutated to H<sub>2</sub>O<sub>2</sub> by SOD and the H<sub>2</sub>O<sub>2</sub> converted to water by ascorbate peroxidase (APx; Asada and Takahashi 1987; Asada 1994, 1999). The production of O<sup>•-</sup><sub>2</sub> is exacerbated under stressful conditions such as exposure to xenobiotics or pollutants, high PAR irradiances, exposure to UVR, and exposure to thermal stress that can overwhelm antioxidant defenses to produce damage to both PS II and carbon fixation (Asada and Takahashi 1987; Asada 1994). Molecular oxygen can also be reduced during photorespiration, the oxygenase or C<sub>2</sub> pathway for ribulose 1, 5-bisphosphate decarboxylase/oxygenase (Rubisco) under conditions where the ratio of CO<sub>2</sub> to oxygen is low, forming glycolate. Although this does not generate ROS in the chloroplast, the subsequent conversion of glycolate to glyoxylate in glyoxisomes does generate H<sub>2</sub>O<sub>2</sub> via the divalent reduction of oxygen by glycolate oxidase (Asada and Takahashi 1987; Asada 1994, 1999). <p> In nonphotosynthetic eukaryotic cells the mitochondrion is the primary site of ROS production. Within this organelle there are two primary sites of O<sup>•-</sup><sub>2</sub> generation in the inner mitochondrial membrane: NADH dehydrogenase at complex I, and the interface between ubiquinone and complex III (Brookes 2005). O<sup>•-</sup><sub>2</sub> produced at these sites is then converted by spontaneous dismutation or by SOD to H<sub>2</sub>O<sub>2</sub> (Brookes 2005). The inner membrane is also permeable to protons (H<sup>+</sup>). There are apparent benefits to H<sup>+</sup> leakage despite the loss of energy as it is associated with a decrease in the production of ROS. Other important sites of ROS production in cells include the endoplasmic reticulum of animals, plants, and some bacteria that contain several cytochromes, collectively known as cytochrome P-450, that can form O<sup>•-</sup><sub>2</sub> (Halliwell and Gutteridge 1999). The substrate for these reactions is commonly an organic xenobiotic, such as herbicides, alcohols, insecticides, and a long list of hydrocarbons (Halliwell and Gutteridge 1999). The wide occurrence of these compounds in nature, their detoxification, and the subsequent O<sup>•-</sup><sub>2</sub> production, can contribute significantly to the oxidative load of organisms. <p> Microbodies are cellular organelles that include peroxisomes and glyoxysomes that contain enzymes involved in the ß-oxidation of fatty acids and photorespiration. The H<sub>2</sub>O<sub>2</sub> synthesized by these microbodies is produced by a two-electron transfer and by the dismutation of O<sup>•-</sup><sub>2</sub> to H<sub>2</sub>O<sub>2</sub> via SOD in both peroxisomes and glyoxisomes (Sandalio and del Rio 1988). <p> <p> <b>REACTIVE OXYGEN SPECIES ARE BOTH GOOD AND BAD <p> Oxidative Damage</b> <p> Breathing oxygen is hazardous to your health! The consequence of respiring oxygen is the production of ROS. Oxidative stress can also represent a significant energetic drain on an organism. For example, recent work has shown an increased susceptibility to oxidative stress during periods of reproduction when energy is diverted away from the costs of antioxidant defenses (Alonso-Alvarez et al. 2004). <p> The reaction of ROS with lipids, especially membrane-associated lipids, is considered one of the most prevalent mechanisms of cellular injury (Halliwell and Gutteridge 1999). The peroxidation of lipids involves three well-defined steps: initiation, propagation and termination (Yu 1994; Halliwell and Gutteridge 1999) ultimately forming ROO<sup>•</sup> (peroxyl radical) which then participates in a chain reaction of lipid peroxidation. The lipid hydroperoxide (ROOH) formed during these reactions is unstable in the presence of Fe or other metal catalysts because ROOH will participate in a Fenton reaction leading to the formation of RO<sup>•</sup> (alkoxy radical). Therefore, in the presence of Fe, the chain reactions are not only propagated but also amplified. Among the degradation products of ROOH are aldehydes such as malondialdehyde (MDA), and hydrocarbons such as ethane and ethylene all of which are commonly measured end-products of lipid peroxidation (Freeman and Crapo 1982; Gutteridge and Halliwell 1990). Lipid peroxidation in mitochondria is particularly cytotoxic, with multiple effects on enzyme activity and ATP production, as well as the initiation of apoptosis (Bindoli 1988; Green and Reed 1998). <p> <i>(Continues...)</i> <p> <p> <!-- copyright notice --> <br></pre> <blockquote><hr noshade size='1'><font size='-2'> Excerpted from <b>Oxidative Stress in Aquatic Ecosystems</b> Copyright © 2012 by Blackwell Publishing Ltd. Excerpted by permission of John Wiley & Sons. All rights reserved. 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