1st PhD Biochemistry Lecture (2023) PDF

1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Free Radicals Lipids Peroxidation Oxidative Stress and Cellular Defenses against ROS (Antioxidants) 1  1...

1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Free Radicals Lipids Peroxidation Oxidative Stress and Cellular Defenses against ROS (Antioxidants) 1  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 1.Free radicals and oxidative stress: Usually, an atom is composed of a central nucleus with pairs of electrons orbiting around it. However, some atoms and molecules have unpaired electrons and these are called free radicals. Free radicals are usually unstable and highly reactive because the unpaired electrons tend to form pairs with other electrons. An oxygen molecule (O2) undergoes four-electron reduction when it is metabolized in vivo. During this process, reactive oxygen metabolites are generated by the excitation of electrons secondary to addition of energy or interaction with transition elements. The reactive oxygen metabolites thus produced are more highly reactive than the original oxygen molecule and are called active oxygen species. Superoxide, hydrogen peroxide, hydroxyl radicals, and singlet oxygen are active oxygen species in the narrow sense. Active oxygen species in a broad sense are listed in Table 1. Only active oxygen species having an unpaired electron, indicated with a dot above and to the right of the chemical formula in the table, are free radicals. Table 1: Major Active oxygen species (radicals and non-radicals) NOTES: Oxidation means (Gain in oxygen; Loss of hydrogen; Loss of electrons). Reduction means (Loss of oxygen; Gain of hydrogen; Gain of electrons). TYPES OF Prooxidants 1-Reactive oxygen species (ROS), represent the collection of number of molecules (non-.-. radicals oxygen species) and free radicals oxygen species (Superoxide O2 Hydroxyl OH... Peroxyl RO2 Alkoxyl RO and Hydroperoxyl HO2 ) derived from molecular oxygen. 2  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Non-Radicals oxygen Species have strong oxidizing potential that favor the formation of strong oxidants (e.g., Hydrogen peroxide (H2O2), Hypochlorous acid (HOCl-), Ozone (O3) Singlet oxygen (1O2) Peroxynitrite (ONOO-) and Transition metals Men+ ,in addition, there is another class of free radicals that are nitrogen derived called ; 2-Reactive nitrogen species (RNS). An examples of radical nitrogen species include Nitric Oxide radical NO.. and Nitrogen dioxide radical NO2. These reactive species are readily converted into reactive non-radical species by enzymatic or non-enzymatic chemical reactions (Peroxynitrite ONOO- Alkyl peroxynitrites ROONO Dinitrogen trioxide N2O3 Dinitrogen - tetroxide N2O4 Nitrous acid HNO2 Nitronium anion NO2 + Nitroxyl anion NO Nitrosyl cation NO+ Nitryl chloride NO2Cl) that in turn can give rise to new radicals. 3-Reactive sulfur species (RSS) are a family of sulfurbased chemical compounds that can oxidize and inhibit thiolproteins and enzymes. They are often formed by the oxidation of Thiols and disulfides into higher oxidation states. Examples of RSS include persulfides, polysulfides and thiosulfate. 4-Reactive carbonyl species (RCS) are molecules with highly reactive carbonyl groups, and often known for their damaging effects on proteins, nucleic acids, and lipids. They are often generated as metabolic products. Important RCSs include 3-deoxyglucosone, glyoxal, and methylglyoxal. RCS react with amines and thiol groups leading to advanced glycation endproducts (AGEs). AGE's are indicators of diabetes Table 2: Examples of ROS and RNS Various active oxygen species are generated in the body during the process of utilizing of oxygen. Because the body is furnished with elaborate mechanisms to remove active oxygen species and free radicals, these by-products of oxygen metabolism are not necessarily a threat to the body under physiological conditions. 3  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 The endogenous sources; These molecules, produced as byproducts during the mitochondrial electron transport of aerobic respiration or by oxidoreductase enzymes and metal catalyzed oxidation, have the potential to cause a number of deleterious events. It was originally thought that only phagocytic cells were responsible for ROS production as their part in host cell defense mechanisms. On the contrary, it has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades. It should be noted that ROS can serve as both intra- and intercellular messengers. Notably, if active oxygen species or free radicals are generated excessively or at abnormal sites, the balance between formation and removal (by antioxidants) is lost, resulting in oxidative stress. Consequently, active oxygen species and free radicals damage DNA, RNA, proteins and lipids, resulting in cell death when the level of ROS exceeds an organism s detoxification and repair capabilities, this is negatively impacting the organism and inducing various diseases. Figure 1: Oxidative stress as a results of un balance between free radical formation and removal (by antioxidants) 4  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Biomarkers of Oxidative Stress The biomarkers that can be used to assess oxidative stress have been attracting interest because the accurate assessment of such stress is necessary for investigation of various pathological conditions, as well as to evaluate the efficacy of drugs. Assessment of the extent of oxidative stress using biomarkers is interesting from a clinical standpoint. The markers found in blood, urine, and other biological fluids may provide information of diagnostic value, but it would be ideal if organs and tissues suffering from oxidative stress 5  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 could be imaged in a manner similar to CT scanning and MR imaging. In recent years, attempts have been made to use electron spin resonance (ESR) techniques for this purpose, but it will take time before such methods can be applied to humans. Because the body is not necessarily fully protected against oxidative damage, some of its constituents may be injured by free radicals, and the resultant oxidative products have usually been used as markers. Many markers have been proposed, including lipid peroxides, malondialdehyde, and 4-hydroxynonenal as markers for oxidative damage to lipids; isoprostan as a product of the free radical oxidation of arachidonic acid; 8-oxoguanine (8- hydroxyguanine) and thymineglycol as indicators of oxidative damage to DNA; and various products of the oxidation of protein and amino acids including carbonyl protein, hydroxyleucine, hydrovaline, and nitrotyrosine. Lipid peroxide was assessed in clinical samples even in relatively early studies, and the analytical methods for this substance have improved. The famous method of Yagi, which measures substances that react with thiobarbituric acid, has been widely used in both clinical and experimental studies. Such substances have been the most frequently used marker of oxidative stress partly because lipid peroxidation (Figure 2) is a very important mechanism of cell membrane destruction. Figure 2: The chain reaction causing lipid peroxidation 2.Lipid peroxidation One of the most relevant classes of oxidizing chemical agents causing oxidation of biomolecules are Reactive Oxygen Species (ROS), which are ubiquitous in biological tissues due to aerobic metabolism or to direct exposure to the atmosphere. While affecting most types of molecules, oxidation occurrence in lipids (lipid peroxidation, LPO) is a problem for both human health and the industries of food, feed, and cosmetic products. 6  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Lipid peroxidation is a type of oxidative degradation of biomolecules, where a peroxide is formed from a lipid substrate. Peroxides are compounds containing a structural formula of the type R-O-O-R’, where O are oxygen atoms in the oxidative state −1, a less common and less stable form of oxygen. Lipids are especially prone to peroxidation, mainly the polyunsaturated fatty acids (PUFAs), whether in the form of triacylglycerides (TAGs) or of free fatty acids (FFAs), but also polar lipids containing these PUFAs (mostly glycolipids, phospholipids, and sphingolipids), and cholesterol, due to the presence of methylene groups adjacent to double bonds. On the Mechanism of Lipid Peroxidation Every LPO reaction can be generically described as a three-step process (Figure 3 A and B): initiation, propagation, and termination. Initiation of LPO refers to the moment when a given oxidative trigger contacts with the lipid molecule (LH, Figure 3A) and causes the loss of a hydrogen atom from a methylene group, thus creating a carbon radical lipid (L ). Here, this part of the process is generically described as LH -> L and is analyzed below A B Figure 3: (A) Main molecules involved in the initiation and propagation of lipid peroxidation. LH: lipid molecule; LOO : lipid peroxyl radical; L : lipid radical; LOOH: lipid hydroperoxide. (B): Main molecules involved in the termination of lipid peroxidation. R-H: proton donor; R. : Radical 7  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 The created lipid radical rapidly reacts with available molecular oxygen (O 2) to form a lipid peroxyl radical (LOO , Figure 3A), acquiring both atoms of oxygen at the carbon radical (L + O2 -> LOO ). Before proceeding to an explanation of the remaining process, it is pertinent to note that, upon formation, the peroxyl radical suffers a rearrangement and becomes a conjugated diene. This is only not true if the lipid molecule being oxidized is a saturated fatty acid (extremely unlikely) or a monounsaturated fatty acid (MUFA) (more likely than saturated, but less likely than others containing methylene groups in between double bonds, namely PUFAs). This information is relevant when discussing the methods to detect ongoing lipid peroxidation. This lipid peroxyl radical has now one of two main fates: propagation, in which it reacts with another lipid molecule containing a methylene group, abstracting a proton from this second molecule (LOO + LH -> LOOH + L ), thus becoming a lipid hydroperoxide (LOOH) and generating a new lipid radical (and the cycle restarts, which can happen indefinitely) (Figure 3A); or termination, in which lipid peroxyl radicals and lipid hydroperoxides are neutralized. Termination of lipid peroxidation begins by neutralizing LOO. In this sense, during the propagation phase, despite the generation of a new L that will fuel more LPO, one of the reactive lipids is “terminated”. This process involves other reactions of LOO molecules not resulting in the oxidation of another lipid, as schematized in Figure (3B) and listed below: (a) It receives a proton from a proton-donor (RH) other than another lipid (LOO + RH -> LOOH + R ), such as an endogenous antioxidant (vitamin A or vitamin E), thus generating a lipid hydroperoxide (LOOH) and another radical (R ), which may be less reactive and have its own pathway of degradation (in case of endogenous antioxidant systems, enzymatically or not, these molecules can be restored without causing further damage); (b) It reacts with another lipid peroxyl radical to form a peroxide bridged dimmer (LOO + LOO -> LOOL + O2); (c) It undergoes peroxycyclization, which mediates the decay of LOO into malondialdehyde (MDA) and 4-hydroxynonenal (HNE), the best-known LPO by-products. Either from propagation or from termination by an antioxidant system, LOOH generated also represents an issue, as it is still an unstable molecule that readily reacts as follows: (a) One LOOH reacts with one L to form a lipid dimer (LOOH + L -> LL) and hydroperoxyl radicals (HO2 ) are released; (b) One LOOH decays into a lipid alkoxyl radical (LO ), and hydroxyl radicals ( OH) are released; lipid alkoxyl radicals (LO ) can undergo β-scission, originating smaller radicals (such as pentane and ethane ) and HNE. 8  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Noticeably, LOO and LOOH (created by an oxidative trigger) not only behave like oxidants, reacting with intact lipids and with each other, but also regenerate free radicals when they finally become stabilized (which further amplifies oxidative damages). Furthermore, if dimmers are not formed, and stabilization occurs instead via fragmentation of the lipid chain, several small toxic metabolites (aldehydes, ketones, and carboxylic acids, among others) are formed. Two of the most important ones are MDA and HNE, which present negative impacts in both organism fitness and product quality and safety, by causing additional damages to biomolecules (proteins and DNA) and the off-flavors associated to edible fats rancidification. On the Initiators of Lipid Peroxidation and Sources of ROS LPO is known to be caused by exposure to oxygen (because of ROS formation) and ROS formed by other chemical pathways, light, or high temperatures. In this lecture, the detailed pathways through which each of these triggers actually induces LPO are described, in order to identify the potential targets of antioxidant systems. All the mechanisms described below can be found schematized in Figure 4. Figure 4. Schematic representation of lipid peroxidation initiators, according to their main features: ROS- mediated vs. ROS-independent mechanisms. PS—photosensitizer; oxTM—oxidized transition metal (Fe3+ or 2+ – Cu ). (a) Production of ROS (Superoxide Anion Radical, O2 ) by Iron (II) Fe2+ and Copper (I) Cu+; (b) – Production of ROS ( O2 ) by Light and Atmospheric Oxygen; (c) Production of ROS by Aerobic Metabolism; (d) Production of ROS ( OH) by Fenton Reaction (Reduced Metals and H2O2 Reaction); (e) Production of ROS ( OH) by Light and Hydrogen Peroxide; (f) Direct Oxidation by Light and a Photosensitizer (PS); (g) Direct 3+ 2+ oxidation by Iron(III) Fe and Copper(II)Cu 9  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Indeed, exposure to ROS is the most prevalent mechanism of LPO. ROS can be generated from atmospheric oxygen in the presence of transition metals (Figure 4a and Section 1) or hydrated electrons (Figure 4b and Section 2). More complex enzymatic pathways related to aerobic metabolism also generate ROS (Figure 4c and Section 3), as well as Reactive Nitrogen Species (RNS), under special circumstances, who have also been shown to cause LPO. In addition, hydrogen peroxide (which naturally occurs in the atmosphere and in organic matrices) is unstable and a continuous source of ROS in the presence of transition metals (Figure 4d and Section 4) or light (Figure 4e and Section 5). Photo-oxidation of lipids is another form of LPO, and it is always mediated by a photosensitizer (a molecule that receives a photon and excites other molecules), which can then promote lipid radicalization directly or via singlet oxygen (Figure 4f and Section 6). Transition metals, besides contributing to the generation of ROS, can react directly with lipids, leading to hydrogen abstraction and initiation of the LPO reactions (Figure 4g and Section 7). Generally, the role of temperature in LPO is the acceleration of the reactions that create the lipid radicals. 1.Production of ROS (Superoxide Anion Radical, O2–) by Iron (II) Fe2+ and Copper (I) Cu+(a) Transition metals, like iron and copper, in their reduced states (such as Fe2+ and Cu+), can be oxidized by atmospheric triplet oxygen, to form superoxide anion radicals and Fe3+/Cu2+. Despite the fact that copper is more reactive (higher reducing potential), it is much less abundant than iron in organic matrices. In general, Superoxide anion radicals have low reduction potential and cannot initiate LPO, but they can be converted to hydroperoxyl radicals under low pH, and hydroperoxyl radicals are strong oxidizers that are capable of initiating LPO. Furthermore, Iron (III) and Copper (II) can further amplify LPO by the direct oxidation by Iron (III) Fe3+ and Copper (II) Cu2+ (Figure 4g). 2. Production of ROS ( O2 – ) by Light and Atmospheric Oxygen (b) Light irradiation onto an organic, wet matrix can generate hydrated electrons, i.e., electrons that are solvated and available to react. These electrons can reduce atmospheric oxygen, generating superoxide anion radicals. This mechanism has been shown to be of great relevance both in health and in food quality. 3. Production of ROS by Aerobic Metabolism (c) Cellular respiration involves the oxidation of organic compounds so that an electron can be abstracted from these molecules and transported in a chain (the electron transport chain, ETC), until being accepted by oxygen, which is converted to water. Sometimes electrons leak from the ETC before reaching the end, causing oxygen molecules to ionize (generating superoxide anion radicals) (Figure 5). In living cells, this ROS is mitigated by Superoxide Dismutase (SOD), which converts it to H2O2. Then, catalase neutralizes H2O2 into oxygen and water (Figure 6). However, during their existence, both superoxide anion radical and 10  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 hydrogen peroxide might induce oxidative damages (via the remaining mechanisms described). FIGURE 5. mtROS production in ETC. The electrons donated from NADH in complex I and FADH 2 in complex II flow to complex III through ubiquinone, and then they are transferred to complex IV via cytochrome c; next, the electrons are passed to the molecular oxygen to form water. During this process, complex I, complex III, and complex IV pump protons to the intermembrane space, creating the proton gradient that drives ATP synthesis. During the process of oxidative phosphorylation, electrons leak and interact with molecular −. oxygen to form superoxide (O 2) in complex I and complex III (which are the major ROS production site in the mitochondria) and complex II. Complex III generates superoxide toward the matrix and the intermembrane space, while complex I and complex II produce ROS only toward the matrix. I, complex I; II, − + complex II; III, complex III; IV, complex IV; V, complex V; Q, coenzyme Q; C, cytochrome c; e , electrons; H , protons; ADP, adenosine diphosphate; ATP, adenosine triphosphate; NADH, reduced nicotinamide adenine + dinucleotide; NAD , oxidized nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; O 2, −. molecular oxygen; O 2, superoxide; H2O, water; H2O2, hydrogen peroxide; SOD, superoxide dismutase; Mn, manganese; Cu, copper; Zn, zinc; mtROS, mitochondrial reactive oxygen species; ETC, electron transport chain. FIGURE 6: | mtROS elimination. The generated superoxide is dismutated to H 2O2 by Mn-SOD at the matrix or Cu/Zn SOD at the intermembrane space. The hydrogen peroxide formed can be degraded by catalase, −. thioredoxin, and glutathione peroxidases to form water. O 2 , superoxide; H2O2, hydrogen peroxide; SOD, superoxide dismutase; Mn, manganese; Cu, copper; Zn, zinc; H 2O, water; O2, molecular oxygen; mtROS, mitochondrial reactive oxygen species. 11  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Reference: Hasna Tirichen , Hasnaa Yaigoub , Weiwei Xu, Changxin Wu1 , Rongshan Li and Yafeng Li. Mitochondrial Reactive Oxygen Species and Their Contribution in Chronic Kidney Disease Progression Through Oxidative Stress. Front. Physiol. 12:627837. doi: 10.3389/fphys.2021.627837. 4. Production of ROS ( OH) by Fenton Reaction (Reduced Metals and H 2O2 Reaction) (d) Fenton reactions are a well-known set of reactions in which ROS are generated by the reduction of H2O2 to a hydroxide anion (OH−) and a hydroxyl radical ( OH) through the oxidation of transition metals. Additionally, Iron (III) and Copper (II) can further amplify LPO by the direct oxidation by Iron (III) Fe3+ and Copper (II) Cu2+ (Figure 4g), (Figure 7). Figure 7: Fenton and Haber-Weiss reaction. Reduced form of transition-metals (M𝑛 ) reacts trough the Fenton reaction with hydrogen peroxide (H2O2), leading to the generation of ∙ OH. Superoxide radical (O2 ∙−) can also react with oxidized form of transition metals (M(𝑛+1)) in the Haber-Weiss reaction leading to the production of M𝑛 , which then again affects redox cycling. 5. Production of ROS ( OH) by Light and Hydrogen Peroxide (e) Hydrogen peroxide itself is not a very strong oxidizer. However, as it is an unstable molecule, it can decay into different ROS by different mechanisms. Further, this molecule exists in biological systems as a product of ROS detoxification and is present in the atmosphere. Upon irradiation with UV light, H2O2 is converted into two hydroxyl radicals. 6. Direct Oxidation by Light and a Photosensitizer (PS) (f) Molecules that absorb light are capable of transferring the photon’s energy to other molecules (photosensitizers) and can initiate LPO if light excites them under low-oxygen- concentration conditions. In these cases, if enough energy was transferred to the photosensitizer (chlorophylls and hemoglobin, among others), hydrogen abstraction occurs from lipids directly. If, however, oxygen is abundant, then it is the most likely receiver of the energy contained in the photosensitized molecule, generating singlet oxygen from triplet oxygen. This form of excited oxygen has been shown to initiate LPO by directly interacting with double bonds in lipids, creating unique peroxidation lipid by-products. 12  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 7. Direct Oxidation by Iron(III) Fe3+ and Copper(II) Cu2+ (g) The more oxidized forms of iron and copper (trivalent and divalent cations, respectively) have a sufficiently high reduction potential to cause a proton to abstract from a methylene group, stealing an electron (being reduced into Fe2+ or Cu+), and leaving a lipid radical as result. Oxidative Triggers Cyclic Regeneration It is noteworthy that the product of two of the mechanisms here described (a and d), (Fe3+ or Cu2+), is the reagent in mechanism g, and the product in mechanism g is the reagent in mechanisms a and d. This leads to an amplification of LPO by means of a redox cycle that constantly regenerates oxidative triggers, and such regeneration is in itself an LPO inducing reaction. This recycling of transition-metal-induced LPO initiation potential is further augmented by other parallel, yet very much possible reactions, in a biologic or nonliving organic matrix: (i) the reactions of Fe3+ or Cu2+ with certain antioxidant compounds (which become pro-oxidants in this case), such as ascorbic acid; and (ii) the reactions of iron and copper cations (whether in their more reduced or oxidized forms) with lipid peroxide, yielding lipid alkoxyl- or peroxyl-radicals (Figure 8) Figure 8. Transition metals (iron and copper) redox recycling and its relevance in lipid peroxidation (LPO). AOx: Antioxidant. 13  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Regulation of ROS Production FIRST: Phagocytic Cells The stimulated production of reactive oxygen species by phagocytic cells (macrophages and neutrophil) was originally called “the respiratory burst” due to the increased consumption of oxygen by these cells. This process is catalyzed by the action of NADPH oxidase, a multicomponent membrane bound enzyme complex, and is necessary for the bactericidal action of phagocytes. While several enzymes are recognized as being able to produce ROS moieties, NADPH oxidase is the most significant. NADPH oxidase activity is controlled by a complex regulatory system that involves the G-protein Rac (Figure 9). In resting cells a membrane embedded heterodimer of two polypeptides (p22-phox and gp91-phox), which also contains two heme groups as well as a FAD group, enables the transfer of electrons from cytosolic NADPH across the membrane to molecular oxygen without NADPH oxidase activity. It is believed that the charge compensation occurs when gp91-phox polypeptide also acts as an H+ ion channel. Upon stimulation, a number of polypeptides (p47-phox, p67-phox and p40phox) translocates to the inner face of the plasma membrane to form a fully active enzyme complex that possesses NADPH oxidase activity. A similar process is believed to take place in non phagocytic cells as well. Figure 9. Schematic illustration of the activation of NADPH Oxidase 14  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 SECOND: Oxidative Stress as a Biological Modulator and as a Signal (Figure 10): Oxidative stress not only has a cytotoxic effect, but also plays an important role in the modulation of messengers that regulate essential cell membrane functions, which are vital for survival. It affects the intracellular redox status, leading to the activation of protein kinases, including a series of receptor and non-receptor tyrosine kinases, protein kinase C, and the MAP kinase cascade, and hence induces various cellular responses. These protein kinases play an important role in cellular responses such as activation, proliferation, and differentiation, as well as various other functions. Accordingly, the protein kinases have attracted the most attention in the investigation of the association between oxidative stress and disease. Figure 10: Oxidative stress and cellular responses Oxidative stress can influence many biological processes such as apoptosis, viral proliferation, and inflammatory reactions. In these processes, gene transcription factors such as nuclear factor-B (NF-B) and activator protein-1 (AP-1) act as oxidative stress sensors through their own oxidation and reduction cycling. This type of chemical modification of proteins by oxidation and reduction is called reduction-oxidation (redox) regulation. The transcription factor NF-B undergoes translocation from the cytoplasm to the nucleus in response to an extracellular signal. This translocation induces its ability to bind to DNA, leading to transcriptional up-regulation of the expression of many genes related to inflammation and immunity. Thus, NF-B seems to be involved in development and 15  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 aggravation of many diseases. Recently, it was also suggested that this factor may be involved in the process of carcinogenesis because it is located upstream to a series of transcription regulation factors and because it possesses the ability to suppress apoptosis. With respect to the role that oxidative stress plays in the activation of NF-B, many new findings have been obtained recently. Stimulation with tumor necrosis factor (TNF)-, phorbol myristate acetate (PMA), interleukin (IL)-1, lipopolysaccharide, viral infection, and ultraviolet light leads to the generation of active oxygen species, which function as a second messenger in the activation of NF-B. The mitochondrial respiratory chain is considered to be the major source of active oxygen species. In cells lacking mitochondria, damage caused by TNF-and NF-B dependent IL-6 production is suppressed. It has also been shown that antimycin A, an inhibitor of mitochondrial electron transport, increases the intracellular generation of active oxygen species and enhances the activation of NF-B. Induction of the expression of thioredoxin by active oxygen species is also involved in the activation of NF-B, since thioredoxin gives NF-B the ability to bind to DNA in a process that is regulated by redox reactions. NF-B seems to be the key transcription factor for elucidating the relationship of oxidative stress to lifestyle diseases and identification of the precise mechanisms involved may lead to the development of new therapies for such diseases THIRD: Cell Cycle Control (Figure 11) : The effect of reactive oxygen species on cellular processes is a function of the strength and duration of exposure, as well as the context of the exposure. The typical cellular response to stress is to leave the cell cycle and enter into G0. With continued exposure and/or high levels of ROS, apoptosis mechanisms are triggered. In cycling cells, p21 is activated in response to stress, such as oxidants or oxidative stress and blocks cell cycle progression. Likewise p27 production leads to G1 arrest of cells. In cycling cells, p53 and p21 respond to oxidants by inducing the dephosphorylation of retinoblastoma (RB). Exposure to oxidants such as H2O2 or nitric oxide also results in dephosphorylation of RB that is independent of p53 or p21. In either case cells are arrested in S-phase. Expression of p27 is controlled in part by the Foxo transcription factors, which are known to control the expression of genes involved in cell cycle progression, metabolism and oxidative stress response. For example, mitogenic stimulation by the PI3K/Akt pathway maintains Foxo3a in the cytoplasm, but in the absence of stimulation Foxo3a enters the nucleus and up- regulates genes for oxidant metabolism and cell cycle arrest, such as p27. Under some conditions Foxo3a can directly activate bim gene expression and promote apoptosis. Thus, Foxo3a promotes cell survival of cycling cells under oxidative stress by enabling a stress response, but induces cell death when conditions warrant. Noncycling cells, such as neurons, also have coping mechanisms to oxidative stress that involve Foxo3a. Foxo3a induces expression of the manganese form of SOD in response to oxidative stress 16  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Figure 11. Schematic Illustration of Reported Interactions of ROS and Mitogenic Cascades. On the Quantification of Lipid Peroxidation Several methods to assess LPO have been used by researchers and clinicians, and have been extensively reviewed. Along the process, the physicochemical changes associated with undergoing and/or terminated LPO can be used by researchers to measure the extent of the process. As seen above, oxygen is consumed during LPO, and oxygen consumption rate has been used as a measure of LPO. Evidently, this method detects LPO indirectly and is a weak proxy for the process, as oxygen consumption by other oxidative processes, by respiration of biological nature, solubility changes, and others, may influence the results. 17  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 As discussed in the previously, upon undergoing oxidation, the originally present lipid (LH) suffers several structural changes leading to different forms, including L, LOO, LOOH, and LO, as well as lipid adducts, such as LOOL, LL, or LR, being R any of a series of biomolecules, including DNA and proteins. Each of these products has been used to monitor lipid peroxidation. However, these species are either short‐lived or not guaranteed to occur, depending on the matrix and oxidative conditions. Besides, Liquid Chromatography coupled to Mass Spectrometry (LC‐MS) techniques used to quantify these products are expensive and time‐consuming, and so are immune‐based assays or High Performance Liquid Chromatography coupled to chemiluminescence (HPLC‐CL) based ones. The spectrophotometric detection of conjugated dienes, however, has been shown to be a rapid, low‐cost method to monitor LPO, especially if used to analyze samples undergoing LPO along time. Upon oxidative stress, and having set the baseline absorbance at 234 nm, the change (increment) of absorbance at this wavelength is directly proportional to the formation of lipid peroxyl radicals. Another approach to LPO quantification is the use of secondary end‐products as indicators of the extent of the reaction (Figure 12). Some methods have been described for the detection of pentane, ethane, or HNE; however, their relevance is essentially in a clinical context, for analysis of human‐derived samples. Regardless, and by far, LPO has been mainly quantified by the amount of MDA in the samples. This is because an easy, low-cost methodology for the spectrophotometric detection of MDA exists: the measurement of 18  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 thiobarbituric acid reactive species (TBARS) formation upon reaction with TBA. MDA forms adducts with TBA that strongly absorb light at 532 nm. This reaction has been exhaustively used in the literature to quantify MDA, assuming it to be an indicator of LPO. As with most spectrophotometric methods, however, TBARS assay has been shown to be unspecific, given its potential to generate false positives (by TBA reacting with aldehydes other than MDA). Other methods have been suggested to quantify MDA (such as isolation and quantification by HPLC prior to reaction with TBA), but they are evidently not as practical, and TBARS remains the gold standard of MDA quantification (and, therefore, of LPO quantification). However, as with most indirect methods (that use secondary end-products as estimators of a given reaction), MDA quantification in itself has been shown to be an unreliable measure of LPO, since MDA is a product of reactions other than LPO and is also very reactive: Depending on the composition of the matrix and conditions of the sample, it will be consumed in the chemical reaction. Nonetheless, TBARS has been one of the most used methods to address the development of LPO in the in vitro models reviewed below. For in vitro systems where no chemical oxidizing agent is added, but LPO occurs through different pathways, lipid peroxides thus formed become the main oxidizing compounds in the medium, and LPO can be quantified by using a reagent prone to be oxidized by lipid peroxides (amount of reagent lost can be converted in amount of lipid peroxides). This is the case of the thiocyanate method which has been used to track LPO in lipid-solution- based LPIP assays. NOTE: At the cellular level, specific ROS can be individually assessed from tissue culture, while at the animal level typically the effects of oxidative stress are measured from blood product (e.g. serum or plasma) or from urine samples. 19  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Figure 12: MDA formation and metabolism. MDA can be generated in vivo by decomposition of arachidonic acid (AA) and larger PUFAs as a side product by enzymatic processes during the biosynthesis of thromboxane A2(TXA2) and 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (blue pathway), or through nonenzymatic processes by bicyclic endoperoxides produced during lipid peroxidation (red pathway). One formed MDA can be enzymatically metabolized (green pathway). Key enzymes involved in the formation and metabolism of MDA: cyclooxygenases (1), prostacyclin hydroperoxidase (2), thromboxane synthase (3), aldehyde dehydrogenase (4), decarboxylase (5), acetyl CoA synthase (6), and tricarboxylic acid cycle (7). 3.Cellular Defenses (Antioxidants) Against ROS Detoxification of reactive oxygen species is paramount to the survival of all aerobic life forms. As such a number of defense mechanisms have evolved to meet this need and provide a balance between production and removal of ROS. An imbalance toward the pro- oxidative state is often referred to as “Oxidative stress”. Cells have a variety of defense mechanisms to ameliorate the harmful effects of ROS. The antioxidant reaction protects biological membranes from injury caused by free radicals and lipid peroxides. As studies the biological targets for these highly reactive oxygen species are DNA, RNA, proteins and lipids. Much of the damage is caused by hydroxyl radicals generated from H 2O2 via the Fenton reaction, which requires iron (or another divalent metal ion, such as copper) and a source of 20  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 reducing equivalents (possibly NADH) to regenerate the metal. Lipids are major targets during oxidative stress. Free radicals can attack directly polyunsaturated fatty acids in membranes and initiate lipid peroxidation. Superoxide dismutase (SOD) catalyzes the conversion of two superoxide anions into a molecule of hydrogen peroxide (H2O2 ) and oxygen (O2 ) (Eq. 1). In the peroxisomes of eukaryotic cells, the enzyme catalase converts H2O2 to water and oxygen, and thus completes the detoxification initiated by SOD (Eq. 2). Glutathione peroxidase is a group of enzymes containing selenium, which also catalyze the degradation of hydrogen peroxide, as well as organic peroxides to alcohols. There are a number of non-enzymatic small molecule antioxidants that play a role in detoxification. Glutathione may be the most important intra-cellular defense against the deleterious effects of reactive oxygen species. This tripeptide (glutamyl-cysteinyl-glycine) provides an exposed sulphhydryl group, which serves as an abundant target for attack. Reactions with ROS molecules oxidize glutathione, but the reduced form is regenerated in a redox by an NADPHdependent reductase. Vitamin C or ascorbic acid is a water soluble molecule capable of reducing ROS, while vitamin E (α-tocopherol) is a lipid soluble molecule that has been suggested as playing a similar role in membranes. The ratio of the oxidized form of glutathione (GSSG) and the reduced form (GSH) is a dynamic indicator of the oxidative stress of an organism. 21  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 Antioxidant classification: Antioxidants can be classified according to: 1-Their types 2-Mode of action 3- Location 4-Solubility 5-Structural dependents 6-Origin It is worth to mention that cellular antioxidants can be mainly classified according to their mode of action into: 1. Prevention of prooxidant formation 2. Interception of prooxidants 3. Breaking the chain of radical reactions 4. Repair of damage caused by prooxidants 22 1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 23  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 References 1-An Introduction to Reactive Oxygen Species, measurement of ROS in Cells, By Paul Held (2015), Laboratory Manager, Applications Dept. 2-What Is Oxidative Stress? By Toshikazu YOSHIKAWA* and Yuji NAITO** Professor* and Associate Professor**, First Department of Medicine, Kyoto Prefectural University of Medicin JMAJ. 45(7): 271–276, 2002. Oxidative Stress 3-Lipid Peroxidation: Chemical Mechanism, Biological Implications and Analytical Determination by Marisa Repetto, Jimena Semprine and Alberto Boveris. http://dx.doi.org/10.5772/45943 24  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 APPENDEX Signal Transduction Reactive oxygen species have a role in a number of cellular processes. High levels of ROS, which can lead to cellular damage, oxidative stress and DNA damage, can elicit either cell survival or apoptosis mechanisms depending on severity and duration of exposure. Nitric oxide ( NO) has been shown to serve as a cell-to-cell messenger, being responsible for such effects as decreasing blood pressure. Intra-cellularly, ROS species, in conjunction with antioxidant enzymes, are believed to play a role in turning enzymes on and off by redox signaling in a manner akin to that of the cAMP second messenger system. Examples include superoxide anion, hydrogen peroxide. The steady state level of O2 - is estimated to be so low, however that its activity is spatially limited. Hydrogen peroxide (H2 O2 ) is normally unreactive with thiols in the absence of catalyzing agents (e.g. enzymes,” multivalent metals etc.), it does react with thiolate anion (S-), to form sulfenic acid, which in turn ionizes to form sulfenate (SO-). This intermediate can be reversed by the action of glutathione. Mitogenic signaling begins at the cell surface with the ligand-dependent activation of receptor tyrosine kinases, which activate important MAP kinase cascades necessary for proliferation. These cascades lead to the generation of H2 O2 from several enzyme catalysts, including the NADPH oxidases. It has been estimated that the production of H2 O2 at nanomolar levels is required for proliferation in response to growth factors. Hydrogen peroxide interacts with both the SOS-Ras-Raf-ERK and PI3K/Akt pathways through several mechanisms and in a does- dependant manner (Figure 3). It has been suggested that small increases of H2 O2 , as a result of Nox1 expression result in increased reentry into the cell cycle, while sustained high levels of H2O2 lead to cell arrest and eventual apoptosis after prolonged arrest. Peroxidoxins serve as important regulators of H2O2 and mitogenic signaling. These thiol-dependent peroxidases are activated and recruited to receptors as part of mitogenic stimulation and serve to limit the effect of ROS-associated stimulation on downstream targets of the mitogen cascade. (Figure 3). Cell Cycle Control Redox Signaling As cells proliferate, they move through a coordinated process of cell growth, DNA duplication and mitosis referred to as the cell cycle. The cell cycle is a tightly regulated process with several checkpoints. Each one of these checkpoints is regulated by proteins and protein complexes that are influenced by the oxidative state of the cell. The relationship between the Redox state and cell cycle control is described in great detail in a review by Heintz and Burhans. In multi-cellular animals most of the cells are not replicating and have withdrawn from the cell cycle either temporarily or permanently via terminal differentiation. The exit of G0 and entry into G1 in response to extracellular growth 25  1st PhD Biochemistry lecture by Asst. Prof. Dr. Zainab N. Alabady/2023 factors is controlled by oxidants. Redox-dependent signaling pathways promote the expression of Cyclin D1 , the key protein for re-entry into the cell cycle. As such, cyclin D1 expression has been reported to be a marker for successful mitogenic stimulation. The key regulatory point in G1 is the restriction point (or R point), where cells become committed to entry into S-phase. At the R point, the retinoblastoma (pRB) protein becomes phosphorylated by Cyclin D/CDK complexes. Interestingly enough studies have shown that there is a redox potential of approximately -207 mV for pRB phosphorylation, above which cells pRB is dephosphorylated and cells cease cycling. It has been noted that in synchronized cells the production of ROS increases during the cell cycle, with peak levels occurring in the G2 /M phase. Reactive oxygen species play a role in apoptosis. NF-kB, which is a collective term to describe the Rel family of transcription factors, inhibits apoptosis by upregulating several antiapoptotic genes. Conversely, the c-Jun N-terminal kinase (JNK) promotes apoptosis when activated for prolonged periods. Prolonged activation has been shown to be caused by exposure to ROS directly as well as by inactivating JNK inhibitors such as MAP Kinase phosphatases. Suppression of TNF-α- induced ROS accumulation seems to be the mechanism by which NF-kB downregulates JNK activation. 26