Oxidative Stress and Cellular Defences

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Oxidative Stress and Cellular Defences

Introduction

Every living aerobic organism on the planet relies on and is constantly reacting with oxygen molecules to produce energy in the form of ATP via oxidative phosphorylation. As advantageous as this process is to our (not to mention a plethora of other lifeforms) continued existence, the reaction also produces reactive oxygen species, such as superoxide and hydrogen peroxide, leading to the propagation of free radicals in the cell. The phenomenon known as oxidative stress occurs when the production of these reactive oxygen species (ROS) is greater than the organism’s ability to detoxify the reactive intermediates; the concomitant outcome being damage to all cellular components, such as DNA, proteins and lipids. Such cellular damage can lead to compromised cellular function, apoptosis and even necrosis with a sufficiently high level of oxidative stress. As such, cellular regulation of the redox state is critical for cell viability, activation, proliferation, and organ function. Aerobic organisms have evolved sophisticated integrated antioxidant systems, which include enzymatic and nonenzymatic antioxidants that are usually effective in blocking harmful effects of ROS1.

Endogenous sources of Reactive Oxygen Species

It is already well established that the major endogenous sources of ROS are localized to mitochondria and can be related to the respiratory chain, substrate dehydrogenases in the matrix, monoamine oxidase and cytochrome P4502. Mitochondria from various aerobic organisms have been recognized as effective sources of H2O23, a molecule that plays prominent roles in a variety of pathophysiological processes, including aging, neurodegeneration, and heart and lung toxicity. The H2O2 produced by mitochondria appears to account for 1–2% of the total O2 consumed in vitro. The superoxide anion radical (O.-), formed during ubisemiquinone autoxidation and, secondarily, NADH dehydrogenase activity, is considered the stoichiometric precursor of mitochondrial H2O24.

Superoxide anions that are generated through the respiratory chain during the transfer of electrons, are thought to be released into the mitochondrial matrix via vector, where they become a substrate for Mnsuperoxide dismutase. This view has been supported by the following observations: (a) high levels of Mn-superoxide dismutase (1.1 x 10-5 M) are found in the mitochondrial matrix5 and, thus, the high rate of superoxide anion production in the mitochondrial inner membrane would have a relationship with the enzyme’s localisation(b) submitochondrial particles produce superoxide anion into the medium as monitored by cytochrome c-6 or adrenaline-based assays3 and (c) H2O2, the product of superoxide anion partitioning, can be readily measured diffusing out from mitochondria by peroxidase-based assays7. Hence, the notion that H2O2 released by mitochondria is the product of the dismutation of superoxide anion within the mitochondrial matrix has generally been accepted.

The layout of complex III in the mitochondrial electron-transport chain involves an inner and outer pool of ubiquinone, opposite the matrix and the intermembrane space, respectively. Ubisemiquinone is formed at both the inner pool that near the matrix and at the outer pool close to of the intermembrane space8. This, along with ubisemiquinone autoxidation as a major source of superoxide anion formation by mitochondria, leads to the partial production and release of superoxide anion towards the intermembrane space9. This mitochondrial superoxide production can be significantly enhanced if the rate of electron transport is limited by the buildup of a large proton gradient in the inner mitochondrial membrane. Such a proton buildup can occur with abundant fuel supply, such as NADH production, or with the functional impairment of one or more of the electron transport complexes, most especially complex I and III10. Mitochondrial superoxide anion generation can then lead to the production of ROS such as H2O2 which, in the presence of ferrous iron by means of the Fenton reaction, results in highly reactive hydroxyl radicals. When these ROS overwhelm the antioxidant systems of the cell, oxidative damage and cell death can occur11

Exogenous sources of ROS

In addition to endogenous sources of ROS, there also exist sources of ROS that can be produced or induced extracellularly. One such source involves local inflammation, which is a host immune response that has evolved as a survival strategy to protect against foreign pathogens, more specifically, to facilitate the process of tissue repair. On encountering pathogens, the innate immune system evokes an acute inflammatory response accompanied by vasodilation, vascular leakage and the emigration of leukocytes12. The production of ROS is important to the progression of a range of inflammatory diseases. ROS are produced by cells which have a role in the host-defence response, such as polymorphonuclear neutrophils, and facilitate endothelial dysfunction via the oxidation of important cellular signalling proteins, such as the tyrosine phosphatases. In addition, the production of superoxide anion by gp91phox in leukocytes is critical for killing engulfed microbes within phagolysosomes13. Whilst an excess of ROS is undoubtedly important in the destruction of pathogens, if ROS levels are not kept under control by cellular antioxidant mechanisms, the result is inflammatory tissue injury14.

Chemical or drug induced oxidative stress is another major source of exogenous ROS, implicated as a toxic mechanism in a number of tissues and organs, such as the nervous and cardiovascular systems. There exist many well-characterised drugs associated with producing oxidative stress in the body, such as antipsychotics, cancer therapies, non-steroidal anti-inflammatory drugs and analgesics. The extent to which the mechanisms underlying drug-induced oxidative stress varies, as different drugs may achieve the effect via different mechanisms. One well understood example is chlorpromazine. Chloropromazine achieves an excited state via photodechlorination, with the subsequent energy transfer to molecular oxygen and the generation of both an excited singlet oxygen and superoxide species15. These species can then react with macromolecules and DNA, triggering toxic or adaptive responses in the skin. There is also evidence of elevation in cellular ROS in response to drug exposure, with evidence implicating ROS and oxidative stress in toxicity even if the mechanisms of ROS generation are characterised less fully16.

Another source of exogenous ROS is ionising radiation. There are two types of radiation: non-ionising which is relatively low energy and elicits its effects mainly through heat, and ionising; which possesses sufficient energy to displace electrons from molecules or atoms, producing ions. Since most living organisms are comprised primarily of water, the ionising radiation interacts with H2O molecules displacing electrons, which leads to a rapid production of ROS resulting in oxidative stress17.

Effects of ROS on biological molecules within cells (DNA damage, lipid modification etc).

The effects of ROS on intracellular biological molecules typically include; DNA damage, lipid peroxidation, oxidation of amino acids in proteins and the oxidative deactivation of specific enzymes via the oxidation of co-factors.

The biochemistry of the DNA damage that can be caused by several ROS has been well-characterised in vitro. Of the different ROS, the highly reactive hydroxyl radical reacts with DNA by the addition to double bonds of DNA bases and by the abstraction of a hydrogen atom from the methyl group of thymine and each of the C-H bonds of 2′-deoxyribose18. This can cause structural alterations in DNA, such as deletions, insertions, base pair mutations, rearrangements, and sequence amplification. ROS are also capable of producing gross chromosomal alterations in addition to point mutations and hence can be involved in the inactivation or loss of the second wild-type allele of a mutated proto-oncogene or tumour-suppressor gene that can occur during tumour promotion and progression, allowing expression of the mutated phenotype19. It would appear that damage to DNA by ROS occurs naturally, with low steady-state levels of base damage being detected in nuclear DNA20.

ROS are also responsible for the oxidative degradation of lipids within the cell, in a process known as lipid peroxidation. In lipid peroxidation, free radicals take electrons from the lipids in cell membranes, resulting in cell damage. It most commonly affects polyunsaturated fatty acids, due to the presence of multiple double bonds in between which exist methylene bridges (-CH2-) in possession of especially reactive hydrogens. Peroxides can decompose to a range of mutagenic carbonyl products21. Evidence has been obtained of these products being genotoxic to lymphocytes and hepatocytes and disruptive to gap-junction communications22

Protein damage is also a major consequence of excess ROS generation, demonstrated in vivo23. It has been postulated that an alteration in the conformation of DNA polymerase could explain the number of close-proximity double mutations occurring secondarily to genetic stresses24.

Cellular antioxidant defences

A biological antioxidant can be defined as any substance present at low concentration when compared to an oxidizable substrate, significantly delaying or preventing the oxidation of the substrate25. The redox homeostasis of the cell is maintained by a sophisticated endogenous antioxidant defence system, comprised of endogenous antioxidant enzymes such as catalase superoxide dismutase, glutathione peroxidase, glutathione, and scavengers, such as coenzyme Q, uric acid, and lipoic acid. The antioxidant defence system must keep levels of ROS to a minimum while simultaneously permitting useful roles of ROS in cell signalling and redox regulation26.

Endogenous antioxidants can be classified into primary and secondary antioxidants. Superoxide dismutase, catalase and glutathione peroxidase are primary antioxidant enzymes responsible for the inactivation of ROS into intermediates27. With the exception of the primary antioxidant enzymes, primary antioxidants are water and lipid soluble. Glutathione ascorbate, uric acid amongst others are water soluble, whilst ubiquinols tocopherols, and carotenoids to name a few are lipid soluble. Secondary antioxidant enzymes such as Glucose-6-Phosphate dehydrogenase, Glutathione reductase, glutathione-S-transferase and ubiquinone act to directly detoxify ROS by decreasing the peroxides level and at the same time continuously supplying the NADPH and glutathione for primary antioxidant enzymes in order to maintain proper functioning28.

Exogenous antioxidants, such as vitamins and minerals, obtained through dietary means also have an important role in the antioxidant defence system of the cell. Vitamin E acts to inhibit ROS generation, preventing the membrane from lipid peroxidation29, and Vitamin B12 and folic acid limits free radical-induced ionising-radiation damage and also improves leukocytes counts, whereas choline and Vitamin C help to prevent DNA damage and hepatocellular carcinoma. Vitamin B6, B12 and folate serve as cofactors for the synthesis of methionine synthase (B12) and cystothionase (B6), also acting as a substrate for methionine synthase. These vitamins play a factor in reducing cardiovascular diseases in both rats and humans.

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