Department of Biochemistry and Molecular Biology, Medical Centre of Postgraduate Education, Warsaw
Head of Department: prof. dr hab. Barbara Czarnocka
Most organisms living on Earth are entirely dependent on the presence of oxygen in the atmosphere. However, the by-products of oxygen metabolism are toxic to living organisms. Reactive oxygen species (ROS) in the cell are produced both during normal cellular metabolism or inflammatory reactions and under the influence of external factors like γ, X and UV radiation, biotransformation of dietary chemicals and some diet components, e.g. transient metal ions (1). Normal cellular metabolism seems to be the primary source of endogenous ROS. An imbalance between the formation of ROS and antioxidant defense leads to increased reactive oxygen species generation and oxidative stress development (2). ROS are radical molecules containing oxygen, for example superoxide (O2?–) and hydroxyl radical (?OH), or non-radical molecules, such as hydrogen peroxide (H2O2) and singlet oxygen (1O2), which may be converted into radical forms. The most reactive ROS, hydroxyl radicals, are responsible for oxidation and fragmentation of nucleic acids, proteins and lipids. They are produced in the metal-catalysed Haber-Weiss and Fenton reactions mediated by the transition metal ions such as iron and the copper (3). Iron is a cofactor for many biological reactions and is an important component of metabolism in various tissues and organs, including the thyroid. Iron deficiency may affect thyroid hormone synthesis by decreasing the activity of the heme-dependent thyroid peroxidase (TPO). In addition, low iron levels reduce deiodinase activity, i.e. it slows down the conversion of T4 to T3, and also causes a raise in circulating concentrations of thyroid stimulating hormone (TSH) (4). With higher levels of TSH and low free T4 and T3 levels hypothyroidism occurs. Iron overload, on the other hand, may promote the persistence of harmful labile iron, which can catalyze the generation of potentially carcinogenic DNA adducts in the cell (5).
Despite the fact that hydrogen peroxide does not react directly with components of DNA, it is a precursor to highly reactive hydroxyl radical (?OH), hypochlorite (ClO–) and singlet oxygen (1O2). Therefore H2O2 may facilitate a mutagenic process and DNA modification leading to cancer development (19). A thyrocyte which generates a great amount of H2O2 is a long-lived cell and that allows it to accumulate mutations in DNA (20). Consequently, oxidative stress has been suggested to contribute to the pathogenesis of thyroid cancer (21, 22).
An antioxidative defense systems, that protect from the formation and effects of reactive oxygen species, function in all living organisms. In the cell the defense against the destructive effects of ROS works on the three levels.
The first level of the system prevents the formation of excessive quantities of ROS. The main component of this level are proteins that bind transition metal ions which thus inhibits Fenton reactions. Iron ions are bound by ferritin, transferrin and lactoferrin, copper ions by ceruloplasmin. Metallothioneins bind a number of different metal ions, as well as albumin, which non-specifically, is capable of binding many metal ions (23).
The second defense level neutralizes ROS. This system includes antioxidant enzymes such as superoxide dismutase (SOD), glutathione and ascorbate peroxidases (GPX, APX1), and glutathione transferase. The other elements of this protection level are small molecule antioxidants that work as direct or indirect free radical scavengers: glutathione, ascorbic acid, cysteine, tocopherols (vitamin E), retinoids (vitamin A analogs), uric acid, carotenoids, bilirubin, ubiquinol, and even glucose and pyruvate (3, 24). The above antioxidative protectors have been found in thyroid gland, e.g. GPX and TPO and are upregulated during the synthesis of thyroid hormones (25). There is also evidence that GPX3 which affects the H2O2 concentration directly interferes with hormone synthesis (26).
The third level of the defense is the elimination of ROS harmful effects on the most important cellular macromolecule – DNA. Oxidative DNA adducts are repaired by enzymes of excision repair systems, which will be described in subsequent chapters.
ROS reactions with DNA cause the most dangerous consequences for multicellular organisms. The ?OH radical molecule is one of the ROS that is extremely reactive in the oxidation of cellular constituents such as nucleic acids, proteins and lipids. ?OH interactions with DNA may lead to considerable damage, such as oxidized bases, base and sugar lesions, abasic sites, DNA-DNA intrastrand adducts, single or double strand breaks and DNA-protein cross-links (2, 27-29).
Among modified DNA products generated by the free radicals a significant part are pyrimidine- and purine-derived lesions (30). Some of these modified DNA bases have considerable potential to affect the integrity of the genome (31). The main products of oxidatively damaged DNA include 8-oxo-7,8-dihydroadenine (8-oxoA); 8-oxo-7,8-dihydroguanine (8-oxoG) and its deoxynucleoside equivalent, 8-oxodG; 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, Tg) and ringopened lesions: 4,6-diamino-5-formamidopyrimidine (FapyA) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) (32-34).
Guanine in the cellular nucleotide pool and also as a component of the nucleoside, nucleotide or polynucleotide (DNA, RNA) is especially susceptible to oxidation by ROS. Its oxidation results in the formation of the modified base known as 8-oxo-7,8-dihydroguanine (8-oxoG). 8-oxoG is the most widely studied DNA lesion and the best marker of oxidative DNA damage due to its mutagenic nature and its high sensitivity to immunological detection. The presence of 8-oxoG residues in DNA leads to G > T transversions (35-37) and one of the consequences could be point mutations. Several model studies confirmed that external oxidative factors such as induce G > T transversions in DNA and the overall frequency of point mutations correlate with the level of 8-oxoG (38, 39). Studies on 8-oxoG are focused on finding a link between the presence of mutations in the DNA molecule and malignant transformation of the cell. Elevated levels of 8-oxoG in the DNA were detected in cancer tissues of different origins (40, 41). Moreover experimental data suggests that 8-oxoG occurrence reflects the early changes in the process of carcinogenesis. The significant role of 8-oxoG in carcinogenesis may also be supported by the fact that in tumor tissues G > T transversions are the most common point mutation within the p53 tumor suppressor gene and other genes associated with tumor development (42).
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