Elevated rates of reactive oxygen species (ROS) have been detected in almost all cancers, where they promote many aspects of tumour development and progression. However, tumour cells also express increased levels of antioxidant proteins to detoxify from ROS, suggesting that a delicate balance of intracellular ROS levels is required for cancer cell function. Further, the radical generated, the location of its generation, as well as the local concentration is important for the cellular functions of ROS in cancer. A challenge for novel therapeutic strategies will be the fine tuning of intracellular ROS signalling to effectively deprive cells from ROS-induced tumour promoting events, towards tipping the balance to ROS-induced apoptotic signalling. Alternatively, therapeutic antioxidants may prevent early events in tumour development, where ROS are important. However, to effectively target cancer cells specific ROS-sensing signalling pathways that mediate the diverse stress-regulated cellular functions need to be identified. This review discusses the generation of ROS within tumour cells, their detoxification, their cellular effects, as well as the major signalling cascades they utilize, but also provides an outlook on their modulation in therapeutics.

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Reactive oxygen species in cancer

We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins.

Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O2-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several categories of reactive species, and a range of stable products whose chemistry is currently being elucidated. Among the reactive products, protein hydroperoxides can generate further radical fluxes on reaction with transition-metal ions; protein-bound reductants (notably dopa) can reduce transition-metal ions and thereby facilitate their reaction with hydroperoxides; and aldehydes may participate in Schiff-base formation and other reactions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to unreactive hydroxides. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and in pathologies such as diabetes, atherosclerosis and neurodegenerative diseases. Protein oxidation may also sometimes play controlling roles in cellular remodelling and cell growth. Proteins are also key targets in defensive cytolysis and in inflammatory self-damage. The possibility of selective protection against protein oxidation (antioxidation) is raised.

/pdf/biochemistry-and-pathology-of-radical-mediated-protein-2z7iccorcx.pdf

Biochemistry and pathology of radical-mediated protein oxidation

Free radicals and other reactive oxygen species (ROS) are constantly formed in the human body. Free-radical mechanisms have been implicated in the pathology of several human diseases, including cancer, atherosclerosis, malaria, and rheumatoid arthritis and neurodegenerative diseases. For example, the superoxide radical (O2·−) and hydrogen peroxide (H2O2) are known to be generated in the brain and nervous system in vivo, and several areas of the human brain are rich in iron, which appears to be easily mobilizable in a form that can stimulate free-radical reactions. Antioxidant defenses to remove O2·− and H2O2 exist. Superoxide dismutases (SOD) remove O2·− by greatly accelerating its conversion to H2O2. Catalases in peroxisomes convert H2O2 into water and O2 and help to dispose of H2O2 generated by the action of the oxidase enzymes that are located in these organelles. Other important H2O2-removing enzymes in human cells are the glutathione peroxidases. When produced in excess, ROS can cause tissue injury. However, tissue injury can itself cause ROS generation (e.g., by causing activation of phagocytes or releasing transition metal ions from damaged cells), which may (or may not, depending on the situation) contribute to a worsening of the injury. Assessment of oxidative damage to biomolecules by means of emerging technologies based on products of oxidative damage to DNA (e.g., 8-hydroxydeoxyguanosine), lipids (e.g., isoprostanes), and proteins (altered amino acids) would not only advance our understanding of the underlying mechanisms but also facilitate supplementation and intervention studies designed and conducted to test antioxidant efficacy in human health and disease.

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Free radicals, oxidative stress, and antioxidants in human health and disease

Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants.

https://scholarcommons.sc.edu/cgi/viewcontent.cgi?article=1147&context=chem_facpub

Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation.

Glycoxidation products (GOPs), such as N epsilon-(carboxymethyl)lysine (CML) and pentosidine, are formed during reaction of glucose with protein under oxidative conditions in vitro. It is uncertain whether these GOPs are derived from oxidation of Amadori adducts on protein or from oxidation of glucose or intermediates formed prior to the Amadori rearrangement. To address this question, we reacted collagen with 250 mM glucose in 200 mM phosphate buffer, pH 7.4, under antioxidative conditions, yielding a protein rich in Amadori adducts, but with only traces of GOPs. This "preglycated" collagen was then exposed to [13C6]glucose under oxidative conditions, producing both natural and [13C2]-CML. At 200 mM phosphate buffer, [13C2]-CML was the major product, even at low (5 mM) [13C6]glucose concentration, indicating a limited role for Amadori compounds in formation of CML in high phosphate. The relative yields of natural and [13C2]-CML varied with phosphate concentration, becoming similar at more physiological (10 mM) phosphate. We conclude that during glycation of proteins at high phosphate concentrations in vitro, GOPs are formed primarily by oxidation of free glucose or rapidly-formed intermediates preceding the Amadori rearrangement, such as carbinolamine or Schiff base adducts. In contrast, at lower phosphate and glucose concentrations in vivo, the Amadori adduct may be the more significant precursor of GOPs. The fact that glycoxidation reactions proceed by multiple routes must be considered in the development of therapeutic approaches for inhibiting the Maillard reaction in diabetes.

Pathways of formation of glycoxidation products during glycation of collagen.

The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.

/pdf/age-dependent-increase-in-ortho-tyrosine-and-methionine-5ebi03oh9r.pdf

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes.

https://scholarcommons.sc.edu/cgi/viewcontent.cgi?article=1164&context=chem_facpub

Oxidized amino acids in lens protein with age. Measurement of o-tyrosine and dityrosine in the aging human lens

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P < 0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal < glycated < or = minimally oxidized < glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

Mary C. Wells-Knecht

Papers

Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation.

Pathways of formation of glycoxidation products during glycation of collagen.

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes.

Oxidized amino acids in lens protein with age. Measurement of o-tyrosine and dityrosine in the aging human lens

Toxicity of Mildly Modified Low-Density Lipoproteins to Cultured Retinal Capillary Endothelial Cells and Pericytes