Antioxidant System in bodyMolecular Biology team

Agents that Damage DNA

Certain wavelengths of radiation ionizing radiation such as gamma rays and x-rays ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield . Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways.

Who is responsible? Major causeReactive Oxygen Species (ROS)Reactive Nitrogen Species (RNS)Oxidants are also generated by different types of radiation, with X-irradiation generating the hydroxyl radical and irradiation with ultraviolet light generating electronically excited stateswith subsequent radical formation. Ultrasound and microwave radiation can also generate reactive oxygen species. Even shear stress, e.g. in homogenization, is known to generate radicals.

Free radicals

Molecular oxygen can be reduced to water. The intermediate steps of' oxygen reduction are the formation of- the superoxide anion radical, hydrogen peroxide and the hydroxyl radical,corresponding to the steps of' reduction by one, two and three electrons, respectively.

Types of DNA Damage

All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions. One of the most frequent is the loss of an amino group ("deamination") resulting, for example, in a C being converted to a U.Mismatches of the normal bases because of a failure of proofreading during DNA replication. Common example: incorporation of the pyrimidine U (normally found only in RNA) instead of T.

http://www.benbest.com/lifeext/aging.html#radical

Other damagesBreaks in the backbone. Can be limited to one of the two strands (a single-stranded break, SSB) or on both strands (a double-stranded break (DSB). Ionizing radiation is a frequent cause, but some chemicals produce breaks as well. Crosslinks Covalent linkages can be formed between bases on the same DNA strand ("intrastrand") or on the opposite strand ("interstrand").Several chemotherapeutic drugs used against cancers crosslink DNA [Link].

How? Mutagenesis by ROS/RON contribute to cancer due to1.Cause structural changes e.g. base pair modification, rearrangement, deletion, insertion and sequence amplifications2.Affect cytoplasmic and nuclear signal transduction3.Modulate the activity of the proteins and genes that respond to stress and which act to regulate the genes that are related to cell proliferation, differentiation and apoptosis

Fig. 1. Reactive oxygen species (ROS) can play a role in cell signaling. Oxidative stress can activate numerous intracellular signaling pathways via ROS-mediated modulation of various enzymes and critical transcription factors. In one scenario, transcription factors activated in response to an increase in ROS or oxidative damage travel from the cytoplasm to the nucleus within a cell and bind to promoter regions of particular genes. As a result, these stress-activated pathways can have a significant impact on gene expression, which will ultimately affect the fate of a cell (e.g., apoptosis, proliferation, cytokines). The balance between ROS production, cellular antioxidant defenses, activation of stress-related signaling pathways, and the production of various gene products, as well as the effect of aging on these processes, will determine whether a cell exposed to an increase in ROS will be destined for survival or death.

Where?Mitochondria more than nuclear DNAIntracelluar source is Mitochondiral electron transport may generate radicals ROSPrevented by low calorie intake and free radical inhibitorsFe and Cu are associated with ROS

Why different types of antioxidants?The half-lives of the major reactive oxygen species are vastly different, underscoring the necessity for different types of defense mechanisms

Highest rate constants for the reaction with target molecules are found for the hydroxyl radical; its reactions are diffusion limited, i.e. they take place practically at the site of generation.

In contrast, some peroxyl radicals are relatively stable, with half-lives in the range of seconds. Such moleculesmay diffuse away from their site of generation and thus transport the radical or oxidant function to other target sites.

Oxidative StressAn imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage, is termed 'oxidative stress

Antioxidant defense involves several strategies, enzymatic and non-enzymatic.

.

Natures Strategy Prevention, Interception and RepairPreventionCytochrome oxidase, which carries outmost of the cellular oxygen reduction, does not release superoxide or other radicals, even though it contains iron and copper ions. Likewise, the three-dimensional structure of the enzyme

Ribonucleotide reductase keeps the radical character of the tyrosyl function in subunit B from spreading to the environment by forming an appropriate 'cage'.

Metal chelation is a major means of controlling lipid peroxidation and DNA fragmentation. Thus, the metal-binding proteins ferritin, transferrin, coeruloplasmin and others, e.g. metallothionein, are of central importance in the control

Prevention (contd. )Protection of cells from incident radiation may occur through specialized pigments, e.g. the melanins for ultraviolet radiation or the carotenoids for electronically excited states such as singlet oxygen. However, these and other strategies are not completely preventative, because they operate by decreasing the yield of a given challenging agent with less than 100 % efficiency.

the intestinal mucosal cells. These cells are exposed to a variety of reactive intermediates and xenobiotics, and the rate of accumulation of products of oxidative damage in these cells is high. The turnover and elimination of whole cells prevents further spread of the challenging species.

2. InterceptionAntioxidant defense involves several strategies, enzymatic and non-enzymatic.

In general, this means transferring the oxidizing equivalents from the hydrophobic phases into the aqueous phases, e.g. from the membrane to the cytosol or from lipoproteins to the aqueous phase of the plasma.

Such intercepting chain-breaking antioxidants are often phenolic compounds. (R,R,R)-a-Tocopherol is probably the most efficient compound in the lipid phase

A prerequisite for efficient interception by the phenolic antioxidants is that the lifetime of the radical to be intercepted must not be too short.

Non-enzymaticIn the lipid phase, tocopherols and carotenes as well as oxy-carotenoids are of' interest, (as are vitamin A and ubiquinols. In the aqueous phase, there are ascorbate, glutathione and other compounds.In addition to the cytosol, the nuclear and mitochondrial matrices and extracellular fluids are protected.

SupplementsAntioxidants from our diet appear to be of great importance in controlling damage by free radicals.it is not clear if supplements should be taken and, if so, how much. Once thought to be harmless, we now know that consuming mega-doses of antioxidants can be harmful due to their potential toxicity and interactions with medications. Remember -- antioxidants themselves may act as pro-oxidants at high levels.

Antioxidant

RDA (adults)

Upper Level (adults)

Comment

Vitamin E

15 mg

1,070 mg natural vitamin E

785 mg synthetic vitamin E

Higher amounts impair blood clotting, increasing likelihood of hemorrhage.

Vitamin C

Women: 75 mgMen: 90 mg

2,000 mg

Higher amounts could lead to diarrhea and other GI disturbances. Extremely high levels may lead to cancer, atherosclerosis, and kidney stones.

Beta-carotene

None

None

Chronic high doses turn your skin yellow-orange, but it is not toxic. However, research indicates it is unwise to consume doses of beta-carotene beyond what is in a multivitamin and your regular diet.

Selenium

55 micrograms

400 micrograms

Higher amounts could cause hair loss, skin rashes, fatigue, GI disturbances, and nervous system abnormalities.

EnzymaticOverall, these low molecular mass antioxidant molecules add significantly to the defense provided by the enzymes superoxide dismutase, catalase and glutathione peroxidases

Antioxidant EnzymesThe antioxidant enzymes are proteins with antioxidant properties. There are three known classes of antioxidant enzymes:Superoxide dismutasesCatalasesGlutathione peroxidasesThere are many forms of each class of protein. In general, cancer cells have low levels of these enzymes, when compared to an appropriate normal cell control.

Primary Antioxidant Enzyme System

SODFeSOD which contains an iron ion and is generally found in some prokaryotes, and CuZnSOD, which is active in the cytoplasm of eukaryotic cells.

CuZnSOD occurs as a dimer of identical 16 KDa subunits. Each subunit is 151 amino acids long, and the total protein weighs 32 KDa

Absence of ZnAlzheimer DiseaseSOD act as pro-oxidant than Antioxidant

SODSuperoxide dismutase is categorized as an oxidoreductase class of enzyme, and has the Enzyme Commission identifier of EC1.15.1.1. SOD is a metalloenzyme, meaning that in addition to amino acids, it contains metal ions. Cu2+ + O2- Cu+ + O2Cu+ + O2- + 2H+ Cu2+ + H2O22 O2- + 2H+ H2O2 + O2

RepairSince prevention and interception processes are not completely effective, products of damage are continuously formed in low yields and hence may accumulate. there are multiple enzyme systems involved inDNA repair and lipolytic as well as proteolytic enzymes capable of serving the functions of restitution or replenishment.

Peroxynitrile (RON)which is formed from nitric oxide and superoxide. It was observed that a seleno-organic compound reacts very efficiently with peroxynitrile

Biomarkers-1Lipid Peroxidation: The appearance of 8-epi-prostaglandin PGF2a (8-epi-PGF2a) in plasma or urine has been suggested by a number of investigators as a reliable index of in vivo free radical generation and oxidative lipid formation. There is very strong evidence from animal studies that 8-epi-PGF2a increase in plasma and urine as a result of oxidative stress, and in human, this product is elevated in smokers. Comparison with other measures of lipid peroxidation, 8-epi-PGF2a is specific product of lipid peroxidation, and is very stable. In addition, its formation is modulated by antioxidant status, and its level is not affected by lipid content of the diet.

Biomarkers-2Protein Oxidation: Oxidative damage can affect proteins giving rise among others to protein carbonyl derivatives, via a variety of mechanisms that include fragmentation and amino acid oxidation. Protein oxidation has major deleterious effects on normal functioning of organism. 2-Oxohistine and nitrotyrosine are thought to be an indicator of protein oxidation induced by peroxyl radical and peroxynitrite, respectively.

Biomarkers-3DNA damage: One of the major products of oxygen radical attack is 8-hydroxy-2'-deoxyguanosine. Numerous publications reported that there is an age-dependent increase in the level of this adduct in human brain tissue. Brunswick Laboratories has developed a LC/MS method to measure 8-hydroxy-2'-deoxyguanosine in urine.