INTRODUCTION:

Free radicals are highly reactive molecules with odd number of electrons. They are short-lived as they are highly reactive. It was not until 1968 when scientist discovered the enzymes super oxides dismutase, the biological free radical research become important¹. Free radical contains one or more pairs of unpaired electrons. Free radicals usually represented by a superscript dot after the molecular formula. The simplest free radically is H¹₁ Free radicals may be formed when covalent bond is broken and one electron from each pair remains with each atom (homolytic fission). The energy for the reaction can be obtained by heat, electromagnetic radiation and other means. Free radicals involved in over 100 diseases which include rheumatoid arthritis, cardiomiopathy, cystic fibrosis, Aids etc. Oxidative stress plays an important part in all the above mentioned diseases. The disease related associated oxidative stress could result from diminished antioxidants such as glutathione for oxidize; MnSOD etc or it can result from increase production of reactive oxygen species or reactive nitrogen species. Free radicals are also involved in the ageing process. The free radical theory of Ageing was explained by Denham Harman in 1956. And it is stated that the damage of tissues is by free radicals.²

AGEING:

Theories of ageing: It is difficult to define. It can explain in general terms, in life before the reproductive phase the efficiency of physiological will cause. It differs in several cellular organisms. Traumatic injury will also decreases with age. It is difficult to separate from disease related to age³.

TECHNICAL ASPECTS OF FREE RADICAL DETECTION:

Short lived free radical detection is difficult. Long lived one is transformed by short lived radicals from any substance that scavenges and it is the one which is used in detecting subsequently. Example, Spectrometry is used in in-vitro⁴. By some methodological studies the problems can be illustrated and they are commonly used to test reactions demonstrating for the assay of O₂free radicals by the production of components or plant cells. EPR spin trapping technique is used to bypass the shortcomings of free radicals. To yield persistent nitroxide the nitrone or nitroso spins are reacts with transient radicals and it can be detected by EPR spectroscopy. EPR technique is having history in detecting •OH and O₂^-.in chemistry and biology.^5,6

COMMONLY USED SPIN TRAPS-AN OVERVIEW:

As the techniques of spin traps are many, selection of a spin trap plays a very important role in the EPR applications. The selection of spin trap includes its ability to trap particularly selected free radicals and that can be easily classified. It works on the principle of signal to noise ratio which very mostly depends on the number of transient radical formed, the rate constant for spin trapping, and the decay and concentration of spin adducts. Few spin traps are available for various kinds of free radicals.

In biological systems DMPO (5, 5-dymethyl-pyrrolinr-N-oxide) has been widely used from ages to trap oxygen radicals. One of the main disadvantages of using DMPO is that the DMPO superoxide adducts quickly and spontaneously destroys to the DMPO hydroxyl adduct and as the result of this formation of oxygen radicals was typically recognized by the monitoring of (SOD) superoxide dismutase inhibition of production of DMPO/OH. A phosphorylated analogue of DMPO which termed as DEPMPO (5-DIETHOXYPHOSPHORYL-5-METHYL-1-PYROLINE-N-OXIDE) was developed years ago for overcoming this kind of disadvantage. As a result to that DMPO/OOH, the DEPMPO superoxide adducts theoretically does not decay to the DEPMPO/OH. The usage of DEPMPO in simultaneous spin trapping of oxygen radicals is hampered in a way because the EPR spectra of DEPMPO adducts are some complicated due to the additional P hyperfine coupling and the existence of diastereomers. So that, in a time when the spectrum signal to noise ratio is decreased, which often destroys in biological systems, and it is wild to perform spectral simulations. There are some other various traps developed for the detection of superoxide or hydroxyl radicals other than the DMPO and its derivatives.

(A 4-POBN (α (4-pyridyl-1-oxide)- N- tetra- butylnitrone) a regularly used spin trap. Finding the oxygen adduct is very difficult in this due to its instability, thus in case of production of oxygen radicals, 4-POBN detects practically hydroxyl radicals only.

Chelator Tiron (n-2-hydroxyethylpiperazine propane sulfonic acid), another species specific spin trap which got the ability of generating semiquinone radicals when treating with oxygen and tiron is acidic in nature and it can lower intra cellular as well as extra cellular pH.^7-11

EPR is specific technique for free radicals and the presence of unpaired electrons can be detected. Either + ½ or – ½ spins are in unpaired electrons and its characteristics is like small magnet. It can form itself in order like anti parallel or parallel when it exposed to the field of external magnet. To display the first derivative spectra ESR spectrometers are used to set up. Rate of change of absorbance is shown by this ESR technique.¹²

The absorbance can be obtained by the condition is:

∆ E = gβH

∆E is the gap between the levels of electrons energy level, β is Bohr magneton constant, H is magnetic field applied and splitting factor is g.

In this spin trapping technique to produce a long lived radical it should react with reactive radical. A Nitroxide radical is produced by the reaction of nitroso compounds.¹³

│

R− N═O + R′^∙→ N─O^∙

R symbolises Reactive radical │ nitroxide radical

Rest of molecule R

Nitroxide radicals are also produced by nitrone traps

H O⁻H O^∙

│ │ │ │

R ─ C ═ N ─ R ′ + R^∙′′ → R ─ C ─ N ─ R′

+ │

R′′

ESR spectra splits nitroxides have triplet 1:1:1 due to the interaction of unpaired electrons with nitroxide group nitrogen nucleus. In the magnetic nuclei the splits arise from the trapped radical and some other nuclei in the spin trap.

NITROXIDES USED IN SPIN TRAPPING:

Nitroxides are used for the measurement of pathological and biological phenomenon which related to ROS generation and Oxidative stress. Nitroxide formed from chiral drugs except hydrogen is trapped if any free radical is obtained where β hydrogen is from N-oxide used as spin trap. Diastereoisomery and diastereotopic are the two faces of molecule when the nitrone itself is Chiral. Cis/trans pairs of nitroxide can occur yield by spin adduct. In biological spin trapping, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO), 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the phosphorylated (DEPMPO) 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide are synthesize. By these nitrones the main interest was focused on superoxide trapping. In the both EMPO and DEMPO family the Cis/trans diastereoisomers of nitrone/SOH adducts and when trapped by DEMPO or DIPPMPO one can assume the same situation for a small and reactive HO^.14,15. Stable nitroxides which are commonly used are five and six membered nitroxides as probes and agents like nuclear magnetic resonance imaging contrasts in some studies, these undergo redox reaction and for catalyzation. To oxidize transition metals the nitroxides are used and these metals are also used to potentiate and mediate the free radical induced damage. Studies explains that nitroxides protect against H₂O₂ induces the cyto toxicity of hamster cells^16,17

OXIDATIVE DNA DAMAGE:

Free radical reactions are also form the DNA protein cross-links. The mostly used DNA free radicals are Guanine, thymine, tyrosine etc. This DNA damage is measured by the technique of (8-OHdG) 8-hydroxydeoxyguanosine, (8-OH-G) 8-hydroxyguanine which is attached to the deoxyribose. By the attack of OH^∙ this DNA damage can form; followed by oxidation of one electron.

OH^∙ -1e^-

Guanine → [8-OHdG]^∙ → 8-OHdG

In DNA (step B)

[8-OHdG]^∙ radical is having alternatives and such products are generated as FAPy-guanine. Reaction conditions are affected to this two products 8-OHdG and FAPy-guanine. Attack of ¹O₂ and RO₂^∙/RO^∙ radicals results hydroxylated guanine. Guanine to xanthine, adenine to hypoxanthine and cytosine to uracil deaminated by several RNS and the formation of 8-nitroguanine in DNA where as Chlorcytosine generates from HOCL by the exposure to ONOO⁻. Formation of apurinic site in the DNA, is by depurination of 8-nitroguanine rapidly.¹⁸

In mammalian chromatin a thymine-tyrosine cross-link is identified in cells exposed to in vitro of free radical generating systems. The DNA protein cross-link mechanism involves in the C3 position of tyrosine ring the thymine allyl radical is added in the DNA vicinity followed by Oxidation. To measure the DNA free radicals the most important technique is HPLC-ECD but in this technique the structural evidence is not provided. 8-OH-dGuo nucleoside is measured by this technique. This nucleoside is having mutagenic properties most studies concentrated on this product. In the European recent trails in the levels of 8-OH-dGuo significant differences measured. The DNA damage is measured by GC/MS technique and it is capable for the measurement of all four DNA base products. 8, 5’-cyclopurine-2’-deoxynucleosides and DNA protein cross-links are the products of sugar moiety. Among the many products 8-OH-Gua, 5-OH-cyt, 5-OHMeUra, 8-OH-Ade and 5-formyluracil can be measured by this GC/MS technique. If the derivatization temperature ranging from 23°C to 120°C not effected to the levels of 5-OH-cyt, 5-OH-cyt and 8-OH-Ade. At 23°C and 60°C similar levels are observed in the case of 8-OH-Gua. DNA from other sources is compared by levels of 5-OH-Cyt, 8-OH-Gua and 8-OH-Ade¹⁹.

Fenton Reaction: Transition metals are used to catalyse the damaging free radicals reaction it is an example of Fenton chemistry. Many organic molecules are oxidises with the mixture of Fe²⁺ salt and H₂O_2.²⁰.

Fe²⁺ + H₂O₂→ intermediate complex (es)→ Fe (iii) + OH^. + OH^-

Fe²⁺ + H₂O₂ are using to the mechanism of action of organic compounds by oxidation from 120 years.

H₂O₂ is used to further reaction to Fe (iii), at physiological pH the Fe (iii) reaction is slower than that the H₂O₂ with Fe²⁺ reaction and it depends upon the ligand.

Fe (iii) + H₂O₂→ intermediate complex (es)→ O₂^.
- + 2H⁺

It generates OH^.By the inhibition of certain super oxide dismutase like Fe (iii) and H₂O₂ they are appears to form O₂^–.²¹

Under certain circumstances Fenton reaction of H₂O₂ and Fe²⁺ forms biological system. The Sum of these is unless adding some other reagents the decomposition of H₂O₂ is catalysed from iron²².

2H₂O₂ → O₂ + 2H₂O

CONCLUSION:

Finally it is concluded that it is possible to trap DNA free radicals by using some stable nitroxides and some techniques which can be mentioned as ESR and EPR. There are some other techniques used for the detection of free radicals are GCMS, DMPO and spin trap. Recently LC/MS technique is also used to measure the nucleosides in the DNA. These free radicals are represented by superscript dot after the molecular and these are highly reactive molecules. It may involve in several diseases and oxidative stress also plays an important role in these diseases. Ageing is process is caused by involving free radicals. When the organism ages the chances of deaths will increase. The lifespan increases due to the early growth of life restriction food intake. RAS1 and RAS2 are the two genes which are used to prolong the life and expression of an over expression. Human disorders of premature ageing will cause by Hutchinson-Gilford and Werner’s syndrome. Free radical will also affect in cancer chemotherapy. Alkylating agents are used to provide chemotherapeutic agents and the other drugs are interfering with metabolic reactions in this cancer therapy some enzymes are used to inhibition and reduction. Anti-steroidal agents are used to treat cancers. Selection of spin trapping plays an important role. DMPO is having some disadvantages i.e. super oxide adducts quickly and spontaneously destroys the DMPO hydroxyl adducts. For detection of super oxide or hydroxyl radicals some other traps are developed. Unpaired electrons can be detected by ESR spin trapping method.

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Received on 10.10.2010 Modified on 20.11.2010

Research J. Pharm. and Tech. 4(4): April 2011; Page 499-501