Role of Wheat Germ Oil in Radiation-Induced Oxidative Stress and Alteration in Energy Metabolism in Rats

Transcription

1 Role of Whet Germ Oil in Rdition-Induced Oxidtive Stress nd Altertion in Energy Metbolism in Rts Thesis For the prtil fulfillment of the requirements for the Mster Degree in Biochemistry Presented by Shereen Mohmed Ebrhim Shedid B.Sc. in Biochemistry 2002 Helwn University Fculty of Science Chemistry Deprtment

2 Role of Whet Germ Oil in Rdition-Induced Oxidtive Stress nd Altertion in Energy Metbolism in Rts Presented by Shereen Mohmed Ebrhim Shedid B.Sc. in Biochemistry 2002 For the prtil fulfillment of the requirement for the degree of M.Sc. in Biochemistry Under Supervision of Prof. Dr. El-Syed M. El-Syed Mhdy Professor of Biochemistry, Fculty of Science, Helwn University Prof. Dr. Helen N. Sd Professor of Physiologicl Chemistry, Ntionl Center for Rdition Reserch nd Technology, Atomic Energy Authority, Ciro Prof. Dr. Ussm Z. Sid Professor of Physiologicl Chemistry, Ntionl Center for Rdition Reserch nd Technology, Atomic Energy Authority, Ciro Dr.Wf Ghoneim Shoush Assistnce Professor of Biochemistry Fculty of Science, Helwn University Helwn University Fculty of Science Chemistry Deprtment

3 0TABLE OF CONTENTS List of figures... List of tbles.... Acknowledgement... Abstrct.... CHAPTER 1 INTRODUCTION... AIM OF THE WORK.. I V VIII X 1 4 CHAPTER 2 2. REVIEW OF LITERATURE 2.1. Generl Concept on Rdition Biologicl Effect of Rdition 2.3. Energy Metbolism 2.4. Cellulr Oxidnts Endogenous sources of ROS Exogenous sources of ROS 2.5. Dmge Cused By Free Rdicls Antioxidnts Oxidtive stress Oxidtive Stress nd Rdition Physiologicl Defense ginst Oxidtive Stress Enzymtic ntioxidnt defenses Non-enzymtic defense Dietry (nutritionl) ntioxidnts Effect of Gmm Irrdition

4 Antioxidnts sttus Enzymes ctivities Minerls Protection Aginst Rective Oxygen Species Toxicity..... CHAPTER 3 3. MATERIALS AND METHODS 3.1. Experimentl Animls Rdition Fcility Preprtion of Whet Germ Oil Experimentl Design Preprtion of Smples Biochemicl Anlysis Determintion of superoxide dismutse ctivity Determintion of ctlse ctivity Determintion of reduced glutthione content Determintion of glutthione peroxidse ctivity Determintion of glucose-6-phosphte dehydrogense ctivity Determintion of cretine phosphokinse ctivitiy Determintion of lctte dehydrogense ctivity Determintion of glutmte dehydrogense ctivity Determintion of thiobrbituric cid rective substnces levels Determintion of xnthine oxidoreductse system ctivities

5 Determintion of minerls content Determintion of totl protein Sttisticl Anlysis CHAPTER 4 4. RESULTS 4.1. Role of whet germ oil (WGO) in the Xnthine Oxidoreductse System of Liver Tissue Liver xnthine oxidse Liver xnthine dehydrogense Role of Whet Germ Oil (WGO) in the Antioxidnt Sttus of Liver Tissue Liver superoxide dismutse Liver ctlse Liver glutthione peroxidse Liver glutthione Liver thiobrbituric cid rective substnces Role of Whet Germ Oil (WGO) in the Metbolic Enzymes of Liver Tissue Liver glucose-6-phosphte dehydrogense Liver glutmte dehydrogense Liver lctte dehydrogense Liver cretine phosphokinse Role of Whet Germ Oil (WGO) in Minerls Level of Liver Tissue Liver totl iron level Liver totl copper level Liver totl clcium level

6 4.5. Role of Whet Germ Oil (WGO) in the Xnthine Oxidoreductse System of Skeletl Muscle Tissue Muscle xnthine oxidse Muscle xnthine dehydrogense Role of Whet Germ Oil (WGO) in the Antioxidnt Sttus of Skeletl Muscle Tissue Muscle superoxide dismutse Muscle ctlse Muscle glutthione peroxidse Muscle glutthione Muscle thiobrbituric cid rective substnces 4.7. Role of Whet Germ oil (WGO) in the Metbolic Enzymes of Skeletl Muscle Tissue Muscle glucose-6-phosphte dehydrogense Muscle glutmte dehydrogense Muscle lctte dehydrogense Muscle cretine phosphokinse Role of Whet Germ oil (WGO) in Minerls Level of Skeletl Muscle Tissue Muscle totl iron level Muscle totl copper level Muscle totl clcium level CHAPTER 5 5. DISCUSSION 5.1. Rdition Effects on Pro-Oxidnts in Liver nd Skeletl Muscle tissues Rdition Effects on Antioxidnt Sttus in Liver 149 6

7 nd Skeletl Muscle Tissues 5.3. Rdition Effects on Some Metbolic Enzymes Activity of Liver nd Skeletl Muscle Tissues 5.4. Protective Effect of Whet Germ Oil... SUMMARY AND CONCLUSION REFERENCES. ARABIC SUMMARY. ARABIC ABSTRACT

8 LIST OF FIGURES Figure 1 Pthwys in the univlent reduction of oxygen to wter leding to genertion of vrious intermedite rective oxygen species 11 Figure 2 Endogenous sources of rective oxygen species 15 Figure 3 Pthwys illustrting the sources of rective oxygen species nd its role in the development of cncer.. 16 Figure 4 Antioxidnt/pro-oxidnt blnce in the cell Figure 5 Pthwys of rective oxygen species (ROS) production nd clernce.. 27 Figure 6 Liver xnthine oxidse ctivity (XO) (mu/mg protein) in different rt groups.. 62 Figure 7 Liver xnthine dehydrogense ctivity (XDH) (mu/mg protein) in different rt groups Figure 8 Liver superoxide dismutse ctivity (SOD) (U/g fresh tissue) in different rt groups Figure 9 Liver ctlse ctivity (U/g fresh tissue) in different rt groups Figure 10 Liver glutthione peroxidse ctivity (GSH-Px) (mg consumed glutthione/min/g fresh tissue) in different rt groups. 75 Figure 11 Liver glutthione content (mg/g fresh tissue) in different rt groups. 78 Figure 12 Liver thiobrbituric cid rective substnces levels (TBARS) (nmol/g fresh tissue) in different rt groups. 81 Figure 13 Liver glucose-6-phosphte dehydrogense ctivity (G-6-PDH) (U/g fresh tissue) in different rt groups. 84 8

9 Figure 14 Liver glutmte dehydrogense ctivity (GDH) (U/g fresh tissue) in different rt groups Figure 15 Liver lctte dehydrogense ctivity (LDH) (U/g fresh tissue) in different rt groups Figure 16 Liver cretine phosphokinse ctivity (CPK) (U/g fresh tissue) in different rt groups Figure 17 Liver totl iron level (Fe) (µg/g fresh tissue) in different rt groups.. 96 Figure 18 Liver totl copper level (Cu) (µg/g fresh tissue) in different rt groups. 99 Figure 19 Liver totl clcium level (C) (µg/g fresh tissue) in different rt groups. 102 Figure 20 Muscle xnthine oxidse ctivity (XO) (mu/mg protein) in different rt groups 106 Figure 21 Muscle xnthine dehydrogense ctivity (XDH) (mu/mg protein) in different rt groups Figure 22 Muscle superoxide dismutse ctivity (SOD) (U/g fresh tissue) in different rt groups. 113 Figure 23 Muscle ctlse ctivity (U/g fresh tissue) in different rt groups Figure 24 Muscle glutthione peroxidse ctivity (GSH-Px) (mg consumed glutthione/min/g fresh tissue) in different rt groups Figure 25 Muscle glutthione content (mg/g fresh tissue) in different rt groups. 122 Figure 26 Muscle thiobrbituric cid rective substnces levels (TBARS) (nmol/g fresh tissue) in different rt groups

10 Figure 27 Muscle glucose-6-phosphte dehydrogense ctivity (G-6-PDH) (U/g fresh tissue) in different rt groups Figure 28 Muscle glutmte dehydrogense ctivity (GDH) (U/g fresh tissue) in different rt groups Figure 29 Muscle lctte dehydrogense ctivity (LDH) (U/g fresh tissue) in different rt groups 135 Figure 30 Muscle Cretine phosphokinse ctivity (CPK) (U/g fresh tissue) in different rt groups 138 Figure 31 Muscle totl iron level (Fe) (µg/g fresh tissue) in different rt groups. 141 Figure 32 Muscle totl copper level (Cu) (µg/g fresh tissue) in different rt groups. 144 Figure 33 Muscle totl clcium level (C) (µg/g fresh tissue) in different rt groups

11 LIST OF TABLES Tble 1 Liver xnthine oxidse ctivity (mu/mg protein) in different rt groups Tble 2 Liver xnthine dehydrogense ctivity (mu/mg protein) in different rt groups.. 64 Tble 3 Liver superoxide dismutse ctivity (U/g fresh tissue) in different rt groups Tble 4 Liver ctlse ctivity (U/g fresh tissue) in 71 different rt groups... Tble 5 Liver glutthione peroxidse ctivity (mg consumed glutthione/min/g fresh tissue) in different rt groups 74 Tble 6 Liver glutthione content (mg/g fresh tissue) in different rt groups.. 77 Tble 7 Liver thiobrbituric cid rective substnces levels (TBARS) (nm/g fresh tissue) in different rt groups. 80 Tble 8 Liver glucose-6-phosphte dehydrogense ctivity (U/g fresh tissue) in different rt groups 83 Tble 9 Liver glutmte dehydrogense ctivity (U/g fresh tissue) in different rt groups 86 Tble 10 Liver lctte dehydrogense ctivity (U/g fresh tissue) in different rt groups 89 Tble 11 Liver cretine phosphokinse ctivity (U/g fresh tissue) in different rt groups 92 Tble 12 Liver totl iron level (µg/g fresh tissue) in different rt groups Tble 13 Liver totl copper level (µg/g fresh tissue) in 11

12 different rt groups 98 Tble 14 Liver totl clcium level (µg/g fresh tissue) in different rt groups Tble 15 Muscle xnthine oxidse ctivity (mu/mg protein) in different rt groups Tble 16 Muscle xnthine dehydrogense ctivity (mu/mg protein) in different rt groups 108 Tble 17 Muscle superoxide dismutse ctivity (U/g fresh tissue) in different rt groups 112 Tble 18 Muscle ctlse ctivity (U/g fresh tissue) in different rt groups 115 Tble 19 Muscle glutthione peroxidse ctivity (mg consumed glutthione/min/g fresh tissue) in different rt groups 118 Tble 20 Muscle glutthione content (mg/g fresh tissue) in different rt groups 121 Tble 21 Muscle thiobrbituric cid rective substnces levels (nm/g fresh tissue) in different rt groups 124 Tble 22 Muscle glucose-6-phosphte dehydrogense ctivity (U/g fresh tissue) in different rt groups 128 Tble 23 Muscle glutmte dehydrogense ctivity (U/g fresh tissue) in different rt groups Tble 24 Muscle lctte dehydrogense ctivity (U/g fresh tissue) in different rt groups Tble 25 Muscle cretine phosphokinse ctivity (U/g fresh tissue) in different rt groups

13 Tble 26 Muscle totl iron level (µg/g fresh tissue) in different rt groups 140 Tble 27 Muscle totl copper level (µg/g fresh tissue) in different rt groups. 143 Tble 28 Muscle totl clcium level (µg/g fresh tissue) in different rt groups

14 ACKNOWLEDGEMENT I would like to express my thnks nd grtitude to Prof. Dr. El Syed M. El-Syed Mhdy, professor of Biochemistry, Fculty of Science, Helwn University for sponsoring the thesis, for his kind encourgement nd vluble dvices throughout the stges of the study. My deep thnks nd grtitude to Prof. Dr. Helen N. Sd, professor of Physiologicl Chemistry, Ntionl Center for Rdition Reserch nd Technology Atomic Energy Authority, for suggesting the subject, for her vluble criticism, scientific help nd kind encourgement through ll the stges of this work. I would like to express my deep grtefulness nd pprecition to Prof. Dr. Ussm Z. Sid, Professor of Physiologicl Chemistry, Ntionl Center for Rdition Reserch nd Technology Atomic Energy Authority, for suggesting the plne nd providing ll the fcilities for the ccomplishment of this work s well s his continuous vluble guidnce nd helpful discussion throughout the stges of the study nd lso for hndwriting this mnuscript. Thnks nd high pprecition lso to Dr. Wf Ghoneim Shoush, Assistnce Prof. of Biochemistry, Fculty of Science, Helwn University for her kind supervision, support nd encourgement throughout the stges of the study. My gret thnk to my collegues in the Rdition Biology Deprtment (NCRRT), prticulrly to my collegues in the unit of physiologicl chemistry, for their vluble ssistnces in completing this work. 14

15 ABSTRACT The liver is essentil in keeping the body functioning properly while musculr strength is importnt in sport s well s in dily ctivities. Exposure to ionizing rdition is thought to increse oxidtive stress nd dmge liver nd muscle tissues. Whet germ oil is nturl unrefined vegetble oil. It is n excellent source of vitmin E, octcosnol, linoleic nd linolenic essentil ftty cids, which my be beneficil in neutrlizing the free oxygen rdicls. This study ws designed to investigte the efficcy of whet germ oil, on rditioninduced oxidtive dmge in rt s liver nd skeletl muscle. Whet germ oil ws supplemented orlly vi gvge to rts t dose of 54 mg/ kg body weight for 14 successive dys pre- nd 7 dys post-exposure to 5 Gy (single dose) of whole body γ- irrdition. Animls were scrificed 7, 14 nd 21 dys post rdition exposure. The results reveled tht whole body γ-irrdition of rts induces oxidtive stress in liver nd skeletl muscles obvious by significnt elevtion in the levels of xnthine oxidse nd thiobrbituric cid rective substnces (TBARS) ssocited with significnt decreses in the content of reduced glutthione, s well s decreses in xnthine dehydrogense, superoxide dismutse, ctlse nd glutthione peroxidse ctivities. Irrdited rts showed lso significnt decreses in cretine phosphokinse, glutmte dehydrogense nd glucose-6- phosphte dehydrogense ctivities while lctte dehydrogense were significntly incresed. Totl iron, totl copper nd totl clcium levels significntly incresed in the liver nd skeletl muscles of irrdited rts group compred to control group. Whet germ oil treted-irrdited rts 15

16 showed significntly less severe dmge nd remrkble improvement in ll the mesured prmeters, compred to irrdited rts. It could be concluded tht whet germ oil by ttenuting rdition-induced oxidtive stress might ply role in mintining liver nd skeletl muscle integrity. 16

17 1- INTRODUCTION Ionizing rdition hs proven to be double-edged sword since its discovery by Dr Roentgen in Rdition is potent mutgen nd crcinogen; however, it is lso used in the dignosis nd tretment of humn diseses. All orgnisms (e.g., bcteri, plnts, or nimls, including humns) re exposed everydy to vrying mounts of ionizing rdition tht interct with molecules, cells, nd tissues, cusing reversible devitions from homeosttic equilibrium or irreversible dmge. Mny spects of ging nd mny diseses re thought to stem from exogenous nd endogenous deleterious gents cting on key components of cells within the body (Anonymous, 1990). The lives of most orgnisms re dependent on brekdown of denosine triphosphte (ATP) to produce energy which is used to power ll body function. Genertion of free rdicls or rective oxygen species (ROS) during metbolism or s result of exposure to gmm rdition beyond the ntioxidnt cpcity of biologicl system gives rise to oxidtive stress (Mikulikov nd Popes, 2001), which leds to decrese in mitochondril ATP synthesis, cusing liver nd muscle dysfunction nd cell deth (Grce et l., 2006). Oxidtive stress plys role in hert diseses, neurodegenertive diseses, cncer nd in the ging process (Sin, 2003). ROS re tightly controlled by ntioxidnt defense systems, including non-enzymtic rdicl scvengers s reduced glutthione nd enzymes tht cn either directly detoxify ROS or indirectly regulte their levels. The mjor enzymtic ntioxidnts re superoxide dismutse, ctlse, nd the glutthione redox system (Meister, 1992). Dietry ntioxidnts oppose the oxidtive dmge nd lower risk of 17

18 degenertive disese (Alslvr et l., 2005) nd thus rises necessity to extrct these ntioxidnts from the plnt mtrices. These plnt-bsed dietry ntioxidnts re believed to hve n importnt role in the mintennce of humn helth becuse the endogenous ntioxidnts provide insufficient protection ginst the constnt nd unvoidble chllenge of rective oxygen species (Fridovich, 1998). Whet germ is high in fiber nd diet high in fiber cn be useful in the regultion of bowel function (i.e. reducing constiption) nd my be recommended for ptients t risk for dibetes, colon nd hert diseses (Mhn nd Stump, 2000). Whet germ oil ws climed ntiinflmmtory nd described s suitble nturl ntioxidnt due to its high content of vitmin E (Prnich et l., 2000). Whet germ oil contins lso octcosnol, long chin ftty lcohol, reported to be helpful in cholesterol mngement (Singh et l., 2006). The oil ws reported lso to be vluble source of essentil ftty cids, including linoleic cid nd linolenic cid, which insufficiency ws observed to cuse tiredness, dry skin, immune insufficiency, norexi, digestion nd crdiovsculr disorders (Mohmed et l., 2005). 18

19 AIM OF THE WORK The present study ws designed to evlute nd to elucidte the reltionship between rdition-induced oxidtive stress in liver nd skeletl muscle tissues of lbino rts nd chnges in some metbolic enzymtic ctivities fter totl body exposure to gmm rdition t 5 Gy pplied s one shot dose nd to evlute the role of whet germ oil ginst rdition-induced disturbnces in energy metbolism. This gol hs been chieved by: 1- Determintion of the rdition-induced oxidtive stress in the liver nd skeletl muscle by mesuring the vritions in the xnthine oxidoreductse system, reduced glutthione level nd the ctivity of the ntioxidnt enzymes (superoxide dismutse, ctlse, glutthione peroxidse) nd chnges in the level of thiobrbituric cid-rective substnces; mrker of lipid peroxidtion. 2- Determintion of the rdition-induced chnges in some metbolic enzymtic ctivities in the liver nd skeletl muscle by mesuring the chnges in the ctivity of lctte dehydrogense, cretine phosphokinse, glucose-6-phosphte dehydrogense nd glutmte dehydrogense. 3- Determintion of rdition-induced chnges in totl iron, totl copper nd totl clcium levels in the liver nd skeletl muscle. 4- Evlution of the protective role of whet germ oil ginst rdition hzrds by compring results obtined for irrdited rts with those obtined for whet germ oil -treted irrdited rts. 19

20 2- REVIEW OF LITERATURE All orgnisms (i.e., bcteri, plnts, or nimls, including humns) re exposed ech dy to some mount of rdition nd 81% of the dose received from rdition comes from nturl sources: 55% from rdon; 8% from cosmic rdition; 8% from rocks nd soil; nd 10% from internl exposures to rdition from the rdioctive mterils in food nd wter consumed in the dily diet. The remining 19% of the dily dose my originte from mn-mde sources: medicl x ry exposure (11%), nucler medicinl exposure (4%), consumer products (3%) nd other sources (<1%). This lst ctegory includes occuptionl sources, nucler fllout, the nucler fuel cycle rdioctive wste, hospitl rdioctive wste, rdioctively contminted sites nd other miscellneous sources (NCRP 1987) Generl Concept on Rdition Rdition is defined s energy in trnsit nd comprises electromgnetic rys; such s X-rys or gmm rys nd prticulte rdition such s neutrons, lph prticles, nd hevily chrged ions. Rdition ffects people by depositing energy in body tissues. The extent of the dmge depends upon the totl mount of energy bsorbed, the time period nd dose rte of exposure nd the prticulr orgn(s) exposed (Yrmonenko, 1988). Rdition my be non-ionizing or ionizing ccording to energy. Non ionizing rdition hs enough energy to excite molecules nd toms cusing them to vibrte fster, which is obvious in microwve oven where the rdition cuses wter molecules to vibrte 20

21 fster creting het. Rdio-wves, infrred nd visible light re ll exmples of non ionizing rdition. Ionizing rdition hs more energy thn non-ionizing rdition; enough to cuse chemicl chnges by breking chemicl bonds. This effect cn cuse dmge to living tissues. The ionizing rditions of primry concern re lph nd bet prticles, gmm nd x rys (Yrmonenko, 1988). Ionizing rdition triggers the formtion of free rdicls, which interct mong themselves nd with criticl biologicl trgets with the formtion of plethor of newer free rdicls. It is generlly believed tht production of these free rdicls is the min mechnism through which rdition induces biologicl dmge t lower rdition doses (Weiss nd Kumr, 1988) Biologicl Effect of Rdition Rdition cn dmge every tissue in the body. The fstest growing tissues re the most vulnerble; becuse rdition s much s triples its effects during the growth phse. Ioniztion of living tissue cuses molecules in the cells to be broken prt. This interction cn kill the cell or cuse them to reproduce bnormlly. Dmge to cell cn come from direct ction or indirect ction of the rdition. 1- Direct ction occurs when the rdition intercts directly with cell's essentil molecules. The rdition energy my dmge cell components such s the cell wlls or the deoxyribonucleic cid (DNA). When rdition intercts with cell wll or DNA, the cell either dies or becomes different kind of cell, possibly even cncerous one. 21

22 2- Indirect ction occurs when rdition intercts with wter molecules, which re roughly 80% of cells composition. The energy bsorbed by the wter molecule cn result in the formtion of free rdicls. Free rdicls re molecules tht re highly rective due to the presence of unpired electrons, which result when wter molecules re split. Free rdicls my form compounds, such s hydrogen peroxide, which my initite hrmful chemicl rections within the cells. As result of these chemicl chnges, cells my undergo vriety of structurl chnges which led to ltered function or cell deth (Weiss nd Kumr, 1988). Depending on the dose rte nd dose of exposure, the effects of rdition rnge from nuse nd vomiting to immune system compromise to deth from either rdition-induced tissue dmge or infection. Potentil sequele of rdition exposure include ctrct formtion, decresed fertility nd fetl tertogenesis. Although it is believed tht the effects of exposure to moderte doses (1 8 Gy) of γ- rdition result in hemtopoietic dysfunction, the net injury is result of dynmic process involving cell killing, ltered cell-to-cell communiction, inflmmtory responses, compenstory tissue hypertrophy nd repir (Stone et l., 2003). Higher rdition doses compound these effects with gstrointestinl nd neurovsculr tissue dmge (Colemn et l., 2004) Energy Metbolism The humn body's min power comes from the mitochondrion which exists in every cell in the body. Energy metbolism consists of series of chemicl rections tht brek down foodstuffs nd thereby produce energy. The energy-rich chemicl compound tht provides virtully ll the energy needed by the body is known s denosine 22

23 triphosphte, or simply ATP. The mitochondri re the production centers for ATP. The energy relesed from the brekdown of ATP is used to power ll body functions. The ATP is used for mny cell functions including trnsport work, moving substnces cross cell membrnes. It is lso used for mechnicl work, supplying the energy needed for muscle contrction. It supplies energy not only to hert muscle (for blood circultion) nd skeletl muscle (such s for gross body movement), but lso to the chromosomes nd flgell to enble them to crry out their mny functions. A mjor role of ATP is in chemicl work, supplying the needed energy to synthesize the multi-thousnds of types of mcromolecules tht the cell needs to exist (Hickmn et l., 1997) Cellulr Oxidnts Cellulr oxidnts, derivtives of oxygen, which re often clled rective oxygen species (ROS), re constntly produced in the cells. - Among cellulr ROS, the most ggressive entities re superoxides (O 2 ) nd hydroxyl rdicls ( OH). Superoxides, through the rection ctlyzed by superoxide dismutse (SOD) re trnsformed into the much less rective hydrogen peroxide moiety (H 2 O 2 ). However, when H 2 O 2 intercts with ions of trnsition metls such s iron or copper, the most rective ROS, hydroxyl rdicls ( OH) re formed (Fenton rection) (Hlliwell nd Gutteridge, 2006). (Fig.1) Besides ROS, cells lso generte rective nitrogen species (RNS) such s nitric oxide (NO ), nitrogen dioxide (NO 2 ) nd peroxynitrite (ONOO ). Nitric oxide nd nitrogen dioxide crry out number of physiologicl functions but excessive NO, NO 2 nd ONOO dmge cell constituents (Moncd et l., 1991). (Fig.1) 23

24 Fig.1: Pthwys in the univlent reduction of oxygen to wter leding to genertion of vrious intermedite rective oxygen species. ROS ply crucil role in few lifesving biologicl mechnisms. Phgocytic cells protect the body from dedly microorgnisms, killing them by producing n vlnche of ROS. When neutrophils nd other phgocytic cells engulf bcteri, they gretly increse consumption of oxygen ("respirtory burst"), which is rpidly trnsformed to ROS tht kill the dngerous intruders. Importntly, by burst of ROS, phgocytes kill not only invding bcteri, but lso cncer cells (Hlliwell nd Gutteridge, 2006). ROS re generted from mny exogenous nd endogenous sources in biologicl rections Endogenous sources of ROS ROS re constntly generted in living cells nd re usully beneficil to the metbolic processes. Mjor sites of endogenous genertion of ROS in the biologicl system re within the mitochondri, microsomes, endoplsmic reticulum, phgocytic cells, endothelil cells, nd nuclei. Sources of free rdicls re the mitochondril electron trnsport chin, the enzymes such s xnthine oxidse, NADPH oxidse, lipoxygense/cyclooxygense nd nitric oxide synthse (NOS) nd utooxidtion of vrious substnces prticulrly ctecholmines (Fig. 2). 24

25 Mitochondri Among the vrious orgnelles in the cell, mitochondri re the mjor source of intrcellulr free rdicls. Mitochondri, the intrcellulr powerhouses which produce the universl energy molecule, ATP, normlly consume O 2 in this process nd convert it to H 2 O 2. Approximtely 2-5% of O 2 used for erobic metbolism in the - mitochondri re converted to ROS such s O 2, H2 O 2 nd OH due to incomplete reduction of the oxygen. It hs been estimted tht more thn 20 billion molecules of oxidnts per dy re produced by ech cell during norml metbolism (Nishiym et l., 1997). Xnthine oxidse Xnthine oxidse is molybdenum nd iron contining hydroxylting enzyme involved in the degrdtion of purine like nucleotides. This enzyme ctlyzes the rection of hypoxnthine to - xnthine forming O 2 nd of xnthine to uric cid forming H 2 O 2 (Leeuwenburgh nd Heinecke, 2001). Peroxisomes Peroxisomes produce hydrogen peroxide s by-product of the degrdtion of ftty cids nd other molecules. In contrst to the mitochondri which oxidize ftty cids to produce ATP nd wter, peroxisomes oxidize ftty cids to produce het nd hydrogen peroxide (Ames et l., 1993). Phgocyte cells Other sources of ROS in the body re phgocyte cells, which help defend the body ginst invding microorgnisms. Destroying of bcteri nd virus infected cells by phgocyte cells releses vriety of ROS, including superoxide nions, nitric oxide nd hydrogen peroxide. 25

26 ROS generted from this source ply centrl role in body's defense (Ji, 1996). Trnsition metls Trnsition metls, such s iron nd copper, ply mjor role in the genertion of free rdicls injury nd the fcilittion of lipid peroxidtion. In myocytes, O 2 is converted to H 2 O 2 in the presence of superoxide dismutse found in the mitochondrion nd in the cytosol. H 2 O 2, non-rdicl, but freely permeble nd highly ROS, is converted to OH in the presence of copper or iron (the Fenton rection). Alterntively H 2 O 2 rects with O 2 to form OH in the Hber- Weiss rection. Glutthione peroxidse ctlyzes the conversion of H 2 O 2 to nonrdicl wter nd oxygen (Fig. 2). The Hber-Weiss rection ccelertes the non-enzymtic oxidtion of molecules such s epinephrine nd glutthione tht genertes O 2 nd H 2 O 2 nd subsequently OH (Hlliwell nd Gutteridge, 2006). Endogenous levels of ROS, which endnger our helth, increse during chronic infection nd inflmmtion, strenuous physicl exercise, hyper-metbolic sttes seen in stress, trum nd sepsis nd during exposure to exogenous sources. Fig.2: Endogenous sources of rective oxygen species 26

27 Exogenous sources of ROS Exogenous sources of ROS include the following: tobcco smoke, which hs brod spectrum of oxidnt, ionizing rdition, which genertes free rdicls in exposed tissues, notbly the highly rective OH rdicl; UV light, which produces singlet oxygen ( 1 O 2 ) nd OH, ozone (O 3 ) nd oxides of nitrogen in polluted ir; industril toxins such s crbon-tetrchloride; drugs such s phenobrbitl, which is known tumor promoter in liver; nd chrcol-broiled foods, which form vriety of crcinogens, notbly benzo()pyrene. Fig. 3: Pthwys illustrting the sources of rective oxygen species nd its role in the development of cncer. The combintion of oxidtive dmge by exogenously nd endogenously produced free rdicls hs ominous consequences for body tissues. The oxidnts induce ltertions in the structures of tissues nd in their functions which mnifest s ging nd chronic degenertive diseses like rthritis, therosclerosis nd cncer (Fig.3) (Ames et l., 1993) Dmge Cused by Free Rdicls Free rdicls cn be beneficil but hrmful if uncontrolled. ROS ffects cellulr clcium metbolism. Uncontrolled rises in intrcellulr 27

28 free clcium cn result in cell injury or deth. Excessive free rdicl formtion cn dmge ll cellulr mcromolecules including proteins, lipids nd nucleic cids. 1- Proteins perform numerous crucil functions in the cell, primrily in the form of enzymes tht medite most biochemicl rections required for cellulr functions. Proteins re mde up of pproximtely 20 different building blocks clled mino cids, which differ in their sensitivity to interctions with ROS. Alterntively, the ROS induced oxidtion of proteins cn led to chnges in the proteins three dimensionl structure s well s to frgmenttion, ggregtion, or cross linking of the proteins. The destructive effects on proteins my ply role in the custion of ctrcts (Bgchi nd Puri, 1998). 2- Lipids tht contin phosphte groups (i.e., phospholipids) re essentil components of the membrnes tht surround the cells s well s other cellulr structures, such s the nucleus nd mitochondri. Consequently, dmge to the phospholipids will compromise the vibility of the cells. The complete degrdtion (i.e., peroxidtion) of lipids is hllmrk of oxidtive dmge. The polyunsturted ftty cids present in the membrnes phospholipids re prticulrly sensitive to ttck by OH nd other oxidnts. In ddition to dmging cells by destroying membrnes, lipid peroxidtion cn result in the formtion of rective products tht themselves cn rect with nd dmge proteins nd DNA (Dhll et l., 2000). 3- DNA is the cell s genetic mteril nd ny permnent dmge to the DNA cn result in chnges (i.e., muttions) in the proteins encoded in the DNA, which my led to mlfunctions or complete inctivtion of the ffected proteins. ROS re mjor source of DNA dmge, cusing strnd breks, removl of nucleotides, nd vriety of 28

29 modifictions of the orgnic bses of the nucleotides. Oxidtive dmge to cellulr DNA cn led to muttions nd my, therefore, ply n importnt role in the initition nd progression of multistge crcinogenesis (Gulm nd Hseeb, 2006). Severe oxidtive stress with lipid peroxidtion, protein oxidtion, DNA dmge nd ATP depletion leds to cell deth by necrosis, which is chrcterized by disruption of the cell membrne nd cellulr orgnelles (Kroemer et l., 1998) Antioxidnts Biologicl orgnisms hve evolved defense mechnisms ginst free rdicls tht re known collectively s ntioxidnts. An ntioxidnt is substnce tht prevents oxidtion. In biologicl systems ntioxidnts cn work in vrious wys, including ctlytic removl of free rdicls, s scvengers of free rdicls or in the form of proteins tht minimize the vilbility of pro-oxidnts such s metl ions (Hlliwell, 1996). Progress in understnding the deleterious effects of ROS on cell components nd structures hs led to the development of protective ntioxidnt supplements. The supplements re used to protect cell structures from oxidtive dmge, cncer nd other ROS-dependent morbid conditions 2.7. Oxidtive Stress Oxidtive stress is the inpproprite exposure to ROS nd results from the imblnce between prooxidnts nd ntioxidnts leding to cell dmge (dmge of lipids, proteins, crbohydrtes nd nucleic cids) nd tissue injury (Sies, 1997); this imblnce my be due to n excess of prooxidnt gents, deficiency of ntioxidnt gents or both fctors simultneously (Fig. 4). 29

30 High oxidtive stress is common in orgns nd tissues with high metbolic nd energy demnds, including skeletl nd hert muscle, liver, lymphtic orgns, nd blood cells (Eli et l., 2004). In liver excess of ROS cn induce heptocyte cell deth by either poptosis or necrosis leding to liver injury nd loss of liver function. Fig. 4: Antioxidnt/pro-oxidnt blnce in the cell Contrcting skeletl muscle genertes n incresed mount of rective oxygen nd nitrogen species (Mlcolm, 2005). Further studies hve demonstrted tht muscles relese incresed mounts of superoxide nion (McArdle et l., 2001) nd nitric oxide (Blon nd Ndler, 1994) into the extr-cellulr fluid nd tht hydroxyl rdicls re formed from hydrogen peroxide (H 2 O 2 ) lso relesed from the muscle cells (McArdle et l., 2004). A lrge number of studies hve reported tht mssive formtion of RNS nd/or ROS is closely ssocited with incresed poptotic processes in the myocytes of nimls nd humns. In such multinucleted cells, this my led to trophy nd reduced muscle strength (Reid et l., 1993). 30

31 2.8. Oxidtive stress nd rdition Rdition injury to living cells is, to lrge extent, due to oxidtive stress. Evidence suggests tht cell s oxidtive stte not only plys role t the time of rdition exposure, but lso hs effects long fter exposure. As the result of irrdition, cells cn produce ROS for severl minutes or even hours fter being exposed. In ddition to ROS production, cells re stimulted to increse their expression of ntioxidnts (Spitz et l., 2004) Physiologicl Defense ginst Oxidtive Stress To protect molecules ginst toxic free rdicls nd other ROS, cells hve developed ntioxidnt defenses. Efficient defense nd repir mechnisms exist in living cells to protect ginst oxidnt species. The ntioxidtive defense system is composed of methods to (i) trnsfer sensitive mteril to comprtments better protected from the ction of rective species, (ii) complex trnsition metls, potentil source of electrons, thereby rendering them uncretive, (iii) inhibit vulnerble processes such s DNA repliction, (iv) repir dmged molecules, (v) initite poptosis (vi) ctivte ntioxidnt enzymes nd finlly (vii) use vriety of direct free rdicl scvengers (Hlliwell nd Gutteridge, 2006). Antioxidnts re substnces tht either directly or indirectly protect cells ginst the dverse effects of xenobiotics, toxicnts, drugs, nd crcinogens. Antioxidnts cn be endogenous (produced by the body) or exogenous (obtined through the diet). Antioxidnt defenses cn be clssified into three ctegories enzymtic, non-enzymtic nd dietry (nutritionl) ntioxidnts. 31

32 1. Enzymtic ntioxidnt defenses tht specificlly metbolize ROS precursors, such s ctlse, superoxide dismutse nd glutthione peroxidse. 2. Non-enzymtic systems (intrinsic molecules such s glutthione, lbumin, bilirubin nd uric cid). 3. Dietry (nutritionl) ntioxidnts re low moleculr weight compounds ingested in the diet such s vitmin C, vitmin E, the crotenoids, flvonoids, other plnt phenolics nd wine phenolics. Antioxidnts cn lso be mnufctured syntheticlly. These belong to the clss of synthetic ntioxidnts. The min disdvntge with these ntioxidnts is their side effect when tken in vivo (Chen et l., 1992). Most of the nturl ntioxidnts re found to hve higher ntioxidnt ctivity when compred with tht of the synthetic ones. Severl rguments suggest tht the ntioxidnt components of fruits nd vegetbles contribute in the defense effect. Epidemiologicl studies nd intervention trils on prevention of diseses such s cncer nd crdiovsculr disese in people hve shown the positive effects of tking ntioxidnt supplements (Ames et l., 1993; Rimm et l., 1993) Enzymtic ntioxidnt defenses Superoxide dismutse (SOD) is metlloenzyme, mening tht in ddition to mino cids, it contins metl ions. Superoxide dismutse helps the body use zinc, copper, nd mngnese. SOD plys different role in keeping cells helthy nd protects the cells cytoplsm, nd their mitochondri from free rdicl dmge. Abnormlities in the coppernd zinc-dependent superoxide dismutse gene my cuse deteriortion 32

33 of motor nerve cells in the brin nd spinl cord (Cluskey nd Rmsden, 2001). The superoxide rdicl (O - 2 ) is formed in the mitochondri s byproduct of electron trnsport or by ionizing rdition (Tortor et l., 1995). It is very dngerous to the cell becuse they stel electrons from neighboring molecules, strting cscde of electron steling, the end result being dmging of the cell. The superoxide dismutse enzyme is the first step tht cells use to stop this process. SOD converts superoxide rdicls to less toxic ROS, hydrogen peroxide (H 2 O 2 ), s follows: 2 O H SOD H 2 O 2 + O 2 The hydrogen peroxide molecule, which is still dnger to cells, is then further processed to nontoxic by-products. Ctlse, n iron-contining enzyme is primry found in peroxisomes except in cells like erythrocytes tht do not contin these orgnelles. In n queous environment, the enzyme ctlse degrdes the hydrogen peroxide s follows: 2 H 2 O Ctlse H 2 O + O 2 The oxygen gs creted in this rection, ccounts for the bubbles seen when hydrogen peroxide is poured on skin injury. Mny bcteri do not hve ctlse, nd the cells re destroyed, which is why hydrogen peroxide is effective ginst bcteri. Glutthione peroxidse is cytoplsmic nd mitochondril enzyme tht is importnt for detoxifying H 2 O 2 in most cells. The body uses glutthione peroxidse to degrde the H 2 O 2 s follows: H 2 O 2 + 2H glutthione peroxidse -- 2H 2 O 33

34 Glutthione peroxidse system consists of severl components including the enzymes glutthione peroxidse nd glutthione reductse nd the cofctors glutthione nd NADPH (Fig. 5). Lipid membrnes re very susceptible to dmge by free rdicls, especilly peroxide rdicls. Glutthione peroxidse prevents destruction of cell membrnes by removing severl clsses of these lipid peroxides. Incresing fruit nd vegetble intke cn improve production of GSH-Px (Drgsted et l., 2004) Non-enzymtic defense The non-enzymtic ntioxidnt defense system includes compounds of intrinsic ntioxidnt properties, such s glutthione (GSH), β-crotene, nd vitmin A. Vitmin E is lipophilic, nd its mjor role is to block the chin rection of lipid peroxidtion (Sies et l., 1992). Pre-tretment with vitmin E hs been shown in rbbits to prevent lung injury cused by hyperoxi (Jcobson et l., 1990). Ascorbic cid (vitmin C) nd α-tocopherol cooperte in cellulr defense ginst ROS. There is blnce between both the ctivities nd intrcellulr levels of these ntioxidnts tht re essentil for the survivl of orgnisms nd their helth. Glutthione is the bse mteril for severl other key ntioxidnt enzyme systems including glutthione-peroxidse, glutthionereductse, nd glutthione-trnsferse. Reduced glutthione (GSH) protects cellulr constituents from the dmging effects of peroxides formed in metbolism nd through other ROS rections. Decresed tissue GSH levels re ssocited with cell dmge, depressed immunity nd the progression of ging, nd my increse the risk of cncer development. In ddition to being essentil in the 34

35 glutthione redox cycle, glutthione cn scvenge ROS nonenzymticlly (Freemn nd Crpo, 1981), s well s detoxify xenobiotics vi the glutthione S-trnsferse rection. Glutthione protects thiol groups of proteins from oxidtion nd mintins vitmin- C levels (Meister, 1992). Fig. 5. Pthwys of rective oxygen species (ROS) production nd clernce.gsh, glutthione; GSSG, glutthione disulfide Dietry (nutritionl) ntioxidnts Dietry (nutritionl) ntioxidnts re low moleculr weight compounds ingested in the diet such s vitmin C, vitmin E, the crotenoids, flvonoids nd other plnt phenolics. Mny epidemiologicl studies hve shown link between diet nd/or nutrition nd the development of disese. Studies hve shown tht high consumption of fruits nd vegetbles hve been ssocited with low risk of cncer, crdiovsculr disese, nd other chronic diseses Effect of Gmm Irrdition The liver is the lrgest internl orgn in the humn body, nd is n orgn present in vertebrtes nd some other nimls. It plys mjor 35

36 role in metbolism nd hs number of functions in the body including glycogen storge, decomposition of red blood cells, plsm protein synthesis nd detoxifiction. Skeletl muscle my be subjected to greter level of oxidtive stress during exercise thn liver nd hert due to incresed ROS production. Therefore, the muscle needs greter ntioxidnt protection ginst potentil oxidtive dmge occurring during nd/or fter stress (Ji, 1995). Among the sub-cellulr orgnelles, mitochondri form some of the key components of cell killing induced by oxidtive stress or rdition (Slyshenkov et l., 1996). During oxidtive stress, the flow of oxygen through muscle cells is gretly incresed t high levels of oxygen uptke. At the sme time, the rte of denosine triphosphte (ATP) utiliztion exceeds the rte of ATP genertion tht cn led to free rdicl genertion nd hs been implicted in ftigue, muscle soreness, myofibril disruption, nd impirment of immune function (Yu, 1994). Gmm rdition lters the metbolism of the most common type of cell found in the liver, the heptocyte. Under norml circumstnces the blood supplies enough oxygen to the liver, but if heptocytes use up more oxygen, oxygen deficits (i.e., hypoxi) cn develop in some liver res. Hypoxi, in turn, my impede the liver cells bility to produce n energy rich molecule ATP, which is generted during the brekdown of nutrients nd supplies energy needed for numerous biochemicl rections. Sufficiently high levels of ATP re essentil to the survivl of ll cells; reduced ATP levels in the liver re one fctor contributing to liver cell deth nd my contribute to development of liver cirrhosis (Crol et l., 2003). 36

37 Antioxidnts sttus The results of extensive studies showed tht exposure to ionizing rdition results in significnt ltertions of ntioxidnt enzymes which depends upon the dose of rdition exposure, the time of biochemicl nlysis nd the tissue nlyzed. Rdition exposure results in chnges in ntioxidnt enzyme levels: most consistently reported is elevtion in SOD (Weiss nd Lnduer, 2000). Irrdition cused incresed lipid peroxide nd decresed GSH levels in the intestine. Intestinl superoxide dismutse nd glutthione peroxidse ctivities were incresed, but glutthione trnsferse ctivity decresed following irrdition (Umit et l., 2000). Lipid peroxidtion is believed to be n importnt cuse of destruction nd dmge to cell membrnes nd hs been shown to be contributing fctor to the development of oxygen rdicls-medited tissue dmge. Commonly mesured prmeter of lipid dmge fter ionizing rdition exposure is thiobrbituric cid rective substnces (TBARS) (Smuni et l., 1997). Plnivel et l., (1998) reported tht rdition represents stte of incresed oxidtive stress, which is minly bsed on the evidence of incresed lipid peroxidtion, or by indirect evidence of reduced ntioxidnt reserve, like SOD nd CAT, in niml models. Sd et l., (1999) reported tht whole body gmm irrdition of rts with frctionted dose of 8 Gy (4 x 2 Gy weekly) produces significnt increse of blood GSH content, GSH-Px nd GSH-reductse ctivities 1hr nd 1dy post-irrdition followed by significnt decrese on the 3 rd dy. (Sd et l., 2003) found tht gmm 37

38 irrdition of rts with 7 Gy induces significnt decrese in liver nd kidney SOD nd CAT ctivities 3, 7, 10 dys post-irrdition. On the contrry lung SOD nd CAT ctivities were incresed. TBARS levels showed significnt increses in ll the tissues. Koc et l., (2003) reported tht totl body irrdition with single dose of 6 Gy resulted in significnt increse in the liver TBARS levels nd decrese of SOD nd GSH-Px ctivities. Klpn nd Menon, (2004) reported tht in irrdited rts the ctivities of SOD, CAT nd GSH-Px were decresed in the blood nd liver tissues. Sid, (2004) showed tht 5 Gy whole body gmm irrdition results in significnt decreses of brin SOD nd GSH-Px ctivities ssocited with n increse in TBARS content 1, 7 nd 14 dys post-irrdition. Also, Sid et l., (2004) showed tht whole body gmm irrdition of rts with 6 Gy (single dose) resulted in significnt increse in crdic TBARS long with reduction in crdic SOD, CAT nd GSH-Px ctivities 1, 2 nd 4 weeks following rdition exposure. Sid et l., (2005) reported tht whole body gmm irrdition of rts with 6 Gy produces n increse in liver nd plsm TBARS nd decrese in GSH content 1 nd 2 weeks post-irrdition. Hldun et l., (2006) found tht Lipid peroxidtion level ws incresed nd the SOD, CAT nd GSH-PX ctivities were decresed in the brin of irrdited rts. Sid nd Azb, (2006) reported tht 7 Gy (single dose) of whole body gmm irrdition induced significnt elevtion in TBARS levels with decrese in reduced glutthione content in hert tissues. Furthermore, gmm rdition t different doses (1, 2 nd 4Gy) ws found to significntly increse TBARS levels wheres the level of GSH 38

39 nd the ctivities of SOD, CAT nd GSH-Px in lymphocytes were significntly decresed. The mximum dmge to lymphocytes ws observed t 4Gy irrdition (Srinivsn et l., 2006). El-Missiry et l., (2007) reported tht in irrdited rts subjected to two doses of 2 nd 4Gy from Cesium-137 source, the TBARS, protein crbonyl contents nd CAT ctivity were significntly incresed in the liver, 5 dys fter irrdition. Prbhkr et l., (2007) reported tht there is depletion of glutthione trnsferse, SOD nd CAT ctivities in the liver of irrdited mice. Irrdition with 5 Gy significntly incresed the MDA level s n end product of lipid peroxidtion. Irrdition lso significntly decresed SOD ctivity nd incresed GSH-Px ctivity, 10 dys fter irrdition, indicting the genertion of oxidtive stress nd n erly protective response to oxidtive dmge (Ibrhim et l., 2007) Enzymes ctivities Glutmte dehydrogense Glutmte dehydrogense (GDH) is n enzyme, present in mitochondri of eukryotes, s re some of the other enzymes required for ure synthesis, tht converts glutmte to α-ketoglutrte, nd vice vers. Under cloric restriction nd low blood glucose, glutmte dehydrogense ctivity is rised to increse the mount of α- Ketoglutrte tht is produced. The product α-ketoglutrte cn be used to provide energy by being used in the citric cid cycle to ultimtely produce ATP. Zyrinov nd Lvrov, (1998) found significnt decrese in the ctivities of glutmte dehydrogense nd sprtte minotrnsferse in the liver of rts 1, 7 nd 15 dys fter gmm whole 39

40 body gmm irrdition. Sd nd Azb, (2001) showed tht whole body exposure of rts to 7 Gy gmm irrdition results in significnt decrese in the ctivity of glutmte dehydrogense in mitochondri of the liver fter rdition exposure nd this ws ccompnied by n increse in its ctivity in the cytosol. Rdition-induced cellulr dysfunction hs been ttributed, t lest in prt; to impirment of mitochondril function s this orgnelle is both mjor source of oxidnts nd trget for their dmging effects, which cn result in reduction of energy production, thereby compromising cell function. Rdition-induce significnt decrese in glutmte dehydrogense ctivity in rt liver due to glyction dmge (Hmelin et l., 2007). Lctte dehydrogense Lctte dehydrogense (LDH) is responsible for metbolism nd biosynthesis of energetic mcromolecules for different essentil functions. Any interference in the ctivity of LDH enzyme leds to biochemicl impirment nd lesions of the tissue nd cellulr function (Khn et l., 2001). Elevted LDH levels cn be cused by number of conditions, including hert filure, hypothyroidism, nemi, nd lung or liver injury. Frnken et l., (2000) reported tht plsm LDH level ws incresed between 2 h nd 24 h fter locl hert irrdition with single dose of 20 Gy while crdic enzyme (LDH) were reduced. Snchez et l., (2002) found relese of enzyme ctivity of LDH to blood plsm in mice exposed to oxidtive stress. Azb et l., (2003) found lso tht whole body gmm irrdition of rts with shot dose 40

41 of 6 Gy produces remrkble increses of serum LDH ctivity 1, 7, nd 14 dys post exposure. Sid nd Azb (2006) reported tht 7 Gy (single dose) of whole body gmm irrdition produced significnt rise in serum cretine phosphokinse nd lctic dehydrogense ctivities on the 7 th dy postirrdition Sid nd Hnfy, (2006) showed tht rts exposed to 1 Gy whole body gmm irrdition exhibited significnt increse LDH ctivity in the serum. Cretine phosphokinse Cretine phosphokinse (CPK) plys n importnt role in cellulr energy metbolism in vertebrtes (Sks et l., 1996). It is specificlly locted t plces of energy demnd nd energy production nd plys n importnt role in the energetic of clcium (C 2+ ) homeostsis nd mitochondril membrne stbility. The muscle cells contin cretine phosphte nd this used to restore ATP levels under the very short-term high-intensity conditions. Cretine phosphokinse mobilize phosphte group from the cretine phosphte nd this is quickly trnsferred to ADP to form ATP. So the muscle cell turns ATP into ADP nd the phosphgen quickly turns ADP bck into ATP. Elevtion of serum CPK is n indiction of muscle dmge (Holger et l., 2005). Frnken et l., (2000) reported tht plsm CPK level ws incresed between 2 h nd 24 h fter locl hert irrdition with single dose of 20 Gy while crdic enzyme CPK ws reduced. Sánchez et l., (2002) found relese of enzyme ctivity of CPK, to blood plsm in mice exposed to oxidtive stress. Sridhrn nd Shymldevi, (2002) observed tht exposure of rts to gmm-rys (3.5 Gy) cused increses in lipid peroxides nd the excessive production of free rdicls 41

42 nd lipid peroxides might hve cused the lekge of cytosolic enzymes LDH. Azb nd Nd, (2004) reported tht exposure of rts to 7 Gy (cute dose) gmm irrdition induces significnt decrese in the ctivities of mitochondril CPK of brin nd hert. In ddition, Sid nd Azb, (2006) reported tht 7 Gy (single dose) of whole body gmm irrdition produced significnt rise in cretine phosphokinse, nd lctic dehydrogense ctivities on the 7 th dy post-irrdition. Glucose -6- phosphte dehydrogense Glucose -6- phosphte dehydrogense (G6PD) is the first enzyme in the pentose phosphte pthwy nd is widely distributed in nture, being found in lmost ll niml tissues nd microorgnisms. It is present in lrge mounts in blood cells, smller mount in liver, kidney, hert nd skeletl muscle nd in only trce mount in serum. G6PD plys key role in protection ginst oxidtive stress especilly in erythrocytes (Beutler, 1978). Svitskii et l., (1985) noticed tht single totl-body exposure of rts to gmm rys in n bsolutely lethl dose cused significnt chnges in the ctivity of G6PD in the brin, liver, myocrdium nd skeletl muscles. Abdy et l., (2003) reveled significnt increse G6PD in blood nd spleen of rts, one hour fter exposure to frctionted doses of whole body gmm irrdition up to cumultive dose of 6 Gy. This increse ws followed by significnt decrese on the 7 th dy post rdition exposure. Bbu et l., (2008) observed tht oxidtive stress induced lipid peroxidtion nd decrese ctlse, glutthione reductse, glucose-6- phosphte dehydrogense enzyme ctivities nd glutthione levels. 42