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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 AGE DEPENDENT EFFECTS OF T-BUOOH ON ITS PHARMACOKINETICS, ANTIOXIDANT ENZYMES AND GLUTATHIONE BY MEI LING CHANG A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Pharmaceutical Sciences) August 1995 Copyright 1995 Mei Ling Chang UMI Number: 9621707 UMI Microform 9621707 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 90007 This dissertation, written by m .M m .G W M ........................................... under the direction of hex. Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillm ent of re quirements for the degree of D OCTOR OF PHILOSOPHY C . Dean of Graduate Studies D a te Jfex. I.?.,.. 1995................. DISSERTATION COMMITTEE Chairperson DEDICATION I DEDICATE THIS TO MY FAMILY FOR ALL THE LOVE AND SUPPORT THROUGHOUT THE YEARS. TABLE OF CONTENTS ACKNOWLEDGEMENTS............................................................................................. IX LIST OF TABLES..............................................................................................................V LIST OF FIGURES...........................................................................................................VI ABSTRACTS..................................................................................................................... X CHAPTER I : INTRODUCTION........................................................................................ 1 1 . Free Radical Theory..............................................................................................1 2. Lipid Peroxidation................................................................................................3 3. t-BuOOH as a neurotoxin................................................................................... 11 4. Antioxidative Defense Mechanisms.....................................................................12 5. Glutathione.........................................................................................................12 CHAPTER II: AGE DEPENDENT EFFECTS OF T-BUOOH ON GLUTATHIONE DISULFIDE REDUCTASE, GLUTATHIONE PEROXIDASE AND MALONDIALDEHYDE IN THE BRAIN............................................... 19 1. Introduction............... 20 2. Materials and Methods......................................................................................21 3. Results.............................................................................................................. 24 4. Discussion.........................................................................................................41 CHAPTER m. PHARMACOKINETICS OF INTRACEREBROVENTRICULAR T-BUOOH IN YOUNG ADULT AND MATURE MICE.......................................................................................................44 1 . Introduction...................................................................................................... 45 2. Materials and Methods...................................................................................... 46 3. Results.............................................................................................................. 48 4. Discussion.........................................................................................................65 iv CHAPTER IV: IN VIVO BRAIN GLUTATHIONE TURNOVER RATE IN YOUNG AND MATURE MICE...........................................................67 1. Introduction...................................................................................................... 68 2. Materials and Methods......................................................................................69 3. Results.............................................................................................................. 71 4. Discussion.........................................................................................................83 CHAPTER V: CONCLUSION OF THE PROJECT.........................................................86 REFERENCES..................................................................................................................87 LIST OF TABLES TABLES PAGE Table 1. Brain MDA values of 2 month old and 8 month old mice............................................................................................................. 37 Table 2. GSH peroxidase activities for 2 month old mouse controls and after t-BuOOH treatment.............................................................39 Table 3. GSH peroxidase activities for 8 month old mouse controls and after t-BuOOH treatment............................................................40 Table 4. Pharmacokinetics parameters of 2 month old mouse t-BuOOH...........................................................................................................56 Table 5. Pharmacokinetics parameters of 8 month old mouse t-BuOOH...........................................................................................................57 Table 6. GSH synthetase activities (nm GSH/min mg protein) of 2 month old mouse and the effects of t-BuOOH...........................................................................................................72 Table 7. GSH synthetase activities (nm GSH/min mg protein) of 8 month old mouse and the effects of t-BuOOH............................................................................................................73 Table 8. GSH specific activity (uci/umole) for 2 month old and 8 month old mice and the effects of t-BuOOH.................................. 74 Table 9. GSSG specific activity (uci/umole) for 2 month old and 8 month old mice and the effects of t-BuOOH..................................75 Table 10. Brain GSH turnover parameters for 2 month old and 8 month old mice....................................................................................... 82 LIST OF FIGURES FIGURES PAGE Figure 1. Production of Oxygen Derived Radicals............................................................ 2 Figure 2. Mechanisms of Cell Injury By Oxidative Stress................................................. 4 Figure 3. Lipid Peroxidation Process..................................................................................6 Figure 4. DNA Damage Products.....................................................................................8 Figure 5. Vitamin E Relationship with Vitamin C and GSH............................................ 10 Figure 6. Antioxidants.....................................................................................................13 Figure 7. Glutathione.......................................................................................................14 Figure 8. GSH synthesis..................................................................................................16 Figure 9. Interaction of GSH with t-BuOOH.................................................................. 17 Figure 10. GSH levels in the cortex before and after T-BuOOH administration............................................................................. 25 Figure 11. GSH levels in the striatum before and after T-BuOOH administration............................................................................. 26 Figure 12. GSH levels in the thalamus before and after T-BuOOH administration.............................................................................27 Figure 13. GSH levels in the hippocampus before and after T-BuOOH administration............................................................................ 28 Figure 14. GSH levels in the midbrain before and after T-BuOOH administration............................................................................ 29 vii Figure 15. GSH levels in the cerebellum before and after T-BuOOH administration............................................................................30 Figure 16. GSSG reductase activities in the cortex before and after t-BuOOH administration.....................................................31 Figure 17. GSSG reductase activities in the striatum before and after t-BuOOH administration.................................................... 32 Figure 18. GSSG reductase activities in the thalamus before and after t-BuOOH administration.....................................................33 Figure 19. GSSG reductase activities in the hippocampus before and after t-BuOOH administration....................................................34 Figure 20. GSSG reductase activities in the midbrain before and after t-BuOOH administration.....................................................35 Figure 21. GSSG reductase activities in the cerebellum before and after t-BuOOH administration....................................................36 Figure 22. Mean 2 month old versus 8 month old t-BuOOH concentration versus time curves in the cortex..............................................49 Figure 23. Mean 2 month old versus 8 month old t-BuOOH concentration versus time curves in the striatum........................................... 50 Figure 24. Mean 2 month old versus 8 month old t-BuOOH concentration versus time curves in the thalamus........................................ 51 Figure 25. Mean 2 month old versus 8 month old t-BuOOH concentration versus time curves in the hippocampus................................... 52 Figure 26. Mean 2 month old versus 8 month old t-BuOOH concentration versus time curves in the midbrain.......................................... 53 Figure 27. Mean 2 month old versus 8 month old t-BuOOH concentration versus time curves in the cerebellum...................................... 54 Figure 28. GSSG levels in the cortex before and after T-BuOOH administration.............................................................................59 Figure 29. GSSG levels in the striatum before and after T-BuOOH administration.............................................................................60 Figure 30. GSSG levels in the thalamus before and after T-BuOOH administration............................................................................. 61 Figure 31. GSSG levels in the hippocampus before and after T-BuOOH administration............................................................................. 62 Figure 32. GSSG levels in the midbrain before and after T-BuOOH administration............................................................................. 63 Figure 33. GSSG levels in the cerebellum before and after T-BuOOH administration.............................................................................64 Figure 34. GSH turnover rate of 2 month old mouse......................................................76 Figure 35. GSH turnover rate of 8 month old mouse......................................................77 Figure 36. HPLC analysis of brain homogenates of 8 month old mouse 2 hours after t-BuOOH administration.........................................................78 Figure 37. Cysteine specific activity of 2 month old mouse from 20 min to 108 hour......79 Figure 38. Cysteine specific activity of 8 month old mouse from 20 min to 108 hour......80 ix ACKNOWLEDGEMENT I would like to express my gratitude to Dr. James David Adams for all his guidance, advice and support throughout my graduate study as an excellent advisor as well as a great friend. I would like to thank my dissertation committee members Dr. Robert Koda and Dr. Murad Ookhtens for all their advice. I would also like to thank all of my friends in the school of pharmacy for making my graduate study a memorable one. X ABSTRACT This project studied compensatory mechanisms of detoxifying t-BuOOH and compared whether 2 month old and 8 month old mice differ in their compensatory mechanisms. In the first part of this dissertation, GSH peroxidase and GSSG reductase in 2 month old and 8 month old mice were investigated. Malondialdehyde (MDA) levels were measured using a thiobarbituric acid assay and were compared between the two groups. GSSG reductase activity increases in young adult mice after t-BuOOH administration, but not in mature mice. GSH peroxidase activity is significantly lower in 8 month old than 2 month old mouse striatum after t-BuOOH administration. Furthermore, MDA levels in 8 month old striatum increase significantly 20 min after t-BuOOH administration. In the second part of this dissertation, the pharmacokinetics o f icv administered t-BuOOH in 2 month old and 8 month old mice in different brain regions were compared. Brains were dissected at 11 time points from 0.5-60 min. The pharmacokinetics parameters were compared using least squares regression analysis. T-BuOOH concentration versus time curve profiles between the two age groups were similar. Pharmacokinetic parameters between the two age groups were similar. Overall, the striatum behaves differently than the rest of the region. The last part of this dissertation examines GSH synthetase activity and GSH turnover rate in the mouse brain during severe oxidative stress induced by t-BuOOH. GSH turnover is monophasic in the 2 month old mice but it is biphasic in the 8 month old mice. Brain GSH synthetase activity 20 min after t-BuOOH administration in 8 month old mice is higher than in 2 month old mice. This data suggest that two age groups have different compensatory mechanisms against oxidative stress. 1 CHAPTER I: BACKGROUND Free radicals have been implicated in many diseases such as stroke, ruptured berry aneurysms, Parkinson's disease, amyotrophic lateral sclerosis and brain trauma. All aerobic cells generate a continual flow of superoxide anions, hydrogen peroxide and hydroxyl radicals due to exposure to oxygen, radiation, ultraviolet radiation, ozone, peroxides and drugs (1). Hydrogen peroxide may be produced in neurodegenerative disorders from the action of monoamine oxidase, tissue hypoxia, release of blood components into the brain extravascular spaces and other conditions (2,3). These unstable radicals can produce chain reactions by interacting with nonradicals such as biological molecules and metals. 1. FREE RADICAL THEORY Free radicals are any chemical species which can exist independently with one or more unpaired electrons. Superoxide anion, hydrogen peroxide, singlet oxygen, hypochlorous acid and hydroxyl radical are examples of reactive oxygen species (4). Superoxide anion is formed by the addition of one electron to oxygen. It can penetrate membranes through anion channels and can react readily with many compounds (5) (Figure 1). Two molecules of superoxide generate hydrogen peroxide in the presence of the enzyme superoxide dismutase (SOD). Figure 1: Production of Oxygen Derived Radicals (Adapted from Reference 5). OVERALL OXYGEN REDUCTION: O, + 4e + 4Hf -______ > 211*0 PARTIAL OXYGEN REDUCTIONS: electron reduction of O, o , + e' — > O,’ to superoxide aidon spontaneous or SOD-catalyzed dismutation of superoxide anion Haber-Weiss reaction Fenton reaction I------------------- + IP — > 0.5 H*0, + 0.5 O, I ---------------------------------------------- — L I 11*0* — > Oil + OH’ + O, + Metal" — > Metal"1 + O, X Metal"1 +11,0, — > Metal* + OH + OH- 1_____________________ A ] lipid hydroperoxides ■ 211*0* ■ 2 REDUCED GLUTATHIONE ' low reactive hydroxy fatly acids 211,0 + O, OXIDIZED GLUTATHIONE hexose monophosphate shunt NADP+ NADPII 3 SOD 2 0 2' + 2 H+ > 02 + H2 0 2 Superoxide dismutase exists in a Mn-dependent form in mitochondria and a Cu/Zn dependent form within the cytoplasm. Hydrogen peroxide can be eliminated by either catalase or glutathione peroxidase (6). eatalaag 2 H2 0 2 ------------ >2 H2 0 + 0 2 GSH peroxidase Hydrogen peroxide can react with Cu+ or Fe2 + to form highly reactive hydroxyl radicals. The copper and iron metals must be in the reduced state form to catalyze the oxidative reaction. 0 2 " + Fe3 * ---------------- >02 + Fe2 + H2 0 2 + Fe2+----------------- > OH + OH- + Fe3 + The accumulation of free radicals can create problems such as GSH depletion, DNA damage, protein damage and cytoskeletal damage (7,8) (figure 2). 2. THEORY OF NEURODEGENERATION No mammalian organs are more vulnerable to chronic oxygen mediated damage than the brain because neurons do not replicate in mature mammals. Loss of neuronal function is critical because replacement by cell division is Figure 2* Hochaulams o£ Cell Injury By Oxidative Stress Direct Damaged Pnrtdna GSH Depletion 5 impossible. Therefore, repair and maintenance in such cells is crucial. Furthermore, the brain contains lower levels of GSH peroxidase than other organs which makes the brain susceptable to oxidative stress. Similar to other cells, neurons have targets susceptible to damage, such as DNA, lipid and protein components. 3. CONSEQUENCES OF FREE RADICALS 3A. LBPID PEROXIDATION Free radicals in the presence of oxygen have a number of toxic effects on the cell, including the initiation of lipid peroxidation. Lipid peroxidation is a common process because almost all biological lipids contain some polyunsaturated fatty acids (PUFA). For example, PUFAS are present in cholesteryl esters, triacylglycerols, phospholipids, and free fatty acids (9). The brain compared to other tissues contains a higher concentration of iron than any other metal (10,11,12,13). Although ferritin and transferrin have a very high affinity for iron at neutral pH, they both release iron when the pH is 6.0 or lower then release of iron can induce oxygen radical formation and lipid peroxidation (14). The brain is a preferential target for a peroxidative process because it has a high content of polyunsaturated fatty acids and lipid peroxidation is a process dependent upon the presence of iron. Damaged brain cells readily release iron to surrounding tissues to catalyze the generation of 6 OH (15). Considerable variation has been reported in the endogenous iron content in different areas of the brain (15). Therefore, regions with high iron content seem to be most vulnerable to membrane lipid peroxidation. Lipid peroxidation can cause extensive damage to subcellular organelles and biomembranes and has been shown to occur in isolated mitochondria, lysosomes and microsomes (1). Lipid peroxidation may generate activated oxygen species such as hydroxyl radicals and superoxide anions. The lipid peroxidation process is iniated after hydrogen abstraction from an unsaturated fatty acid (16) (Figure 3). The lipid radical formed reacts with molecular oxygen if present. The resultant products of these processes are monohydroperoxides of lipids (LOOH). In the presence of metal ions, the lipid monohydroperoxides break down to form lipid alkoxyl radicals. These can undergo cleavage of Carbon- Carbon bonds which may result in the formation of unsaturated fatty acid aldehydes and alkyl radicals which can further initiate new radical chain reactions. One of the most popular methods to measure lipid peroxidation is by the use of the thiobarbituric acid reaction. This procedure is simple and sensitive. Thiobarbituric acid does not react with some byproducts of the peroxidative breakdown of polyunsaturated fatty acids such as 4- hydroxyalkenals (17,18). Thiobarbituric acid does react with malondialdehyde (MDA), a volatile end F ig u re 3: L lp ld P e ro x id a tio n P ro cess (A dapted £rom R efe ren c e 1 2 ). ( l ) INITIATION (2) PflOPAQATION mi Lli LOO* O* Lll ( 3 ) SINGLET OXYGEN INITIATION L O O I I LO O K ( 4 ) RE-INITIATION LOOM + Fe2* LOOM + Fe3* F e3* + OIF + LO* F e2f * II* + LOO* ( 5 ) PRODUCT REMOVAL LOOH + 2 GSH ► LOH + GSSG + IIjO ( 6 ) TERMINATION LOO*/L*/LO* + 1 1 1 LOOII LOO* LOO* + LOO* + I* LOOH/LH/LOH + I* L=0 + LOH + Oa LOO-I 8 product of oxidative lipid degradation to yield a fluorescent red adduct. 3B. DNA DAMAGE The toxicity of t-BuOOH has been attributed to DNA damage mediated by a Fenton reaction that generates a reactive hydroxyl radical. NADH + Fe3 * ------------------ > Fe2 * + NAD + H * t-BuOOH + Fe2 * -------------------> OH' + ButO + Fe3 * DNA damage is considered to be a useful indicator of carcinogenicity. DNA is associated with certain regions of the nuclear membrane. Therefore, nuclear membrane peroxidation may damage DNA and disrupt many critical functions. DNA may also interact with reactive compound generated in lipid peroxidation due to the proximity of the nuclear membrane to DNA. Oxygen radicals such as superoxide anions and hydroxyl radicals may damage DNA by altering nucleoside bases or disrupting the sugar phosphate backbone (1) (Figure 4). Attack on the sugar moeity leads to sugar fragmentation, base loss and strand break, resulting in a terminal fragmented sugar residue (1). 3C: PROTEIN OXIDATIVE DAMAGE Oxygen free radical attack on proteins yields many possible products: (1). cross linked reaction products with cellular components, (2). fragmentation products, (3). site specific lesions that affect functionality (9). Most protein F ig u re /<; D N A Damage P ro d u cts (A dapted from R efe ren c e 1 ), I a i I ' o n i / } ,-L »— I— a o "• i« m 4hiIo* n « • r I .. , „ — >**' Sugar dam age N __ . I * K ? V ? He-*".. - r ‘ i l-r o C H O ■ f e f e X. f e - CC 3e 3: J" J- 3- l ■ dH M I V vO 10 structures contain cysteinyl residues either in the free reduced form or in the oxidized disulfide form (2). With the administration of t-BuOOH, protein sulfyhdryl groups (protein-SH) become more oxidized (protein-SSG). 3D: VITAMIN E DEPLETION Vitamin E, a lipid soluble antioxidant, is essential in the brain (6). The primary role of vitamin E is the quenching of free radicals and terminating lipid peroxidation (16) (Figure 5). The vitamin E radical can be reduced back to vitamin E with ascorbate and GSH. The side chain of vitamin E is in close contact with the unsaturated portions of PUFA (6). This provides a barrier that sterically impedes the attack of free radicals on PUFA. 4. AGE RELATED RESPONSE TO FREE RADICALS Free radical damage is important in the aging process (19). Decreased levels of GSH and mitochondrial cytochromes have been reported in aged rodent brain (20,21,22). Protein oxidation products are increased in both aged rodent and human brain (23). There is an age related redistribution of ferritin from oligodendrocytes to astrocytes (6). The content of human brain membrane lipids is also decreased with age. Lipid peroxidation is also increased resulting in accumulation of lipofuscin (age pigments) in aged brain (24,25). Data from our laboratory show that 2 month old mice were less F ig u re 5 : VLtaaLn E R e la tio n s h ip w ith V itam in C and G S 1 I (A dapted from R eferen ce 1 6 ). i-T o c o p h e ro l 3 -ro c o p h e ry l chrom anoxy— r a d i c a l a -T o c o p h a ry l q u In a n e -> 0 Inters T r iM r i 7 o c o p h e ro l reg en e ion l y t t i e i O ehydro-Z seail- dehydro a s c a r - b lc a c id G SM syn th etases G3II 4 Ascorbic acid re d u c ta s e A tc o rb a te u c s a s e s N A D PII OSM HADII C y stein e Qlucamic acid G lycine 12 susceptible to oxidative stress in the brain compared to 8 month old mice, as evidenced by an increase in intracellular levels of GSSG in 8 month old mice in every brain region. This altered the GSH/GSSG ratio due to an increased GSSG and decreased GSH. This alteration may make neurons more susceptible to oxidative stress, in combination with the low amount of GSH they have. Oxidative stress also causes depletion of protein sulfhydryl groups in some mouse brain regions (27). ICV t-BuOOH significantly increased vitamin E levels in the striatum perhaps as a compensatory mechanism to defend against oxidative stress. 5. T-BUOOH AS A MODEL FOR OXIDATIVE STRESS Tertiary butyl hydroperoxide (t-BuOOH) is a lipophilic organic hydroperoxide which is reduced by GSH peroxidase (26). It is stable in solution and can readily permeate throughout the brain and cells. There is no reported toxicity of its byproduct which is tertiary butanol (27). One way to examine oxidative stress in the brain is through intracerebroventricular (ICV) injection of the lipophilic organic hydroperoxide, t-butyl hydroperoxide (t-BuOOH). This route of administration allows t- BuOOH to permeate throughout the brain (26,27). This model compound may provide a useful simulation of hydrogen peroxide which has been reported to be neurotoxic (4,10). 13 Hydrogen peroxide (HjOJ serves as a substrate for GSH peroxidase and causes oxidative stress but does not penetrate into the brain following ICV injection. Hydrogen peroxide may be produced in neurodegenerative disorders from the action of monoamine oxidase, tissue hypoxia, release of blood components into the brain extracellular spaces and other conditions (10,21). Previous work has shown that t-BuOOH damages many cell types in the brain, including astrocytes, endothelial cells, pericytes, oligodendrocytes, and many neurons including dopaminergic, GABAergic, and cholinergic (28). t-BuOOH inhibits many enzymes such as GSH peroxidase, GSSG reductase and catalase (29). In addition, dose response curves show the range of toxic doses of t-BuOOH (27,28,29). It has also been reported that older rodents have lower quantities of enzymes available to detoxify oxygen radicals (21,22,30). Age relationships has been reported that 8 month old mice are more susceptible to free radical species in the brain than 2 month old mice due to the higher intracellular levels of GSSG in every region of the 8 month old mice. Depletion of protein sulfhydryl groups has also been shown in some brain regions after t-BuOOH administration. 6. ANTIOXTDATIVE DEFENSE MECHANISMS Since active oxygen species is produced continuously in vivo, major 14 antioxidative defense mechanisms (31) (Figure 6) are created by the body to prevent or reduce free radical chain reactions. Defense mechanisms are divided into enzymatic, such as superoxide dismutases (SOD), catalases,and glutathione peroxidases, and nonenzymatic, such as tocopherols (vitamin E), ascorbate (vitamin C), vitamin A and glutathione (GSH) (31). Without these defense mechanisms, many major pathways of cell regulation can be disrupted. Free radicals can inactivate many of the antioxidative enzymes such as peroxidases. 7. GSH AS A MAJOR ANTIOXIDANT AGAINST T-BUOOH Glutathione (L-gamma glutamyl L-cysteinylglycine) (32) (Figure 7) is a tripeptide, which has some remarkable characteristics. It is found intracellularly in high concentrations existing as reduced GSH with only a fraction as GSSG. It has a sulfhydryl group (due to cysteine) and a gamma glutamyl bond (the bond that links cysteine to glutamate). The sulfhydryl group is responsible for the catalytic and reactive properties of GSH. It is able to participate in oxidation-reduction reactions and thus exists in two forms- the reduced form, glutathione, and the oxidized form glutathione disulfide (33). The gamma glutamyl bond prevents enzymatic breakdown of GSH. The only enzyme capable of breaking the bond is gamma glutamyl transpeptidase. Brain GSH levels range from 1-3 mM (34,35,36,37). GSH is present in low levels in neuronal somas but found in high levels in astrocytes and nerve F ig u r e 6: A ntio x id aiifcs (A d ap ted from R e fe re n c e 31). Vitamin £ e n o o p l a s m k h e t i c u l C m ( N U C L E U S Vitamins C and £ Calalase Cu/Zn SOD t w o b i l a y e r o r a l l CELLULAR MEMBRANES PERO XISO M ES •O o LTSO S O M E S C Y T O P L A S M MITOCHONDRION y — GSH r Glutathione Peroxidase Vitamin E / ■Vitamin C Vitamin E SQO+ Glutalhiona Peroxidase ♦ GSH Figure 7: Glutathione (Adapted fron Reference 32). GLUTAMATE CYSTEINE GLYCINE 17 terminals (37,38). GSH is found in micromolar concentrations in the brain endothelium and astroglia (38). GSH levels in the brain change as rodents grow and mature. GSH peroxidase activity has been reported to decrease with age whereas GSSG reductase activity has been reported to increase with age (39,40,41). GSH can be depleted directly by conjugation with electrophiles and indirectly by the addition of inhibitors of GSH biosynthesis and regeneration (42,43). GSH participates in a variety of biochemical reactions such as the enzymatic elimination of hydrogen peroxide and organic peroxides by GSH peroxidase and detoxification of foreign compounds (44). GSH is synthesized via the following pathway (31) as shown in (Figure 8). Both enzymes are found in the brain (31). GSH synthesis is regulated by the availability of cysteine. The amount of cysteine available is limited by feedback inhibition of gamma glutamylcysteine synthetase by GSH. GSH protects microsomal incubations against both NADPH and ascorbate induced lipid peroxidation. Some reports have shown that the addition of GSH produces a 40% decrease in NADPH induced peroxidation of rat liver microsomes (32). The classical selenium dependent GSH peroxidase and certain GSH-S-transferases have been shown to reduce lipid hydroperoxides to lipid alcohols and repair DNA (45,46). Glutathione disulfide (GSSG) is formed in the cell through the reduction of hydrogen peroxide or other hydroperoxides by glutathione peroxidase or the Figure 8: GSII Synthesis (Adapted from Reference 32). Mg TH I0NIN6 Livsr ATP '» » . P Gfcj|*m»i« Glycine I '* 'I |C ysti.neJ- ■ > igtuUmytonaainc *------- » -|g Sh | ATP ATP igfcjcys lynihetasa q sh synlheiasa Q l u t a lh lo n e l y n l h a i l i lio m III IhrM am in o a c i d p r a c u r a o r a : g lu t a * m a l a , c y a i a ln a , a n d g ly c in e . 19 reduction of protein mixed disulfides (Figure 9). Oxidation of GSH can be an index of the induction of oxidative stress (26,47). The capacity of each cell to maintain its redox status is decreased with increasing oxidative stress and thus it is vital for GSSG to be actively transported out of the cell (48). GSSG has been used as an index of oxidative stress. Reports have shown that patients develop hemolytic anemia due to oxidative stress as a result of gamma- glutamylcysteine synthetase and GSH synthetase deficiency (25). GSH also reduces protein disulfides and prevents the oxidation of cysteinyl residues of certain enzymes which can lead to changes in the enzyme catalytic activity. The reaction is as followed: Protein-SSG + GSH< >Protein-SH + GSSG Almost all nonprotein cysteine is stored as GSH because cysteine autoxidizes readily to cystine, a process which creates free radicals. Figure 9: Interaction of GS1I with t-BuOOII C1I3 C II3 1 1 H ,C — C — OOH + 2 GSH — I I 3 C — C — OH + GSSG -I- IKO I I CHj CHj CHAPTER H: AGE DEPENDENT EFFECTS OF T-BUOOH ON GLUTHIONE DISULFIDE REDUCTASE, GLUTHATHIONE PEROXIDASE AND MALONDIALDEHYDE IN THE BRAIN. 22 OBJECTIVE Previous work show that t-BuOOH administered by ICV increases brain GSSG levels and decreases brain GSH levels (27) and that age is a factor in this response. The purpose of the study is to examine the activities of critical enzymes related to GSH in the brain under conditions of acute oxidative stress. In addition, this study seeks to compare differences in protection by GSH peroxidase and GSSG reductase in two different age groups. In this report, we investigate whether malondialdehyde (MDA) levels increase following t- BuOOH administration. MDA products may be produced from the peroxidation of lipids, DNA or proteins (18). 23 MATERIALS AND METHODS Young adult (2 month old) and mature (8 month old) male C57 Bl/6 mice, each weighing approximately 30 g were used. Each mouse was anesthetized by injecting 200 mg/kg of ketamine and 2 mg/kg of xylazine intraperitoneally (ip) prior to t-BuOOH administration. All mice were cared for according to University of Southern California and NIH guidelines. For controls, mice were given 5 ul of 0.9% saline ICV. Treated mice were given 5 ul of t-BuOOH as a 70% solution ICV. Some of our previous work involved the administration of equal doses of t-BuOOH, based on body weight, to the two age groups (27). The brains from 2 and 8 month old mice were not significantly different in weight (about 0.4 g each). Therefore, we decided to use the same amount of t-BuOOH in the 2 age groups, in order to induce the same amount of oxidative stress in the brains. Five ul (109.7 mg/kg) of ICV t- BuOOH killed 7/11 of the 8 month old mice by 2 hours, whereas 0/5 of the 2 month old mice died. Brains were removed at appropriate times and were dissected into cortex, striatum, thalamus, hippocampus, midbrain and cerebellum. For GSSG reductase activity assessment, brains were dissected on ice at 0,2, 5 and 20 min after t-BuOOH administration. Brain regions were homogenized in 1 ml of 100 mM sodium phosphate buffer (pH 7.6) and centrifuged at 105,000 x g for 1 hour at room temperature. The supernatant 24 (100 ul) was added to 100 ul of 0.3 uM of GSSG and 100 ul of 0.1 uM NADPH. The loss of NADPH was measured by spectrophotometer at 340 nm as a measure of GSSG reductase activity (49). GSSG reductase activity was calculated according to the following formula: C=change in A/ E where E of NADPH at 340 nm is 6270 M'1 A is the absorbance read from the spectrophotometer The oxidation of 1 uM/min of NADPH under this condition is used as a unit of GSH reductase activity. The specific activity is expressed as units per mg of protein. Protein lowry was done with the supernatant (50). Brain sample supernatant (50 ul) was added to 50 ul of solution containing 75 ml of 10 M sodium hydroxide in 1 liter of water. To this solution, 300 ul of water was added. Reagent D is a combination of 196 ml of 2% sodium carbonate, 0.1 M sodium hydroxide, 2 ml of 1% CuS04 • 5H2 0, and 2 ml of 2% Na-K tartrate. 1.5 ml of this solution was added to the sample and 0.15 ml of Folin phenol reagent was added. All tubes were read at 750 nm against water. For the thiobarturic acid (TBA) assay (51), brains were dissected at 0 and 20 min. Brain regions were homogenized in 1 ml of the stock solution. Stock solution was made containing 200 ml of water, 750 mg TBA, 30 g of trichloroacetic acid and 4.12 ml of concentrated HC1. The homogenates were 25 heated at 100 C for 15 min. After 5 min, this homogenates were centrifuged briefly for 4 min. The absorbance of the supernatants was assessed at 535 nm. A standard curve was obtained using 10 ul-900 ul of tetramethoxypropane as a source of MDA. GSH peroxidase was measured by the Paglia and Valentine method, 1967 (52). Brain regions were dissected, homogenized in 50 ul of 100 mM sodium phosphate buffer with 2 mM azide and 4 mM EDTA. This homogenate was centrifuged at 12,000 x g for 15 min. An aliquot of the supernatant was added to a cuvette, along with 30 ul of 5 mM GSH, 30 ul of 0.3 mM NADPH and 10 ul of GSSG reductase. The cuvette was then incubated for 10 min, until the addition of 30 ul of 0.1 mM H2 0 2 . H2 0 2 is a substrate for only the selenium dependent form of GSH peroxidase. The loss o f NADPH was measured over time at 340 nm. Statistical analysis was done with Student's t-test or Anova with Newman Keuls test. Regression analysis was done with the SAS statistical analysis software program by SAS Institute Inc. Differences were significant at p less than or equal to 0.05. All data values were expressed as means and standard deviations. 26 RESULTS The trend is that GSH is depleted in the mouse cortex, hippocampus and midbrain after t-BuOOH (though not significantly). Two month old mice are able to increase GSH in the thalamus and the cerebellum after 20 min of t- BuOOH administration though not significantly. Eight month old mice are able to increase GSH in the striatum after 20 min of t-BuOOH administration though not significantly (Figure 10-15). Control GSSG reductase activities are similar to previous reports (39,40,47) and tend to be higher in 8 month old mice than 2 month old mice (Fig 16-21). This age related change in GSSG reductase activity is similar to other reports (39). In the 2 month old cortex, thalamus and cerebellum, enzyme activity increases significantly by 2 min following t-BuOOH administration (Fig 16,18,21). The general trend is that GSSG reductase activity in young adult mice increases by 20 min after t-BuOOH. In 8 month old mouse brain regions, there are no significant changes in GSSG reductase activities (Fig 16-21). However, there is a trend for GSSG reductase activities to decrease after t- BuOOH administration. Control 2 month old thalamus levels of MDA are the highest followed by midbrain, striatum, cerebellum, cortex and hippocampus (Table 1). Control 8 month old thalamus MDA levels are the highest followed by midbrain, striatum, hippocampus, cortex and cerebellum (Table 1). In every region, MDA values in 2 month old mice tend to be higher than in 8 month old COR TEX G S H L E V E L S (UMOLE/G) 2- it* 101 OSII l a v n l n In Lite c o r t a x b e f o r e mill a l t e r t-DuOOII a t l m l u l a t rnl: I a n . * B l y n l F lc n n l 1y (11 ( f tjlenl: 1 iom 3 inniiMi o l d c o n t r o l v a l u e s (JXO .O SI by AIIOVA w ll.li tleWMnn-Keiiln t o n t . ** 0I911 I f .leant I y Ul f fprirnl: from II 1 1 1 0 1 1 t .l1 o ld c o n t r o l v a lu e a ||><0 .U5) by A IIO V A w ith llawnan-Ksula Leak. ■ ■ 2 monihB 0 m o n lh s 0 to 0 2 B 20 • 4 P l y t i r f t I I I ttiMI l i t v i i l i * Im Ilia* *»l c I iiI .iim f # t t f t i t o m i l l n l t u i l- l h i U l J J l r tilm l it I ttl i t« I I m m * 1 I ki'ttnl I y ill llni c u t 1 1 mu ) nit hi Hi iilil u m i t . i o l v i l u i a | |i < 0, H5 I liy AIIIIVa i/IIIi Ihn/ini.iiKiitiJii lim l, * 1 (I I tjtil ( I |y it I I f m mil I i mu II tuuiil li ulil uiililliit VHluvt f |*<U* 051 Itjr i/tLli JfuKfiiaii-lleiilii Limt. o 3 O H :c \n o 2 i - o 0 1 H 2 m u iillia I5 2 J U iiioiiIIis B au ro 00 TIM E (M IN ) ^ 1 - |2 l HUII Ipvnln In tlm tlinlnmtio lin ln to and a l t a r t-lliiOUII nilinl nl n il nl. I u n . * H I i|ii I I li'iinl I y ilI I f hi mil. 11 nm 1 : 11 old u o n li o l valuoa | |i<0. tl 5 1 1 > y AIIOVA wllli IlnwmiiiiKuiiln Innl. " tllgui f Inaiil I )' ill f riitniil. finm II monl.li old c o n tr o l valuoa ||i<0.05) liy AIIOVA wllli llowman-Kaiila l o a t . 2 2 m o n th s 0 m o n th s 0 0 t B 20 4 65 f i g u r e IJ i Knit le v o ln In th e lilpjioonmpiiB b e fo r e end a f t e r t-llllUUII ntlml n I n l r nl I o n . * B l g n l f l o n n t I y d i f f e r e n t from ?. month o l d c o n t r o l v a l u e s ||i<(l.llli| l»y AIIOVA w llli llnwmiin-Kenln t n n t . •* B ig u if Irani ly ill (f o r o u t from II mouth o ld c o n t r o l valuon |p < 0 .0 9 | by AIIOVA w ith llewmau-Keuie t e a t . ■ ■ I 2 m onths IS Z 1 0 m o n th s ® * 6 2d T IM E 4 MID8RAIN G S H L E V E L S (UMOLE/G) f i g u r e H i Will Ipvnln In 1.1m m iilhtnln b e fo r e nml n i t e r t-lluUOII m l r r tln I nl. f n l I o n . * HI qn I f I rnnl ly il1ffr*»nnl from 7 tmmlli «i|«l control vniuon (|<«l.nri| liy nM llVA wllli ffpwrnrtii-l’euie Irnl;. M F t i tj ii I f Irnnt I y d I f I r»t mil: fiom ft month o ld c o n t r o l vnluen (|K U .0 5 | by AtlUVA w ith llewinnii-Kfliiln t e n t . i i u- o a ■ ■ I 2 m o n th s [ 5*21 U m uiilhs 20 IIM L (M IN ) F ly u ra ISi linn lo v n ln lii t.lio ooroliol linn Im toro and a l t e r t-UuOUII mltnl ii I nl tnl l mi. • !! I ijii I I Im m l I v i l l f l m m i l: tloni 7 inniilli o l d c o n t r o l valllon I <0 . IIS I liy AMIIVA w 11 Ii IlnwinnnKonln I n n l . ** llltjnl f lea n t I)’ d lllm iM il: from II month o ld u u iitru l v a lu e s ||i< U .0 5 | liy .. w llli Ilnwimiu-Kuulii Lout. o 2 - l/i o a s 20 IIMIT (M IN ) 17 F ig u re 16 i GSSG re d u c ta s e a c t i v i t i e s (nm oles NADPIl/min ing p r o t e i n ) w ere m easured i n th e c o r t e x i n c o n t r o l s and a f t e r 5 u l o f t-BuOOII a d m i n i s t r a t i o n . * Significantly different from 2 month old control values (p<0.05) by ANOVA with Newman-Keuls test. h > r - o < W J q ; O (_> 40 J5- J 0 - 25 20 15 U a id m o 10- 1/7 V) O 5- 0 mm 2 months EZ1 a M O N TH S ■ I 10 timi£ u > U J F ig u re 17: GSSG re d u c ta s e a c t i v i t i e s (nm oles NADPH/min mg p r o t e i n ) w ere m easured in th e s tr ia tu m i n c o n t r o l s and a f t e r 5 u l o f t-fiuOOU a d m i n i s t r a t i o n . * Significantly different from 2 month old control values (p<0.05) by ANOVA with Newinan-KeulB test. £ > O < ISlU p i o < 1— " S m o in V) o mm 2 MONTHS £SZJ B M O N TH S 100- 90- 70- 60- 30- 10- 0 - lo 2 0 B TIME F ig u re 18: GSSG r e d u c ta s e a c t i v i t i e s (nm oles NADPH/min mg p r o t e i n ) w ere m easured in th e thalam us in c o n t r o l s and a £ t e r 5 u l o f t-BuOOH a d m i n i s t r a t i o n . * Significantly different from 2 month old control values (p<0.05) by ANOVA with Newman-Keuls test. 9 0 m m 2 MONTHS ( S21 8 M O N TH S ao s o a TIME to U l HIPPO CAM PUS GSSG REDUCTASE ACTIVITY l Figure 19: GSSG reductase activities (nmoles NADPH/min mg protein) were measured in the hippocampus in controls and after 5ul of t-BuOOII administration. * Significantly different from 2 month old control values (p<0.05) by ANOVA with Newinan-Keuls test. 40- 30- 20 - 10 - 0 - o 1 t w m 2 MONO IS 1521 U M0N1HS 10 TIME u > C T \ F ig u re 20s GSSG re d u c ta s e a c t i v i t i e s (nm oles NADPH/min mg p r o t e i n ) w ere m easured in th e m id b rain i n c o n t r o l s and a £ t e r 5 u l o f t-BuOOH a d m i n i s t r a t i o n . * Significantly different from 2 month old control values (p<0.05) by ANOVA with Newman-Keuls test. n . , < m i _ 60- £ j" 50 O < Z 1 U J 4 0 - i!l/l S y JO- 5 a K 2 0 - o tn (/) O ' 10- o- I m m 2 MONTHS E 2 J o MONTHS 2 0 TIME F ig u re 2 i t GSSG r e d u c ta s e a o t i v i t i e n (umolen HAOl’ll/m ln my p r o t e i n ) were m easured in t|ie c e re b e llu m i n c o n t r o l s and a f t e r 5 u l o f t-IJuOOll ad m in ls ti.fi Lion . * Significantly different from 2 month old control values (p<0.05) by ANUVA with Newmau-Keulo teat. 90 75 U 5 < 00- =!UJ B § o s 30- O m t/» . j s - O 0 - I M M 2 M ON 1 1 IS ISZ1 » . 20 TIME O J 00 0 Table 1: Brain MDA values of 2 month old and 8 month old mice 39 Region Control 2 20 min-2 Control 8 20 min-8 Cortex 0.43±0.20 0.51±0.26 0.24±0.06 0.81±0.65 Striatum 0.49±0.44 0.82±0.27 0.29±0.05 0.52±0.16b Thalamus 0.79±0.42 0.88±0.41 0.40±0.05 0.52±0.26 Hippocampus 0.29±0.15 0.46±0.20 0.25±0.05 0.29±0.12 Midbrain 0.51±0.43 0.61±0.29 0.37±0.05 0.41±0.10 Cerebellum 0.47±0.22 0.40±0.14 0.23±0.03 0.34±0.23 2 Month old mouse MDA values (nmoles MDA/mg of tissue) for controls (control 2) and 20 min (20 min-2) in the cortex, striatum, thalamus, hippocampus, midbrain and cerebellum after t-BuOOH treatment3 and 8 month old mouse MDA values (nmoles MDA/mg of tissue) for control (control 8) and 20 min (20 min-8) after t-BuOOH treatment.*. * Young (2 month old) and mature (8 month old) mice received 5 ul of t- BuOOH. MDA formation was measured at 535 nm by spectrophotometer. Results are expressed as means ± SD in terms of nmoles/mg tissue. b Significantly different from control value (p<0.05) by Student's t-test. mice. Control brain MDA levels are similar to other reports (45,51). MDA levels do not significantly increase in 2 month old mouse brain regions after t- BuOOH administration. In the 8 month old striatum, MDA levels 20 min after t-BuOOH administration are significantly higher than control levels. The time, 20 min, was chosen because previous work showed that t-BuOOH induces lipid peroxidation at this time (26). Control mouse brain GSH peroxidase activities in Table 2 are similar to rat brain GSH peroxidase activities (52,53,54). Control activities of GSH peroxidase are higher in the 8 month old cerebellum than in the 2 month old cerebellum (Table 3). In addition, control midbrain activities from both age groups exhibit the highest peroxidase activities. In contrast to the 2 month old mouse GSSG reductase activity, GSH peroxidase activity decreases after t- BuOOH administration. This is significant only in the midbrain of young adult mice 5 min after t-BuOOH treatment. However, the 8 month old striatal GSH peroxidase activity is significantly lower than the 2 month old striatal activity following t-BuOOH administration. 41 Table 2: GSH peroxidase activities (nmoles NADPH/min*mg tissue) of 2 month old mouse controls and after t-BuOOH treatment.* Region Control 2 min 5 min 20 min Cortex 19±4.2 18±4.3 14±3.7 12±4.9 Striatum 22±6.2 29±5.3 28±3.8 21±2.1 Thalamus 33±11 22±2.0 25±6.7 28±5.9 Hippocampus 26±5.5 26±8.8 19±2.4 18±4.5 Midbrain 34±8.3 29±5.1 17±7.6b 26±7.9 Cerebellum 8.1±2.1 5.2±0.68 6.5±2.0 7.7±1.4 “ Young (2 month old) and mature (8 month old) mice received 5 ul of t- BuOOH. Selenium dependent GSH peroxidase activity (nmoles NADPH/min*mg protein) was measured by following the loss of NADPH in a spectrophotometer at 340 nm and using hydrogen peroxide as a substrate. Results are expressed as means ± SD. b Statistically different from control values (p<0.05) by ANOVA with Neuman-Keuls test. 42 Table 3: GSH peroxidase activities (nmoles NADPH/min*mg tissue) of 8 month old mouse controls and after t-BuOOH treatment.* Region Control 2 min 5 min 20 min Cortex 13±4.0 21±12 14±5.3 20±5.8 Striatum 20±3.8 22±2.5C 22±2.1c 23±7.7 Thalamus 20±7.2 26±2.6 32±9.4 23±2.9 Hippocampus 23±12 23±5.9 22±4.4 21±5.8 Midbrain 27±10 24±7.7 24±6.6 29±6.8 Cerebellum 23±5.6C 21±0.87c 18±1.9C 27±5.6° c Statistically different from young age group values at the corresponding time point by Student's t-test. 43 DISCUSSION Control 8 month old mice tend to have higher brain GSSG reductase activities than control 2 month old. However, young adult mice have greater protection from oxidative stress because GSSG reductase activities increase after t-BuOOH treatment. It has been reported that high intracellular GSSG levels activate GSSG reductase (39,40). It may be that the very rapid increase in GSSG levels in 2 month old mice activates GSSG reductase. In the 8 month old striatum and hippocampus, GSSG reductase activities tend to decrease after t-BuOOH treatment. This may explain why 8 month old mice have elevated GSSG levels for about 20 min after t-BuOOH administration (28). Mature (8 month old) mice cannot increase the activity of GSSG reductase to reduce GSSG perhaps due to the irreversible binding of the protein moiety of the enzyme. t-BuOOH has been shown to inhibit GSSG reductase (26). This inhibition probably involves the oxidation of a protein sulfhydryl group. Young (2 month old) mice do not suffer from this inhibition. On the contrary, they can increase the activity of the enzyme. High GSSG and NADP+ have been reported to elevate GSSG reductase activity. MDA is a product of the free radical induced peroxidation of lipids, proteins or DNA (30). Our previous work with conjugated dienes has shown that lipid peroxidation is induced by t-BuOOH only in the midbrain (26). Lipid peroxidation occurs only in the midbrain probably due to the high amount of iron in the midbrain. The current study found that MDA levels increase only in the 8 month old striatum 20 min after t-BuOOH administration. This may indicate that 8 month old mice suffer more from oxidative stress than 2 month old mice. The fact that MDA levels did not increase in the midbrain may show that the conjugated diene assay is better for detecting lipid peroxidation than the MDA assay. We have recently found that t-BuOOH damages cellular nuclei (27). This damage may result from the known ability of t-BuOOH to peroxidize DNA (55) which can produce MDA. Deoxyribose, released from peroxidizing DNA, can react with TBA. This is a desirable reaction since deoxyribose is released during DNA peroxidation. Therefore, increases in striatal MDA levels may be the result of DNA peroxidation by t-BuOOH. The standard deviations for MDA levels in 2 and 8 month old mice seem very different. Therefore, differences between the 2 month old control and 8 month old control variances were tested using the variance ratio test. The control variances between the two age groups are significantly different in every region except the hippocampus. This may imply that there is a different mechanism involved between the two age groups in the control of MDA levels in the brain. GSH peroxidase did not exhibit an increase in activity with the administration of t-BuOOH. An inhibition of activity was, however observed in the midbrains of 2 month old mice, and perhaps in the striatums of 8 month old mice between 2 and 5 min after t-BuOOH treatment, at least in comparison to 45 2 month old mice. This is indicative of the inhibition of the enzyme by t- BuOOH as reported by others (53,54) and may be due to the oxidation of a protein sulfhydryl group. GSH peroxidase is probably inhibited by t-BuOOH in some brain regions but recovers by 20 min. It seems possible that in this system, the selenium dependent GSH peroxidase may be responsible for the detoxification of t-BuOOH. Activities of the selenium dependent form of GSH peroxidase have been shown to be much higher than activities of the selenium independent form in the rat brain (45,56). This GSH peroxidase data combined with the MDA data indicate that the midbrain and striatum may be more susceptible than other regions to damage from oxidative stress induced by t- BuOOH. This may be due to the presence of dopamine in these regions, which might be released following t-BuOOH administration. If dopamine is released, it will oxidize with the production of oxygen free radicals. Both the striatum and midbrain contain high levels of iron that may catalyze the formation of free radicals from t-BuOOH, which will damage tissue. It is interesting to note that the behavior of GSH peroxidase and GSSG reductase differ in response to t-BuOOH. GSSG reductase has the ability to increase in activity in the young adult mice. However, GSH peroxidase does not. One explanation of this response may be due to the kinetic mechanisms involved in the regulation of their activities. GSH peroxidase has a low Km (20-50 uM) for most peroxides (53) and is inhibited easily by t-BuOOH (53). 46 GSSG reductase on the other hand, has a high Km (50-130 uM) for GSSG (39,40), but is inhibited by large amounts of GSH and NADPH (also by t- BuOOH, Ki=l 1 mM) (39,40). Under normal cellular conditions, GSSG reductase is inhibited by GSH and NADPH with Ki's in the micromolar range (39). With the administration of t-BuOOH, GSH and NADPH may be depleted and consequently GSSG and NADP+ may be elevated. This activates GSSG reductase activity. However, it is still unclear at this stage how the different substrate ratios of reducing/oxidizing species affect the kinetics of activation and inactivation of mouse brain GSSG reductase activity. Future work (in progress) may clarify how the GSSG/GSH and NADPH/NADP+ ratios and t- BuOOH inhibition correlates with the observed age related differences in GSSG reductase activity in the brain. 47 CHAPTER HI: PHARMACOKINETICS OF IN TRACEREBRO VENTRICULAR T-BUOOH IN YOUNG ADULT AND MATURE MICE. 48 OBJECTIVE Pharmacokinetics parameters of in vivo t-BuOOH in the brain and its distribution in the different regions have not been reported. To compare whether two age groups differ in their ability to eliminate the toxin, the current work compared t-BuOOH pharmacokinetics parameters in six different regions of 2 month old and 8 month mice. EXPERIMENTAL SECTION ANIMALS AND TREATMENT- 2 month old (young adult) and 8 month old (mature) male C57 Bl/6 mice, each weighing approximately 30 g were used. Each animal was anesthetized by injecting 200 mg/kg of ketamine and 2 mg/kg of xylazine ip before t-BuOOH administration and throughout the experiment. All mice were cared for according to University of Southern California and NIH guidelines. For controls, mice were given 5 ul of 0.9% saline ICV. Treated mice were given 5 ul of t-BuOOH as a 70% solution ICV (109.7 mg/kg). This amounts to identical brain doses of t-BuOOH in the two age groups, since their brains weigh the same amount (0.4 g). Mice were decapitated at appropriate times and brains were removed. Brains were dissected in a minute on ice into cortex, striatum, thalamus, hippocampus, midbrain and cerebellum. For GSH and GSSG quantification, brains were dissected at 0,2, and 5 min following t-BuOOH administration. Preliminary studies showed that peak amounts of GSH oxidation occurred at 2 or 5 min. Brain regions were homogenized with 1 ml of either 0.198 g DTNB (bis (3-carboxy-4-nitrophenyl)) disulfide in 100 ml of buffer 1 or 125 mg of N-ethylmaleimide (NEM) solutions in buffer 2 to prevent the artifactual oxidation of GSH (57). 750 ul of potassium buffer pH 7.5 is added to 50 ul of DTNB, 100 ul of GSSG reductase and 3 ul of GSH and 100 ul of NADPH. This is then measured in spectrophotometer at 412 nm. 50 For t-BuOOH quantification, brains were dissected at 0.5, 1, 2, 3, 4, 5, 8,15, 20 ,30 and 60 min after t-BuOOH administration and were placed in dry ice. Brain regions were homogenized using a homogenizer in acetonitrile and centrifuged for 5 min at 10,000 x g at room temperature. The supernatant was mixed with 30 mM pH 6.5 ammonium sulfate buffer and injected onto an HPLC system with an electrochemical detector (58). DATA ANALYSIS t-BuOOH concentration versus time data were analyzed by least square regression analysis. Pharmacokinetics analysis was done following curve fitting with the RSTRIP polyexponential curve stripping program (59). The elimination half life (t1 / 2 ) was determined from fitted curves. Clearance (Cl) was calculated as Cl= F*Dose/AUC. Dose is the total amount administered icv to the brain. F is the fraction of the dose delivered to the specific tissue/ actual total dose delivered to the brain. AUC is the area under the curve of t-BuOOH concentration from time zero to 60 min. Statistical analysis was done by Anova with Newman Keuls test. Regression analysis was done with the SAS statistical analysis software program by SAS Institute Inc. Significant p values were less than or equal to 0.05. All data values are expressed as means and standard deviations. 51 RESULTS The mean 2 month old versus 8 month old t-BuOOH concentration (mg/ml) versus time profiles for each region are presented in figures 22-27. The initial 8 month old mouse cortex, striatum and midbrain t-BuOOH concentration are higher than the 2 month old mouse. However, the initial 8 month old mouse hippocampus and cerebellum t-BuOOH concentration are lower than the 2 month old mouse. The initial thalamus t-BuOOH concentration is the same in both groups. The two age groups t-BuOOH concentration versus time profiles are similar in every region. t-BuOOH concentrations in every region in both age groups decrease by 10 min and disappear by 60 min. The pharmacokinetics parameters for t-BuOOH are presented in tables 4-5. Area under the curve (AUC) values between 2 month old mice and 8 month old mice for all tissues studied tend to be similar. However, AUC values between different regions tend to be different. AUC values are highest in the striatum of the two age groups. These data demonstrate that both age groups accumulate t-BuOOH at the same concentration in every region. This t-BuOOH accumulation is sufficient to damage many cells as reported earlier (26,27,28). The t-BuOOH half lives in 2 month old mice and 8 month old mice are highest in the hippocampus, followed by the midbrain, cortex, thalamus, cerebellum and striatum. The half life values differ from region to region in both age groups, indicating that each region may 1 -n iiO O I I O O H c n N T n A T I O M (m n /m l) o M o o M o » M lit ° 9. a ’ o in o «n o n. <_n to a I - T I i iO O I V C O N C ttN T H A T IO N (n iB /m l) M • * * M M I : £ «n •— * IT -I O “ l E l E l n n a « tv » s D 3 j \ N £ 3 2* . i 3 U l in K R - • i < i i cj a I! if i! if i ; a 1! a ii H II II to to ri n o • V V o o U l In * 1 N U J k .I .V U J.N 'iU N U J H U U » U * I TIMZ (=&:) 1-UliOOII CONCENTRATION (m(|/nil) 55 Tirera 25: iiaer 2aassh old verses S north ole sire t-HaCQH essrestrahion verses hiss esrves is the hippoeasras far n—3. 2 MONTH VS 8 MONTH HIPPOCAMPUS 3 -i ■ S month observed S month calculated s 2TTionth observed ---* : month calculated 1 - C o 0 r 20 30 40 50 TIME (min) I-EiiOOH CONCENTRATION (m\M) 56 ? i g t t r a 2 5 : M ean 2 s n n td i a i d v a r n u s 8 a o s r h a i d s i c s t-3 aO O H c a n s: » « r s u s t i . a n e s r r a s i n %da s i d S r a i a 2 a r n—3 . 1 M O N T H VS S M ON TH MTPB'R ATN 2 - * 8 m cnth observed -* " ■ 8 m cnth calcui£t2d ° 2 m cnth observed — 2 m cnth csicsistsd 0 10 20 30 40 50 60 70 TIME (min) i s t z a n i a u 57 J ijs s a 27: ! « s Z = = — old versoa S aessi: old sxea t-3c00S ooaeastratiaa vmrsss r-an cs srrm a is. Sh« cambttl 7 .^a das a»3. 2 M O N T H VS 8 M O N TH nTRVftFT.T.TTM ^ 1. 0 - es Z c 5- 2 G 2 C U c c Q.8- ■ 8 mcnth observed 8 mcnth calculated 8 2 mcnth observed 2 mcnth calculated 0 10 20 30 40 SO 60 70 TIME (min) 58 TABLE 4: 2 MONTH OLD MOUSE PHARMACOKINETICS PARAMETERS OF t-BUOOH REGION AUC,MIN*MG/ML tj/2,MlN CL,ML/KG* MIN CORTEX 13.7±10.0 16.0±1.53 2.46±1.23 STRIATUM 32.6±28.7 3.53±0.36 2.20±1.94 THALAMUS 13.5±4.85 7.05±6.29 2.27±0.99 HIPPO 10.4±4.59 21.1±7.80 0.70±0.31* MIDBRAIN 8.30±2.70 20.1±6.42 2.74±1.05 CERE 4.13±1.28 4.50±0.51 2.23±0.69 Values are presented as means ± SD with n=3. * represents statistically significant from 8 month old mice. 59 TABLE 5: 8 MONTH OLD MOUSE PHARMACOKINETICS PARAMETERS OF t-BUOOH REGION AUC,MIN*MG/ML 1 1/ 2 ,MIN CL,ML/KG* MIN CORTEX 6.95±2.08 11.5±7.54 2.88±1.01 STRIATUM 13.0±1.85 3.88±1.70 2.23±0.30 THALAMUS 11.8±4.80 11.2±8.34 2.35±0.95 HIPPO 9.44±0.60 19.3±2.66 2.68±0.17 MIDBRAIN 9.39±1.39 14.1±1.72 3.43±0.48 CERE 3.53±2.93 5.14±1.78 5.23±4.34 Values are presented as means ± SD with n=3. 60 have a faster mechanism of elimination. The half life values between the two age groups are very similar. Clearance values for 2 month old and 8 month old mice tend to be similar except for the thalamus. Clearance values vary from region to region in both age groups. Two month old mouse clearance values are highest in the cerebellum, followed by the midbrain, hippocampus, cortex, thalamus and striatum. Eight month old mouse clearance values are highest in the cerebellum, followed by cortex, midbrain, hippocampus, thalamus and striatum. The correlation between t-BuOOH levels and GSSG levels was also done. It might be expected that when t-BuOOH levels are high, GSSG levels should also be high. As t-BuOOH levels decline, GSSG might also decline. However, t-BuOOH levels were highest within the first seconds of administration. GSSG levels were maximal in 2 month old mice at about 2 min, and in 8 month old mice about 5 min (figure 28-33). Perhaps there is a delay in the toxicity of t-BuOOH such that accumulation of GSSG is not maximal until a few min after administration. This delay may be related to t-BuOOH induced inhibition of GSSG reduction or GSSG translocation out of the cell. CORTEX G S S G L E V E L S (NMOLE/G) I F ly iics 211$ (lilllll Invnln In Llio u o r to x lio lo ro mid a l t a r L-UuOUII 1 .! iliiti nt I on. * fl I <|ii I I lonnl I y illffo ren l: from 7 moll III olil c o n t r o l valuuu ( jtcll. Il'i | l>y AIIIIVA w llli llnwiiinii-Kaul n I mil.. ** (ilijni f .Icanl. I y d lf liir n n t Irnm II inonl li o ld c o n t r o l valu aa ( |i < 0 . 0 5 | liy /IIIUVA with Itaw n u iii-K au la tout. 4 0 0 ■ H 2 m o n th s 0 m o n th s 100- 20 o t i m e ; ; t 4 31 STRIATUM G S S G LE V E L S (NMOLE/G) Flguti* 79f t'ASiS | ov**Im In l.lm n tr ln tiim b o l o r c mid n f t o r t-lliiOUII mltnl n I nl.i nl. I «iti. * f? I *|ii I f J rmil. I y illrffrGMl finin 7 mmil!» o ld c o n t r o l valuao |(><O.I1ri| by AtlOVA w llli Howiium-RanIn l o u t . ** ? » I ijii I f Icmti. I y <11 f foriMil: from II inoitllt o ld c o n t r o l v a I u o o (|i<U.U5| by ANUVA w llli (lawman-Kciila t o o t . 5 0 0 4 0 0 - 3 0 0 - 200 - 100 - 0 - -------- , f 4 M O 2 m onlii9 ^ 1 0 m o n th s ♦ » • * . 1 1 < x > M 0 2 B 20 t i m e ; ; % ^ Flyura JOi IIO BO Invulo In Ilia Llialanua lioforu anil a lt a r t-IIUUOII lllllul II I ill l ill I m i . * III i / n 1 11 •mu I I |r i l l f f n i i i i i l f i i i i H 1 m n i i l . l i n l i l u o n t i u l v a lu a a | (•< I I . Il!i I I i / AIKiVA l i l l l i I lii ll in i i II K m l I I I I m i L . ** III ijui f 1 i i i i i i I I y ill I f it rn ii L t mm II nuiiil li nlil u u iitto l Valuaa ||i<(1.05| ly /lllllV A 1 1 1 Hi lliniMaii-Ktiiil u lout. ■H 2 in o n llia fS Z I I I m o n llia 3 0 0 200 100- Q J I -fl_ . 0 80 n 8 tim in ; : 4 HIPPOCAMPUS G S S G L E V E L S (NMOLE/G) F ly tita 111 Unfit! Invnln In Llio lil|niuonm|iiio Im luro nntl a f t e r t-lluUUII otlinl nl nl t nl I m i. * !< I (f III 11 nnnl 1 i l l f f n r a i i l : f rum 1 munt.li n lil u o n lr o l v a lu e s ||i<ll.ll'i| Ity AIIOVA w llli Ilnwimiii-Kotiln l.iint. • * II lijiil 11 until I y tlllln r m il. lunn II moiil.li o ld u o iltr u l valu aa (1><0.U5) by AIIUVA w llli llewmaii-Keiila t a e t . 3 0 0 * tm t 2 inuiilha 1S?1 U inonllis ' 200 - 100- / < y > O a o 20 I MIDBRAIN G S S G L E V E L S (NMOLE/G) F ig u r e 32* GSSU l e v e l s in th e m idbraln b e fo r e anil a f t e r t-BuOOIl aiiini n 1 e t r n t Ion • * S i g n i f i c a n t l y i l i f f e r e u t from 2 month o ld c o n t r o l v a lu e e (|i<0.U 5| by AIIOVA w ith llawmaii-Reule t e s t . ** S i g n i f i c a n t l y ill f fnruiit from 0 inonLli o ld c o n t r o l v a lu e e (|><U.05) by AIIOVA w ith llewman-Keule t e a t . 3 ( M M 2 m o n th s ^ 3 . 0 m o n th s * 3 0 0 - 2 0 0 - • T 1 0 0 - i T 0 - I f f I I f f 0 2 0 20 TIME CEREBELLUM F lyn ro ] ] | ISIilSl! Irvtiln In t.lio cotobnlliiitt b o fo to and a l t a r t-lluUUII ndinl ti I n l in i Inn. * Klgni r lm n l ly d i r i m e n t Crum 2 mantli o ld c o n t r o l valu aa | (|<U. on I by AIIOVA w llli Ilnwmiiii-Kouln tim t. ** Ulyiii lJnanl. ly d l r i n m n t from I) month o ld c o n t r o l valu aa (p < 0 .0 5 ) by AIIOVA w ith llawnan-Kaula t e a t . 400 w m m 2 m o n th s 1 5 3 B M O N T H S o uj* 3 0 0 - O 200 - £> 1 0 0 - (/) CD 20 0 o 2 a\ (Ti t i m e ; ; 4 67 DISCUSSION There are no reports which show pharmacokinetics of t-BuOOH in the brain. However, there are reports on hydrogen peroxide pharmacokinetics parameters. The reported half life of 0.1 mM hydrogen peroxide in the peroxisome is about 2.8 min (60,61). The half life of hydrogen peroxide has been reported to be between 1-18 min depending on the organ (61,62). Pharmacokinetics parameters of t-BuOOH vary from region to region. The mechanism of elimination, alteration of enzyme activities, formation of lipid peroxidation products, and oxidation of GSH also vary from region to region. Our data, in agreement with a previous report (63), show that all brain regions do not behave similarly and that t-BuOOH does not penetrate homogeneously into all regions. Striatal t-BuOOH levels 30 sec after ICV injection are higher than in any other region. The cortex and thalamus demonstrate equal levels of t-BuOOH. The hippocampus, midbrain and cerebellum demonstrate equal levels of t-BuOOH. Overall, the striatum behaves differently from the rest of the brain regions. The striatum, region nearest the site of injection, shows the highest AUC values, in 2 month old and 8 month old mice. Two month old and 8 month old mice half lives, clearance values and volume o f distribution values are the lowest in the striatum. Even though the striatum eliminates t-BuOOH the fastest, the high levels of t-BuOOH in the striatum may alter many enzyme 68 activities through protein oxidation as shown before. With t-BuOOH administration, GSH is oxidized most extensively in the striatum. Since the half lives in the cortex, hippocampus and midbrain are long, more cytotoxicity may occur in these regions. It has been reported that a consequence of oxidative stress is depletion of protein sulfhydryl groups (PSH). Cortical and midbrain PSH groups are oxidized following t-BuOOH administration (62,63). Furthermore, lipid peroxidation, by conjugated diene measurement, is also found in the midbrain following t-BuOOH treatment. In addition to the long half life of t-BuOOH, the midbrain is more vulnerable to lipid peroxidation perhaps due to the high level of iron in the zona reticulata (29,47,63). Comparison of 2 month old and 8 month old mice shows that the cortical half life of t-BuOOH is significantly different between the two age groups. Although the pharmacokinetics parameters are quite similar between the two age groups, 2 month old mice are better at detoxifying the toxin. Perhaps, 2 month old mice, after t-BuOOH, are able to enhance antioxidant enzyme activities. Maintaining high GSSG reductase activity may be vital after t-BuOOH. Our preliminary evidence shows that 2 month old mice are able to increase GSSG reductase activity by 20 min after t-BuOOH (data not shown). Furthermore, GSH is always maintained at high levels after t-BuOOH administration by the 2 month old mice. These data demonstrate that both 2 month old and 8 month old mice have similar pharmacokinetics values for t-BuOOH. This indicates that age dependent toxicity differences are not due to t-BuOOH differential buildup in the brain. This differential toxicity may result from t-BuOOH induced modification of many different antioxidant enzyme activities or other factors. 70 CHAPTER IV: IN VIVO BRAIN GSH TURNOVER RATE IN YOUNG AND MATURE MICE 71 OBJECTIVE The purpose of this study is to measure brain GSH synthetase in 2 month old and 8 month old mice. In vivo GSH turnover rate between the two age groups were also measured. Furthermore, this study also examines the effect of t-BuOOH on the GSH turnover rate. 72 MATERIALS AND METHODS Young adult (2 month old) and mature (8 month old) male C57 Bl/6 mice, each weighing approximately 30 g were used. Each subject was anesthetized by injecting 200 mg/kg of lcetamine and 2 mg/kg of xylazine ip before toxin administration. All mice were cared for according to University Of Southern California and NIH guidelines. For controls, mice were given 5 ul of 0.9% saline ICV. Treated mice were given 5 ul of t-BuOOH as a 70% solution icv (109.7 mg/kg). Brains were removed at appropriate times and were dissected into cortex, striatum, thalamus, hippocampus, midbrain and cerebellum. For GSH synthetase assay, brains were dissected at 0,2,5 and 20 min. GSH synthetase activity was determined by measuring the formation of ADP (64). Mice brains were homogenized in 300 ul of 100 mM tris HC1 buffer. For ADP determination, 75 ul of reaction mixtures containing 100 mM Tris HC1 buffer pH 8.2, 50 mM KC1, 5 mM glutamyl aminobutyrate, 10 mM ATP, 5 mM glycine, 20 mM MgCl2 , 2 mM EDTA and 25 ul of brain homogenates were incubated for 30 min at 37 degrees celsius. This solution is then treated with 0.02 ml of 10% sulfosalicylic acid and a solution containing 0.5 mM phosphoenolpymvate, 0.2 mM NADH, 1 unit of pyruvate kinase, 40 mM MgCl2 , 50 mM KC1 and 250 mM potassium phosphate buffer pH 7.0. The treated solution is then centrifuged and the amount of ADP was calculated from 73 the change in absorbance at 340 nm after the addition of 10 unit (0.1 ml) of lactate dehydrogenase. Protein content was measured by the method of Lowry (50). Ten uCi of 3 5 S cysteine in 4 ul of 0.9% of saline solution was administered icv to young and mature mice. Mice were sacrificed after appropriate times (20 min, 1 hr, 2 hr, 6 hr, 12 hr, 24 hr, 36 hr, 60 hr, 84 hr and 108 hr) and brains were removed. To determine GSH and GSSG specific activity, brains were homogenized in 400 ul of perchloric acid and bathopenanthrolinedisulfonic acid (BPDS). The brains homogenates were centrifuged for 5 min at 10,000g. 200 ul of the supernatant was derivitized with 80 ul of 500 mM iodoacetic acid in 0.2mM m-cresol purple solution to form s-carboxy methyl derivatives. The acidic solution which was pink in color was brought to pH 8-9 by adding 80 ul of 2 M KOH and 2.4 M KHC02. This solution was allowed to incubate in the dark at room temperature for 10 min. The derivatives were conjugated with 300 ul of 5% of 1-fluorodinitrobenzene in ethanol and stored overnight in room temperature and isolated with high pressure liquid chromatography (65). The HPLC allowed quantitation of GSH, GSSG and determination of “ S-GSH and 3 5 S-GSSG. 3 aminopropyl Spherisorb 4.6 x 200 mm, 5 um column was used. The HPLC system with a Shimadzu gradient system with two LC-600 pumps, SCL-6B controller, SIL-6B autosampler, UV visible detector in a series with a flow through B-Ram radiation detector equipped with a 1 ml liquid scintillation cell connected to a computer for UV and radioactive peak integration. To determine rate constants for GSH turnover, 3 5 S total GSH specific activity versus time was plotted. RSTRIP software package (microMath, Scientific Software, Salt Lake City, UT) was used to determine nonlinear least squares iterative minimization. Fractional turnover rate k, is the ratio of 0.693 over half life, k, = 0.693 / 1 1 /2 Turnover time t, is defined as the inverse of fractional turnover rate. t« = 1 / k. The slope of the curve of (pool size)/(initial pool size) over time/(tumover time) is defined as the turnover rate. 75 RESULTS Brain control GSH synthetase activities were lower than reported control liver GSH synthetase activities (64). Control GSH synthetase activities were similar in every brain region and do not appear different in the two age groups (Table 6 and 7). Two month old mouse GSH synthetase activities at 20 min after t-BuOOH were significantly lower than 8 month old activities in every region. In the 8 month old cortex, thalamus and midbrain, GSH synthetase activities at 20 min after t-BuOOH were significantly higher than 8 month old control values. The effect of t-BuOOH on the GSH specific activities at 2,5 and 20 min after t-BuOOH administration was examined (Table 8 and 9). 3 5 S GSH specific activity increased by 5 min after t-BuOOH administration in the 2 month old mice but not in the 8 month old mice when compared to the pooled 2 month old and 8 month old controls by Student's t test (p<0.05). The 2 month old mouse specific activity decreased to control levels by 120 min. The 8 month old 3 5 S GSH specific activity remained the same throughout 120 min after t-BuOOH administration. 3 5 S GSSG specific activity increased by 5 min after t-BuOOH administration in the 2 month old mice. Figure 34 and 35 show the plot of specific activity of 3 5 S GSH from 20 min to 108 hr after administration of 3 3 S cysteine. Figure 36 shows HPLC analysis of brain homogenates of 8 month old mouse 2 hours after t-BuOOH 76 TABLE 6: 2 MONTH OLD MOUSE GSH SYNTHETASE ACTIVITIES (nM GSH/MIN MG PROTEIN) AND THE EFFECTS OF T-BUOOH. REGION CONTROL 2 MIN 5 MIN 20 MIN CORTEX 0.34±0.12 0.32±0.09 0.26±0.09 0.41±0.23 STRIATUM 0.57±0.48 0.83±0.43 0.82±0.70 0.35i0.14 THALAMUS 0.57±0.41 0.54±0.24 0.49±0.36 0.66±0.47 HIPPO 0.51±0.24 0.29±0.11 0.21±0.13 0.34±0.22 MIDBRAIN 0.74±0.34 0.52±0.32 0.28±0.12 0.34±0.09 CEREBELLUM 0.55±0.58 0.80±0.59 0.61±0.45 0.29±0.08 Four mice per group were used in these experiments. Brains were dissected as described in the experimental section. GSH synthetase activity was determined by measuring the formation of ADP in controls, 2 min, 5 min and 20 min after t-BuOOH administration. The amount of ADP was calculated from the change in absorbance at 340 nm after the addition of 1 unit of lactate dehydrogenase. 77 TABLE7: 8 MONTH OLD MOUSE GSH SYNTHETASE ACTIVITIES (NM GSH/MIN MG PROTEIN) AND THE EFFECTS OF T-BUOOH. REGION CONTROL 2 MIN 5 MIN 20 MIN CORTEX 0.60*0.27 0.84±0.61 0.48*0.24 1.81*0.49*+ STRIATUM 0.45±0.14 0.88*1.01 0.30*0.06 1.18*0.13* THALAMUS 0.33*0.21 0.66*0.49 0.66*0.44 1.70*0.12*+ HIPPO 0.34±0.19 0.39*0.06 0.62*0.38 0.81*0.29* MIDBRAIN 0.49*0.38 1.10*0.88 0.72*0.32* 1.91*0.43*+ CEREBELLUM 0.71*0.38 0.84*0.84 1.23*0.67 1.33*0.51* Four mice per group were used in these experiments. Brains were dissected as described in the experimental section. Times are the times following t- BuOOH amdinistration when brains were removed. GSH synthetase activity was determined by measuring the formation of ADP. The amount of ADP was calculated from the change in absorbance at 340 nm after the addition of 1 unit of lactate dehydrogenase. * Significantly different from 2 month old mice by Student's t-test (p<0.05). + Significantly different from 8 month old control values by Anova with Neuman-Keuls test (p<0.05). 78 Table8: GSH specific activity (pci/umole) for 2 month old and 8 month old mice and the effects of t-BuOOH administration. 2 mon-control 0.31±0.20 8 mon-control 0.34±0.04 2 mon-2min 0.18±0.17 8 mon-2min 0.31±0.25 2 mon-5min 0.55±0.06 8 mon-5min 0.33±0.21 2 mon-20min 0.39±0.15 8 mon-20min 0.24±0.19 2 mon-120min 0.31±0.14 8 mon-120min 0.34±0.21 To determine GSH specific activity, brains (n=4 per group) were homogenized in perchloric acid and derivitized with iodoacetic acid to form s-carboxymethyl derivatives. These derivatives were conjugated with 1 fluorodinitrobenzene and characterized with HPLC (64). Times are the times after t-BuOOH administration when brains were removed. 79 Table 9: GSSG specific activity (pci/umole) for 2 month old and 8 month old mice and the effects of t-BuOOH. 2 mon-control 0.54±0.23 8 mon-control 0.39±0.14 2 mon-2 min 0.41±0.34 8 mon-2 min 0.48±0,35 2 mon-5 min 0.71±0.03 8 mon-5 min 0.51±0.18 * 2 mon-20 min 0.59±0.14 8 mon-20 min 0.55±0.32 2 mon-120 min 0.31±0.22 8 mon-120 min 0.34±0.17 To determine GSSG specific activity, brains (n=4 per group) were homogenized in perchloric acid and derivitized with iodoacetic acid to form s-carboxymethyl derivatives. These derivatives were conjugated with 1 fluorodinitrobenzene and characterized with HPLC (64). Times are the times after t-BuOOH administration when brains were removed. * Significantly different from 2 month old mouse values at 5 min (p<0.05). F ig u re 34: GSli tu rn o v e r r a t e o f 2 month o ld mouse. 10 p e l o f 35S c y s te in e was a d m in is te re d to m ice (n -4 p e r g ro u p ). From 20 min to 108 hour l a t e r , b r a in s were removed and an a ly z e d in term s o f CSH s p e c if ic a c t i v i t y . D ata p o in ts re p re s e n t means 1 SEM. 2 Month Old GSH Turnover Rate >* > • m m u < o 0) Q. < n x (0 o 1 m observed calculated .1 .01 0 20 40 60 80 100 120 Time ; 4 G SH Specific Activity F ig u re 35: GStl tu rn o v e r r a t e o f 8 month o ld mouse. 10 /ic l o f 3SS c y s te in e was a d m in is te re d to m ice (n -4 p e r g ro u p ). From 20 min to 108 h r l a t e r , b ra in s were removed and a n a ly z ed in term s o f GSH s p e c if ic a c t i v i t y . D ata p o in ts r e p r e s e n t means ± SEM. 8 Month Old GSH Turnover Rate .01 H 20 40 60 80 100 120 0 Time (hour) 82 Figure 36: HPLC a n a ly s is of h r a is honogenatas o f 8 month o ld mouse 2 h o u rs a f te r c - 2uCQH a d m in is tr a tio n . T ra ce 1 shows d a ta o f th e T JV peaks a t 363 nm. _ T race 2 shows daca o f th e ra d io a c tiv e peaks o f c y s t e i n e , GSH and GSSG. :-s (u Sf a H i 1 I 3 9 C s J9 r * t ES La teSTf fef-Qif*. IQ WTOLE tains iNw ivoii.ass uar*-»v.___ aea * h 9 2 c?* a T r w * I a - i Shir N * a » * © • Stirt i i m m Stoo i id* 1 ‘S W *1 Z T c x a L i i'57 e>3t • r a il xilS m u » t s 5 s 3? 1 2 : Z Z i-i2 U; ;s t3IH ostra Silii bp = : J lu m * 1 1 S -3S (13 - aA C k er^u n a t» M 0 * « d ■ * w » A ll ti^ie m iB C O ^a 14 ISA E t * N o t c Ta.*«c i 3 am1 in«l a- Ku« RS* R S N or*. e.xe Tioo Shift 5* ioe. gotan. tn«oar*l S m . ZTotal T ;m i« iw c X a ---------- - .A I53 - is :a il lS Ui u 70a m fcS 3 — * 1 1 U W -3 S 3 |*wluw rm . » Ml toqSM Cl - gooks • scnsis-sw a. aa« 7A A 11.S 01 m * jr! •v m« t ».*c; ) . administration. Figure 37 and 38 show the plot of specific activity o f3 5 S cysteine from 20 min to 108 hr. The cysteine activities decline sharply and remain low. These data show that the precursor activities are not significant for the analysis of the GSH specific activities. Half lives of GSH turnover were estimated by nonlinear least squares iterative minimization analyses of 3 5 S GSH specific activity. The half life of 2 month old brain GSH calculated from the GSH specific activity time curve was 59.5 hr (Table 10). The half life of 8 month old brain GSH calculated from the GSH specific activity time curve was 79.1 hr. The turnover time for 2 month old brain GSH was 85.7 hr. The turnover time for 8 month old brain GSH was 114 hr. The fractional turnover rate for 2 month old brain GSH was 0.01 hr'1 . The fractional turnover rate for 8 month old brain GSH was 0.01 hr'1 . The turnover rate for 2 month old mice was 0.68. The turnover rate for 8 month old mice was 0.62. I F ig u re 37: C y ste in e s p e c if ic a c t i v i t y o f 2 month o ld mouse from 20 min to 108 hou r. D ata p o in ts re p r e s e n t means ± SEM. 2 moiltli old cysteine specific nctivily 0.4 0.3- 2 monlh observed 0.2 - 0.1 - 0.0 ■ Or 40 80 100 120 60 20 0 Time (hour) CD F ig u re 38: C y ste in e s p e c if ic a c t i v i t y o f 8 month o ld mouse from 20 min to 108 h o u r. D ata p o in ts r e p r e s e n t means ± SEM. 8 m o n t h o ld c y s t e in e s p e c i f ic a c t i v i t y 0.4 0.3- 8 month observed 0.2 - 0.1 0.0 0 20 40 60 SO 100 120 Time (hour) o o U l 86 Table 10: Brain GSH turnover parameters for 2 month old and 8 month old mice. 2 months old 8 months old * 1 /2 59.5 to 79.1 hr turnover time(tt) ss.? hr 114 hr fractional turnover rate (kt) 0.01 hr'1 0.01 hr'1 turnover rate 0.68 0.62 Calculations for turnover parameters are described in the experimental section. 87 DISCUSSION We have reported that icv t-BuOOH decreases brain levels of GSH and increases brain GSSG levels (26). To prevent neuronal degeneration, stimulation of GSH turnover through stimulation of GSH synthetase or GSSG reductase activity may be vital during oxidative stress. T-BuOOH administration may increase GSH synthetase activity. We also found that GSSG reductase activity increases in young mice after t-BuOOH administration but not in mature mice (28). Turnover of GSH is defined as the process of formation and utilization of GSH (69). The formation process is due to the synthesis of GSH from amino acids. These amino acids can come from diet, degradation of proteins and peptides and reduction of GSSG or protein disulfides. The utilization process is due to GSH efflux, oxidation, conjugation and degradation. The liver synthesizes and uses GSH and also exports a huge quantity of GSH into the blood (46). The turnover of GSH in the liver is fast compared with other organs (66). Brain has low GSH turnover and may take GSH from plasma (67). Brain GSH synthetase activity 20 min after t-BuOOH administration in 8 month old mice is higher than in 2 month old mice. Since 8 month old brain GSSG reductase activity does not increase after t-BuOOH treatment, perhaps increased GSH synthetase activity is a possible mechanism for increasing the detoxification of t-BuOOH. However, in 2 month old mice, 88 activation of GSSG reductase may prevent activation of GSH synthetase. The rat brain GSH turnover rate was determined to be 70 hr using 1 4 C glycine (7). The rates of rat GSH turnover in different organs were reported to be very different from the brain (66). Slow non biphasic GSH turnover was found in the lung, skin, forestomach, colon, heart, muscle and blood with half lives of 35-116 hr. The liver and the kidney had rapid turnover rates with half lives of 1-5 hr. 3 5 S cysteine is distributed to every region o f the mouse brain after icv administration. 3 5 cysteine disappears very quickly in both age groups in the first hour. Radioactivity appears rapidly in the GSH pool after icv administration of 3 5 S cysteine (a major precursor o f GSH). GSH turnover is monophasic in the 2 month old mice but it is biphasic in the 8 month old mice, the rapid phase and the slow phase, month old mice absorb higher concentration of cysteine than the 2 month old mice. One possibility of this occurrence is that the 2 month old mice absorb the cysteine in the cytoplasm and immediately incorporate it to the mitochondria and protein pools. Meanwhile the 8 month old mice are retaining the cysteine in the cytoplasm. Eight month old brain GSH specific activity rose rapidly and did not become linear until 20 hr had passed. The specific activity o f GSH decreases over time perhaps due to its catabolism and export out o f the brain. The 2 month old brain GSH specific activity rose rapidly and then declined. 89 However, at 24 hr, GSH radioactivity declined linearly. We chose 24 hr to be the point of t-BuOOH administration. GSH exists in at least two pools, 85-90% in the cytoplasm and 10-15% in the mitochondria (66,67,68,69,70,71). The mitochondrial GSH content in the brain isreported to be 5.89±0.48 nmoles/mg of protein (68). This is about 15% of the total GSH (1.8 uM) in the brain (68). The GSSG content in the brain is reported to be 0.045±0.009 nmoles/mg of protein (68). This is about 0.8% of the total mitochondrial GSH (66). Mitochondrial GSH is a residual pool of GSH which may not be easily depleted by neurotoxins. The mitochondrial pool of GSH may have a profound effect in that it may control hydrogen peroxide formation and ultimately lipid peroxidation. Mitochondrial GSH is vital for cellular defense against endogenous or exogenous oxidant stress (48). If mitochondrial protection from hydrogen peroxide is inhibited, cytoplasmic hydrogen peroxide might cause peroxidation of mitochondrial lipids. Complete depletion of mitochondrial GSH results in gradual loss of cell viability (65). Studies have shown that during oxidative stress in hepatocytes, there is a slow flow of GSH from mitochondria to cytoplasm (67,68). This suggests that this mechanism functions to conserve mitochondrial GSH during cytoplasmic GSH depletion caused by oxidative stress (68). Furthermore, the turnover of GSH depends on several factors such as other compartmental pools. 3 S S cysteine can be incorporated into the GSH pool or the protein pool. Protein turnover was not calculated in this study so there is a strong possibility that the latter part of the curve does not reflect GSH turnover but protein turnover. GSH can be hydrolyzed by extracellular transpeptidase and can be exported elsewhere. The slow phase o f GSH turnover might be due to sequestration of mitochondrial GSH. Two month old mice were able to increase the GSH specific activity during oxidative stress but not the 8 month old mice. This increase in GSH specific activity may be due to release of GSH from intracellular pools or reduction of protein mixed disulfides. The ability of the 2 month old mice to increase the turnover rate of GSH protects them from oxidative stress. 91 CHAPTER V: CONCLUSION OF THE PROJECT 2 month old mice and 8 month old mice have different compensatory mechanisms in detoxifying t-BuOOH. 1. 2 month old mice are able to respond quicker than 8 month old mice in that GSSG levels increase after 2 min of t-BuOOH administration. 2. 2 month old mice after t-BuOOH administration are able to increase GSH in the thalamus and the cerebellum though not significantly. 3. 2 month old mice are able to maintain lipid peroxidation level under control. 4. Furthermore, GSH specific activity of 2 month old mice increaes after 5 min of t-BuOOH administration. 5. 2 month old mice GSSG reductase increases 20 min after t-BuOOH administration. 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