relative importance of intracellular glutathione peroxidase and

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played by free radicals in cardiovascular diseases selenoenzyme, glutathione peroxidase (GSH-Px).8 in humansl-a and laboratory animals.s Since cardiacĀ ...
RELATIVE IMPORTANCE OF INTRACELLULAR GLUTATHIONE PEROXIDASE AND CATALASE IN VIVO FOR PREVENTION OF PEROXIDATION TO THE HEART

BY THOMAS W SIMMONS. I SIRAJ JAMALL

R eprinted from Cardiov ascular Resear ch Vo I ume XXIII, N o . 9, pages7 74-7 1 9, Sep t emb er 1989

CoPYRTGHT @)19E9 RESEARCH CARDrovAscuLAR ALL RIGHTS OF REPRODUCTIONOF THIS REPRINT ARE RESERVED IN ALL COUNTRIES OF THE WORLD

LONDON BRITISH MEDICAL ASSOCIATION TAVTSTOCKSQUARE,LONDONWCl

Cardiovascular Research, 1989, 23. 17+-179

Relativeimportanceof intracellularglutathione peroxidaseandcatalasein vivo for preventionot' peroxidationto the heart THOMAS W SIMMONS," I SIRAJ JAMALL From theToxicologyProgram, St.John's University, Jamaica,New York, USA ABSTRACT The relative importance in vivo of catalaseand the selenoenzymeglutathioneperoxidase for protection against peroxidation was assessedin the rat heart. Each of these enzymes was modulated by feeding animals a low selenium diet either unsupplementedor supplementedwith 0.5 parts per million of selenium, with or without the catalaseinhibitor, 3-amino-1,2,4-triazole, in their drinking water. After 8 weeks, selenium deficient rats had 887o reductions in cytosolic and mitochondrial glutathione peroxidase activities. These reductions were accompaniedby increased peroxidation in heart homogenatesand mitochondrial suspensions.Since increased mitochondrial peroxidation only occurred when both the cytosolic and mitochondrial glutathione peroxidase activities were compromised, these selenoenzymesappearto work in tandem and reductionsin both are a prerequisitefor increasedperoxidation in this organ. Peroxidationdid not occur in aminotriazole treated animals even though cytosolic catalase activity was inhibited by 65-807o. Moreover, inhibition of catalase activity did not exacerbate the level of peroxidation in selenium deficient animals depleted of glutathione peroxidase activity. Because increased peroxidation was only associatedwith reductions in glutathione peroxidase activity irrespective of catalase activity, the selenoenzyme appears to be more important for detoxification of hydrogen peroxide in the heart. There has been a burgeoningof interest in the role played by free radicals in cardiovasculardiseases laboratory animals.s in humansl-a and Hypoxanthine/xanthine oxidasegeneratedsuperoxide (HzO) havebeen anion(O2.-)andhydrogenperoxide implicated as major contributors_to myocardial heart.67Mammalian damagein the postischaemic tissuespossessan enzymaticdefencesystemwhich protectsagainstthesereactiveoxygenspecies. Inthis system,superoxide dismutase(SOD)converts02'- to

H2O2which can be metabolisedby catalaseand the glutathioneperoxidase(GSH-Px).8 selenoenzyme, Sincecardiactissuecontainslow levelsof catalase relative to glutathioneperoxidasecomparedto other tissues such as liver, it has been postulatedthat catalase plays only a minor role, if any,^ in detoxificationofhydrogenperoxideintheheart.e-rl On the other hand, thereis also evidenceto suggest that catalasefunctions as a major pathway..for detoxificationof hydrogenperoxidein thisorgan."-''

upresenr address: Department or Biophysics, Environmerrar ft::iifii3,ul,?I,i#i1.1#i"0"",ff:::i"rji:Tf -;---,-.---,---.; -,--. Health Sciences Center, University of Rochester Schoot oi

glutathione peroxidase and catalase *li:::".:oth Address for correspondenceand reprints: Dr I Siraj Jamall, Invivostudiesinourlaboratoryusingcadmiumand -cardiic Toxic substances Control Division, Technical Services. selenium --.:-..-.-; enzymatic to modulate -" the Departmentof Health Services,7141744 P Street, Sacramento, .--, ^ anti-oxidant defence system suggest that glutathione CAg4z34_732o, USA. peroxidase than catalase for is more important Key words: glutathione peroxidase; catalase; peroxidation; detoxificationof hydrogenperoxide.ll However, only aminotriazole;heart; cytosol: mitochondria. glutathione peroxidase, and not catalase, activity wai depleted in these studies. In order to test this Submittedt2 February 1988 Accepted tI April 1989 hypothesis more directly, rats were fed a low or high Medicine, Rochester,Nv r+6+2, dsa.

774

Cardiac anti-oxidant enzymes

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selenium diet with or without the catalase inhibitor, 3-amlno-t,2,4-triazole (3AT). In this way we were able to assess the relative importance, in vivo, of sustained alterations of intracellular glutathione peroxidase and catalase activities in the myocardium. Methods EXPERIMENTAL

ANIMALS

AND

TREATMENT

Male, weanling Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing between 40 and 50 g on receipt were randomly assigned to one of four treatment groups (5-6 rats per group). The animals were individually housed in polypropylene cages with wire bottoms in our (AAALAC accredited) animal facilities (22 + 1"C,45 + 5Vorelative humidity, 12 h light cycle). All rats were given a tortula yeast based feed low in selenium (less than 0.02 parts per million (ppm) by analysis) which was purchased premixed from DYETS (Pennsylvania) and consisted of torula least30Vo, sucrose3OVo,com starch29Vo, tocopherol stripped corn oil 67o, Bemhardt-Tomarelli salt mix (without added zinc and copper) 4Vo and AIN-76re vitamin mix lVo. The concentration in the basal diet of copper was 5.0 (SD 1.2) ppm, of zinc was 33.4(4.7) ppm and of iron was 128.0(21.3) ppm by analysesin pentuplicate. This diet is in accordance with the recommendations of the American Institute of Nutrition for laboratory rats.ra Animals received deionised drinking water either unsupplemented, or supplemented with 0.5 ppm selenium as sodium selenite or O.2Vo(w/v) 3AT alone, or both selenium and 3AT together. Rats were examined daily and weighed weekly. Age matched rats (n : 9) fed a commercial chow (Purina no 5001, containing 0. 13 ppm selenium and 8 ppm copper by analysis) showed the following levels of enzymes in their heart: (1) Cytosol: glutathione peroxidase 112 (SD 8.0) nmol NADPH oxidised.min-r.mg-r protein; CuSOD 172(38.2) Sigma units.mg-r protein; catalase 1.89(0.32) k.s-r'mg-r protein x l0-3. Q) Mitochondria: glutathione peroxidase 35. 6(6. 7) nmol NADPH oxidised.min-r.mg-r protein; MnSOD 33.0(5.5) Sigma units'mg-r protein. NECROPSY

AND

TISSUE

HOMOGENISATION

At the end of 8 weeks, animals were killed by decapitation under light ether anaesthesia. Dietary 3AT treatment allowed us to study the effects of a sustained reduction of cardiac catalase activity. The heart was excised, weighed, blotted dry and minced using stainless steel scissors. A lOVo (w/v) homogenate in 0.25 M sucrose was prepared using a motor driven pestle. Cytosolic Teflon and mitochondrial fractions were obtained by differential centrifugation according to Murfitt et al.ts All

biochemical assays were completed within 72 h of tissue homogenisation. GLUTATHIONE

PEROXIDASE

ASSAY

The activity of the selenoenzymewas assayedby the method of Paglia and Valentinero as modified by Burk et al." Oxidation of NADPH by hydrogen peroxide (0.25 M) was followed spectrophotometrically at room temperature (25 + 2"C) using a Perkin-Elmer Lambda 3A spectrophotometer equipped with a R-IOOA recorder. Enzyme activity is expressed in nmol NADPH oxidised.min I.mg-r protein. CATALASE

ASSAY

Catalase was assayed in heart cytosolic fractions according to the method of Cohen et al.t8 Catalase activity was calculated from the first order rate constant k : log (SJS,) x 2.303/5, where So is the initial substrate concentration, S, the final substrate concentrationand the reaction time (t) is 180 seconds. Catalaseactivity is expressedas k.s-r.mg-r protein x l0-r. PROTEIN

DETERMINATION

Protein was measured according to the dye binding method of Bradfordre using Coomassie Biue. Bovin serum albumin was used as a standard. PEROXIDATION

Peroxidation was quantitated,in lOVo (w/v) tissue homogenates and mitochondrial suspensions using thiobarbituric acid (TBA) using absorbanceat 532 nm (,4,532)for the TBA chromophore accordins to the method of Ohkawa et al2o as modified bv Jairall and Smith.2r Values for tissue peroxidation (TBARS) were not normalised using sample protein or standardisedagainst malondialdehyde. Since each of these variables only ^^partially represents the composition of TBARS," " reporting data per mg protein or as malondialdehyde equivalents might misrepresent the extent of peroxidation. STATISTICAL

ANALYSIS

The data obtained were analysedby two way analysis of variance (ANOVA) for comparison among the four treatment groups. Where interactions were significant (p