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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=irab20 International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine ISSN: 0020-7616 (Print) (Online) Journal homepage: www.tandfonline.com/journals/irab19 The Effect of X-irradiation on Vitamin E Deficient Rat Liver Mitochondrial ATPase and Cytochrome c Oxidase Selma Korkut To cite this article: Selma Korkut (1978) The Effect of X-irradiation on Vitamin E Deficient Rat Liver Mitochondrial ATPase and Cytochrome c Oxidase, International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 34:4, 385-389, DOI: 10.1080/09553007814551031 To link to this article: https://doi.org/10.1080/09553007814551031 Published online: 03 Jul 2009. Submit your article to this journal Article views: 68 View related articles https://www.tandfonline.com/action/journalInformation?journalCode=irab20 https://www.tandfonline.com/journals/irab19?src=pdf https://www.tandfonline.com/action/showCitFormats?doi=10.1080/09553007814551031 https://doi.org/10.1080/09553007814551031 https://www.tandfonline.com/action/authorSubmission?journalCode=irab20&show=instructions&src=pdf https://www.tandfonline.com/action/authorSubmission?journalCode=irab20&show=instructions&src=pdf https://www.tandfonline.com/doi/mlt/10.1080/09553007814551031?src=pdf https://www.tandfonline.com/doi/mlt/10.1080/09553007814551031?src=pdf INT. J. RADIAT. BIOL ., 1978, VOL . 34, NO. 4, 385-389 The effect of X-irradiation on vitamin E deficient rat liver mitochondrial ATPase and cytochrome c oxidase SELMA KORKUT cekmece Nuclear Research and Education Centre, P. K.1 Havaalani, Istanbul, Turkey (Received 13 February 1978 ; accepted 5 May 1978) 1 . Introduction Although vitamin E plays an essential role in metabolism, the function and mechanism of this compound in the living system is not yet fully understood . The non-enzymatic biological antioxidant function of vitamin E is the earliest accepted hypothesis (Tappel 1965) . The necessity of vitamin E at the cellular level stimulated biologists to examine its role in radiobiology . The concentra- tion of antioxidant vitamin E in tissue and subcellular organelles has been suggested as a major factor regulating the rate of lipid peroxidation in animal tissue after ionizing irradiation (Dawes and Wills 1972), but investigations into the role of vitamin E in radiation protection have led to conflicting views . Haley, McCulloch and McCormick (1954) found that daily intramuscular injections of water-soluble vitamin E into male mice had no effect on their survival times . Contrary to this finding Huber and Schroeder (1962) found that male mice survived longer if pretreated with vitamin E before exposing them to ionizing radiation . It has also been demonstrated that pretreating mice with vitamin E reduces the effect of ionizing radiation in vivo by reducing the permeability and the blood flow in capillaries, and hence the oxygenation of the tissues (Sakamoto and Sakka 1973). Recently Hoffer and Roy (1975) showed that the absence of vitamin E in the diet enhances the fragility of the membrane of erythrocytes of mice exposed to whole-body X-irradiation . The aim of the present study was to decide whether diets either deficient in or supplemented by vitamin E play a role in the effect of whole-body X-irradia- tion on rat liver mitochondrial ATPase and cytochrome c oxidase activities . 2. Materials and methods Two-month-old male albino rats (Rattus norvegicus) weighing about 200 g were used in mitochondrial ATPase and cytochrome c oxidase activity tests. The first group of 36 rats was fed a semi-synthetic diet deficient in vitamin E (Cowthorne, Diplock, Muthy, Bunyan, Murrel and Green 1967) ; the second group of 36 rats was given the same diet supplemented with 17 .64 mg dl-«- tocopherol acetate per 100 g of diet (Hoffer and Roy 1975) . A control group of 36 rats was given a standard (normal) synthetic diet . After 3 weeks on their respective diets, each group was divided into two . Half the subjects in each group were exposed to 600 rad total-body X-irradiation at a dose rate of 24 rad min . The X-ray source was a General Electric Maximar 250 Therapy Unit 3 86 Correspondence operating at 170 kV and 10 mA . The incident beam was filtered through an 0 .5 mm copper/1 . 0 mm aluminium filter . The absorbed doses were measured using a Victoreen Ionizing Chamber . The rats were decapitated immediately after irradiation . The liver mito- chondrial fraction was isolated (Lucena and Depocas 1966) and the mitochon- drial protein was estimated according to Cleland and Slater (1953), with bovine serum albumin as the standard . Isolated rat liver mitochondria were solubilized with digitonin (Schnaitman, Erwin and Greenawalt 1967) before mitochondrial ATPase (Gorrod, Alifano, Papa and Quagliariello 1967) and cytochrome c oxidase (Cooperstein and Lazarow 1951) activity tests were carried out . The ATPase activity is expressed in terms of the amount of inorganic orthophosphate liberated in 15 min, in micromoles, per milligram of mitochondrial protein . The cytochrome c oxidase activity is expressed as the first-order rate constant of oxidation of cytochrome c per 0 . 1 milligram of mitochondrial protein (Smith 1955) . The (x-tocopherol level of liver mitochondria was determined in rats fed on diets deficient in and supplemented by vitamin E, and standard diets . After 3 weeks on their respective diets, each group (eight' 2-month-old male albino rats) was divided into two . Half the subjects in each group were exposed to 600 rad total-body X-irradiation at a dose rate of 24 rad/min. Immediately after irradiation the rats were decapitated and the livers removed . Most of the liver mitochondrial fraction of each rat was isolated, and the non-saponifiable lipids in the mitochondrial fraction separated on a silicic acid column as described by Oliveira, Weglicki, Nason and Nair (1969) . Quantification of a-tocopherol in this fraction was carried out by paper chromatography after being developed in a solvent system of ethanol and water (1 : 1) . Colour was produced with Emmery-Engel reagent and was measured at 540 nm in a Beckman DU spectro- photometer . 3 . Results The inner membrane of mitochondria is particularly important as a site for the localization of vitamin E (Oliveira et al. 1969) and the attachment of ATPase- Mg (Coleman 1973) and cytochrome c oxidase (Vanderkooi, Senior, Capaldi and Hayashi 1972) enzyme systems . In the present experiment, ATPase and cyto- chrome c oxidase activities of isolated rat liver mitochondria were determined after digitonin solubilization . Isolated intact mitochondria show very low or no ATPase (Kielley 1955) and cytochrome c oxidase (Wojtczak and Zaluska 1969) activity ; thus maximum enzyme activity can be reached only by detergent- solubilized or sonicated mitochondria. Stimulation of liver mitochondrial membrane fragility in the vitamin E-deficient group (Vos, Molenaar, Searl-van Leeuwen and Hommes 1972) may produce higher ATPase and cytochrome c oxidase activity, although maximum enzyme activities were not changed, as compared to the control group . Tables 1 and 2 show that liver mitochondrial ATPase activity was stimulated, and cytochrome c oxidase activity inhibited, in the irradiated vitamin E deficient group . Intracellular vitamin E in liver is found mostly in the mitochondrial and microsomal fractions (Grinna 1976) . As found by Green (1972), tissues such Correspondence 3 87 as liver and blood absorb more of the administered doses of vitamin E than other organs, but are depleted more rapidly . Table 3 shows that after the rats had been on their respective diets for 3 weeks, the liver mitochondrial vitamin E level decreased in those fed a diet deficient in vitamin E . The results given in table 3 indicate that 600 rad whole- body X-irradiation does not affect the oz-tocopherolcontent of liver mitochondria in rats fed on diets deficient in and supplemented by vitamin E, and standard diets . Table 1 . Liver mitochondrial ATPase activity of non-irradiated and irradaited rats . Table 2 . Liver mitochondrial cytochrome c oxidase activity of non-irradiated and irradiated rats . Table 3 . «-Tocopherol level of isolated rat liver mitochondria . 100 mg of liver mitochon- drial non-saponifiable lipids obtained from the whole liver of each rat . The results are the averages of four experiments . Diet and irradiation of rats Number of rats Number of experiments /tg (x-tocopherol/100 mg non-saponifiable liver mitochondrial lipids Standard 4 4 7 . 89 Standard, irradiated 4 4 7 . 96 Vitamin E deficient 4 4 2 .42 Vitamin E deficient, 4 4 2 .47 irradiated Vitamin E supplemented 4 4 8 . 05 Vitamin E supplemented, 4 4 8 .02 irradiated Diet Cytochrome c oxidase activity Irradiated Non-irradiated Standard 2 . 150 ± 0.148 2 . 117 ± 0 . 163 1 Vitamin E deficient 0 . 658 ± 0 . 172 2 . 119 ± 0 .410 Vitamin E supplemented 2 . 137 ± 0 . 152 2 . 144 ± 0 . 256 Diet ATPase activity Irradiated Non-irradiated Standard 14 . 77 + 0 . 16 15 . 34 + 0 .71 Vitamin E deficient 24 . 51 + 0 . 43 15 . 13 + 0.17 Vitamin E supplemented 15-39+0 . 11 14-86+0 .56 388 Correspondence 4. Discussion The results given in tables 1 and 2 show that in the irradiated, vitamin E deficient group, liver mitochondrial ATPase was stimulated and cytochrome c oxidase inhibited after irradiation . Irradiation of isolated liver mitochondria in vitro indicates that an increase in the radiation dose resulted in an increase in lipid peroxidation (Wills and Wilkinson 1966) . However, the results given in table 3 show that no differences in liver mitochondrial vitamin E level were ob- served after X-irradiation of rats fed on diets deficient in and supplemented by vitamin E, and standard diets . These findings suggest that liver mitochondrial vitamin E does not act as an antioxidant in radiation-produced oxidation reactions in mitochondria. Changes in liver mitochondrial ATPase and cytochrome c oxidase activity in the irradiated, vitamin E deficient group cannot, therefore, be attributed to the stimulation of oxidation reactions as a result of depleted anti- oxidant vitamin E in mitochondria. Glavind, Hansen and Faber (1965) found that high doses of ionizing radiation in vivo reduce the level of water-soluble antioxidants in liver, while the fat-soluble antioxidants were unaffected . They concluded that the water-soluble non-protein antioxidants act as free radical scavengers in radiation-induced oxidation reactions . Tables 1 and 2 indicate that in rats fed on vitamin E supplemented and standard diets, liver mitochondrial ATPase and cytochrome c oxidase were not influenced by whole-body X-irradiation . Sanner and Pihl (1969) concluded that enzymes embedded in the lipoprotein of the membrane structure are ex- pected to be only slightly affected by radiation-produced water radicals . In accordance with this hypothesis, the present work suggests that, due to the bind- ing of ATPase and cytochrome c oxidase to the mitochondrial inner-membrane structure, their activities were not altered by moderate doses of X-rays . It was reported that the primary biological role of vitamin E is as a membrane compositional and functional stabilizer (Lucy 1972) . According to this view, vitamin E stabilizes membranes by specific physico-chemical interaction between its phytyl side-chain and the fatty acyl chain derived from arachidonic acid . Results of the present work suggest that the liver mitochondrial inner- membrane structure may be altered by a diet deficient in vitamin E, and conse- quently mitochondrial inner-membrane-bound ATPase and cytochrome c oxidase are more easily influenced by X-irradiation under the conditions used . ACKNOWLEDGMENTS I wish to thank Professor Peter Alexander of the Chester Beatty Research Institute for his encouragement, and Professor Atif Sengun, Department of Radiobiology, Istanbul University, Professor Hedi Fritz-Niggli, Strahlen- biologisches Institut der Universitat Zurich, and Professor Peter L . Pedersen, Department of Physiological Chemistry, The Johns Hopkins University for their advice . REFERENCES CLELAND, K. W ., and SLATER, E . C., 1953, Biochem . Y., 53, 547. COLEMAN, N. 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VOS, J ., MOLENAAR, I., SEARLE-VAN LEEUWEN, M ., and HOMMES, F. A., 1972, Ann. N.Y. Acad. Sci ., 203, 74 . WILLS, E. D., and WILKINSON, A. E ., 1966, Biochem . Y., 99, 657 . WOJTCZAK, L ., and ZALUSKA, H., 1969, Biochim. biophys . Acta, 193, 64 . page 1 page 2 page 3 page 4 page 5
