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Ann. N.Y. Acad. Sci. 1048: 369–372 (2005). © 2005 New York Academy of Sciences.doi: 10.1196/annals.1342.041

Differences in Antioxidative Response of Rat Hippocampus and Cortex after Exposure to Clinical Dose of �-Rays

ANA TODOROVIC′, JELENA KASAPOVIC′, SNEŽANA PEJIC′,VESNA STOJILJKOVIC′, AND SNEŽANA B. PAJOVIC′ Laboratory of Molecular Biology and Endocrinology, “Vin�a” Institute of Nuclear Sciences, Belgrade, Serbia and Montenegro

ABSTRACT: Ionizing radiation increases intracellular production of reactiveoxygen species, which can damage cell structure and function. The brain is par-ticularly vulnerable to oxidative injury, and in an area-dependent manner. Inorder to elucidate differences in enzymatic antioxidative response of rat hip-pocampus and cortex, we measured activities of CuZnSOD, MnSOD, and CATin those two brain regions, isolated 1 h and 24 h after exposure to 2 Gy of �-rays. Our results indicate that lower MnSOD activities and inducibility, foundin the hippocampus, are probably some of the main reasons for the particularlygreat oxidative vulnerability of this brain region.

KEYWORDS: hippocampus; cortex; radiation; rat; superoxide dismutase;catalase

Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic or-ganisms, coming from both exogenous and endogenous sources. Exposure to ioniz-ing radiation increases production of ROS and can put irradiated cells into a state ofoxidative stress, which has been implicated in an enormous variety of natural andpathological processes.1 Mammalian brain contains both enzymatic and nonenzy-matic antioxidants against free radical damage. Most abundant antioxidant enzymesare superoxide dismutases (CuZnSOD, MnSOD), which catalyze the dismutation ofO2

•− to H2O2, and enzymes that further convert H2O2 into water (GpX and CAT).As the central nervous system appears to be at particular risk for oxidative damageand especially in an area-dependent manner, we expected that possible regional dif-ferences in inducibility of SODs and CAT in hippocampus and cortex could helphighlight the complex phenomenon of their differing radiosensitivity.

Heads of four-day-old female rats were irradiated with 2 Gy γ-rays, using 60Coas a source of radiation. Animals have been sacrificed 1 h or 24 h after irradiation,hippocampus and cortex were isolated, and homogenates were prepared. Enzyme ac-

Address for correspondence: Dr. Snežana B. Pajovic′, Laboratory of Molecular Biology andEndocrinology, Institute of Nuclear Sciences “Vin�a,” P.O. Box 522, 11001 Belgrade, Serbiaand Montenegro. Voice/fax: 381-11-2455-561.

pajovic@rt270.vin.bg.ac.yu

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FIGURE 1. Relative values of MnSOD (A), CuZnSOD (B), and CAT (C) activity inhippocampus (�) and cortex (�) as a function of time after irradiation with 2 Gy of �-rays.Relative values for enzyme activities are expressed as percent of corresponding controls,which are considered to be 100%.

371TODOROVIC′ et al.: ANTIOXIDANT RADIATION RESPONSE IN RAT BRAIN

tivity of SOD was determined by the method of Misra and Fridovich2 before and af-ter the inhibition of CuZnSOD with KCN.3 The activity of CAT was determined bythe method of Beutler,4 and protein concentration by the method of Lowry et al.5

The results were analyzed by Student’s t-test. Differences between means were con-sidered significant at P < 0.05.

Our results show that, when compared with controls, 2 Gy of γ-rays induce sig-nificant decrease in CuZnSOD activity in hippocampus and cortex, measured 1 h(hippocampus: 12.46 ± 1.60 versus 22.10 ± 2.90 Units/mg protein, P < 0.01; cortex:12.80 ± 1.29 versus 21.35 ± 3.59 Units/mg protein, P < 0.05) and 24 h (hippocam-pus: 13.58 ± 1.72 versus 19.76 ± 1.77 Units/mg protein, P < 0.05; cortex:14.26 ¹ 1.69 versus 19.50 ± 1.96 Units/mg protein, P < 0.05) after exposure. Mn-SOD activity in both examined brain regions was also significantly decreased 1 h af-ter irradiation with 2 Gy γ-rays (hippocampus: 3.99 ± 0.65 versus 6.48 ± 0.75 Units/mg protein, P < 0.05; cortex: 5.62 ± 0.53 versus 6.98 ± 0.49 Units/mg protein,P < 0.05), but 24 h after exposure activity of this enzyme was recovered in the de-gree that depends on the particular brain region: 24 h after irradiation MnSOD activ-ity in the hippocampus achieved almost control values (4.91 ± 0.44 versus5.38 ± 0.48 Units/mg protein, P > 0.05), while at the same time point, MnSOD ac-tivity in the cortex significantly exceed the MnSOD activity of the relevant controls(6.62 ± 0.55 versus 4.91 ± 0.37 Units/mg protein, P < 0.05). We also found that sin-gle dose of 2 Gy �-rays had no effect on the catalase activity in hippocampus andcortex. The activity of this enzyme remained stable 1 h (hippocampus: 11.77 ± 0.62versus 11.84 ± 0.55 Units/mg protein, P > 0.05; cortex: 11.97 ± 0.31 versus12.52 ± 0.42 Units/mg protein, P > 0.05), as well as 24 h (hippocampus: 8.82 ± 0.68versus 8.75 ± 0.61 Units/mg protein, P > 0.05; cortex: 9.76 ± 0.67 versus10.04 ± 0.58 Units/mg protein, P > 0.05) after irradiation.

Although MnSOD is responsible for only 20% of SOD activity in the brain (about80% is due to CuZnSOD activity), it’s known that neurons may tolerate a depletionof cytosolic SOD but are highly vulnerable to depletion of mitochondrial SOD.6 Thisis in agreement with general absence of CuZnSOD radioinducibility in the brain andmay further confirm the connection between lower MnSOD inducibility and greaterradiosensitivity of hippocampus. Our results also show that, in both examined brainregions, a dose of 2 Gy had inhibitory effect on CuZnSOD activity and no effect onCAT activity. Absence of regional differences in radiation effects on CuZnSOD andCAT activities indicate that these enzymes are not implicated in different radiosen-sitivity of cortex and hippocampus.

On the other hand, MnSOD activity in both brain regions is radioinducible, andthis effect is more pronounced in cortex. Lower activities and inducibility of Mn-SOD in hippocampus are probably some of the main reasons for the particularlygreat oxidative vulnerability that characterizes this brain region.

REFERENCES

1. HOLECEK, V. et al. 2002. Free radicals and antioxidants in cerebrospinal fluid in centralnervous system diseases. Cesk. Fysiol. 51: 129–132.

2. MISRA, H.P. & I. FRIDOVICH. 1972. The role of superoxide anion in the autoxidation ofepinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247: 3170–3175.

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3. GELLER, B.L. & D.R WINGE. 1983. A method for distinguishing Cu,Zn- and Mn-con-taining superoxide dismutases. Anal. Biochem. 128: 86–92.

4. BEUTLER, E. 1982. Catalase. In Red Cell Metabolism, a Manual of Biochemical Meth-ods. E. Beutler, Ed.: 105–106. Grune and Stratton. New York.

5. LOWRY, O.H. et al. 1951. Protein measurement with Folin phenol reagent. J. Biol.Chem. 193: 265–275.

6. LINDENAU, J. et al. 2000. Cellular distribution of superoxide dismutases in the rat CNS.Glia 29: 25–34.