62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 UNCORRECTED PROOF Original Contribution APOLIPOPROTEIN E DEFICIENCY PROMOTES INCREASED OXIDATIVE STRESS AND COMPENSATORY INCREASES IN ANTIOXIDANTS IN BRAIN TISSUE THOMAS B. SHEA,* †‡ EUGENE ROGERS,* ‡§ DAVID ASHLINE, ‡§ DANIELA ORTIZ,* and MIN-SHYAN SHEU  *Center for Cellular Neurobiology and Neurodegeneration Research; † Department of Biological Sciences; ‡ Department of Biochemistry; § Department of Health and Clinical Sciences, University of Massachusetts at Lowell, Lowell, MA, USA; and  Nutrix/AST Products, Inc., Billerica, MA, USA (Received 11 June 2002; Revised 25 June 2002; Accepted 27 June 2002) Abstract—The epsilon 4 allele of the apolipoprotein E gene (ApoE) is associated with Alzheimer’s disease (AD). The extent of oxidative damage in AD brains correlates with the presence of the E4 allele of ApoE, suggesting an association between the ApoE4 genotype and oxygen-mediated damage in AD. We tested this hypothesis by subjecting normal and transgenic mice lacking ApoE to oxidative stress by folate deprivation and/or excess dietary iron. Brain tissue of ApoE-deficient mice displayed increased glutathione and antioxidant levels, consistent with attempts to compensate for the lack of ApoE. Folate deprivation and iron challenge individually increased glutathione and antioxidant levels in both normal and ApoE-deficient brain tissue. However, combined treatment with folate deprivation and dietary iron depleted antioxidant capacity and induced oxidative damage in ApoE-deficient brains despite increased glutathione, indicating an inability to compensate for the lack of ApoE under these conditions. These data support the hypothesis that ApoE deficiency is associated with oxidative damage, and demonstrate a combinatorial influence of genetic predisposition, dietary deficiency, and oxidative stress on oxidative damage relevant to AD. © 2002 Elsevier Science Inc. Keywords—Free radicals, INTRODUCTION The epsilon 4 allele of the apolipoprotein E gene (ApoE) is linked with an increase in, and an earlier age of onset, of sporadic and familial Alzheimer’s disease (AD) [1]. Oxidative damage is elevated in the frontal cortex of AD patients, and the extent of this damage correlates with the presence of the E4 allele of ApoE; lipid peroxidation was significantly elevated in AD cases homozygous for the ApoE4 allele vs. age-matched controls or AD cases homozygous for E3 [2]. In addition, activities of enzy- matic antioxidants including catalase and glutathione peroxidase were also higher in AD cases with at least one epsilon 4 allele of ApoE, while superoxide dismutase activity was unchanged [2]. Supplementation with cer- tain agents such as Ginkgo biloba extract and the neu- rosteroid dehydroepiandrosterone were able to protect control individuals and AD cases bearing one E4 allele and an E3 allele against lipid peroxidation, but were unable to protect E4 homozygotes. These findings prompted Ramassamy and colleagues [2] to advance the hypothesis that the ApoE4 genotype and reactive oxy- gen-mediated damage are linked in the frontal cortex of AD patients. Studies with transgenic mice lacking ApoE suggest that deficiencies in this protein may lead to synaptic loss and cytoskeletal compromise [3]. ApoE-deficient mice also demonstrate increased susceptibility to lipid peroxidation under conditions that promote oxidative stress [4]. Since ApoE is normally secreted by astro- cytes following neuronal injury to redistribute lipid breakdown products [5– 8], deficiency in ApoE func- tion may be crucial following accumulation of reactive oxygen species (ROS) and resultant membrane com- promise [9]. Increased levels of glutathione are present in the central nervous system (CNS) of ApoE- deficient mice, which may represent an attempt to Address correspondence to: Dr. Thomas B. Shea, University of Massachusetts at Lowell, Center for Cellular Neurobiology and Neu- rodegeneration Research, Department of Biological Sciences, Lowell, MA 01854, USA; Tel: (978) 934-2881; Fax: (978) 934-3044; E-Mail: thomas&unbar;shea@uml.edu. Free Radical Biology & Medicine, Vol. 33, No. ●, pp. 0000 – 0000, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(02)01001-8 1 rich2/2b-frb/2b-frb/2b1902/2b6991d02g destepha S=4 8/7/02 13:55 Art: Original contribution AQ: 2