ARTICLE |
Correspondence to: Mark A. Smith, Inst. of Pathology, Case Western Reserve U., 2085 Adelbert Road, Cleveland, OH 44106.
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Summary |
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Formation of carbonyls derived from lipids, proteins, carbohydrates, and nucleic acids is common during oxidative stress. For example, metal-catalyzed, "site-specific" oxidation of several amino acid side-chains produces aldehydes or ketones, and peroxidation of lipids generates reactive aldehydes such as malondialdehyde and hydroxynonenal. Here, using in situ 2,4-dinitrophenylhydrazine labeling linked to an antibody system, we describe a highly sensitive and specific cytochemical technique to specifically localize biomacromolecule-bound carbonyl reactivity. When this technique was applied to tissues from cases of Alzheimer disease, in which oxidative events including lipoperoxidative, glycoxidative, and other oxidative protein modifications have been reported, we detected free carbonyls not only in the disease-related intraneuronal lesions but also in other neurons. In marked contrast, free carbonyls were not found in neurons or glia in age-matched control cases. Importantly, this assay was highly specific for detecting disease-related oxidative damage because the site of oxidative damage can be assessed in the midst of concurrent age-related increases in free carbonyls in vascular basement membrane that would contaminate biochemical samples subjected to bulk analysis. These findings demonstrate that oxidative imbalance and stress are key elements in the pathogenesis of Alzheimer disease.
(J Histochem Cytochem 46:731735, 1998)
Key Words: Alzheimer disease, carbonyls, 2,4-dinitrophenylhydrazine, oxidative damage
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Introduction |
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Although oxidative stress and consequent free radical damage are involved as a major cytopathological mechanism in a number of diseases and in normal aging, methods to document free radical involvement have not grown at the same rate as hypotheses. This gap has stemmed primarily from problems in quantifying free radicals in vivo, because they are rapidly removed by antioxidants or quenched by reaction with biopolymers. In addition, normal metabolism generates abundant free radicals, such that detecting them without knowing the balance of oxidants and antioxidants is not biologically meaningful. This aspect has been circumvented by methods to detect excess radical production. Although detection can be performed using exogenous agents that are specifically modified by free radicals, such as spin traps (
Modifications of proteins and polynucleotides take two forms: (a) adduction reactions by highly reactive intermediate products of lipid peroxidation or glycation, and (b) direct oxidative modification of the macromolecules. In the case of proteins, powerful oxidizing agents such as the hydroxyl radical directly modify amino acid side-chains, resulting in a diverse array of altered amino acids that are used to assess oxidative damage (
Here we report a highly specific and sensitive method to cytologically define oxidative damage that is based on the DNP-H reaction with free carbonyls but involves subsequent immunocytochemical enhancement. The technique is demonstrated using tissue sections from cases of Alzheimer disease and controls, because glycoxidative and lipoperoxidative damage are well documented in the pathological lesions of Alzheimer disease, neurofibrillary tangles and senile plaques (
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Materials and Methods |
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Tissue
The hippocampus extending from the entorhinal cortex to the full hippocampus from six cases of Alzheimer disease (ages 7690), three age-matched controls (ages 7078), and three younger controls (ages 3264) was fixed in Methacarn (methanol:chloroform:acetic acid 60:30:10) at 4C for 24 hr. Tissue was dehydrated through ascending ethanol solutions and xylene and then embedded in paraffin (~60C). Sections of 6 µm sections were prepared with a microtome (Leica), placed on Silane-coated glass slides (Sigma; St Louis, MO), and sequentially deparaffinized in xylene and rehydrated in descending ethanol. Endogenous peroxidase activity was inactivated by treating with 3% H2O2 in methanol for 20 min to block artifactual staining from endogenous peroxidase activity (
Dinitrophenyl Labeling
Sections were covered with 0.10.001% DNP-H in 2 N HCl. All incubations were done in a humidified plastic box. After a 1-hr incubation at room temperature (RT), sections were exhaustively rinsed in TBS, followed by a 30-min incubation in 10% normal goat serum (NGS) to block nonspecific binding sites. After rinsing with 1% NGS/TBS, a rat monoclonal antibody (LO-DNP-2; Zymed, San Francisco, CA) to dinitrophenyl (DNP) was diluted 1:100 in 1% NGS/TBS and incubated with the sections at 4C for 16 hr. Sections were then rinsed with 1% NGS/TBS followed by goat antiserum to rat IgG (BoehringerMannheim; Indianapolis, IN) diluted 1:50 with 1% NGS/TBS. After rinsing in 1% NGS/TBS, rat peroxidaseanti-peroxidase complex (ICN) (1:250) in the same buffer was incubated with the section at RT for 1 hr, after which it was rinsed with 1% NGS/TBS. Peroxidase activity was localized by development for 510 min with 0.015% H2O2 in 50 mM Tris-HCl, pH 7.6, with 0.75 mg/ml 3,3'-diaminobenzidine (Sigma). Development of the sections was directly monitored for maximal contrast under the x10 objective of a Zeiss Axioskop 20 microscope.
Chemical and immunochemical controls were used to define carbonyl-specific binding. Chemical reduction of free carbonyls and Schiff bases was performed by incubating sections with 25 mM sodium borohydride (NaBH4) in 80% methanol for 30 min at RT before incubation with DNP-H. Specific reduction of Schiff bases, while leaving carbonyls intact, was performed with 50 mM sodium cyanoborohydride in 0.1 M phosphate buffer, pH 6.0, for 1 hr at RT. Immunochemical specificity was demonstrated by omission of the antibody to DNP or the DNP-H treatment. Immunoabsorption of the antibody to DNP was performed by incubating the antibody (1:100) with 5 mM pyruvate 2,4-dinitrophenylhydrazone (stock 1 mg/ml in ethanol) at 4C for 16 hr and comparing the resulting immunoreactivity with unabsorbed antibody that had been similarly treated with 5% ethanol.
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Results |
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DNP-H derivatizes free carbonyls on biomacromolecules, leading to DNP adduction to the carbonyl-containing biomacromolecule. In contrast, reaction of DNP-H with Schiff bases formed between exogenous carbonyl compounds and proteinaceous lysine side-chains leads to release of soluble dinitrophenylhydrazones with no fixation of the DNP moiety to the tissue. By examining the sites of DNP adduction from reaction with DNP-H, localization of oxidative damage at the cytological level can be achieved. Without subsequent antibody enhancement, DNP-H showed only faint yellow staining of the large blood vessels in the brains of aged individuals. No staining of other brain cells or of the pathological changes of Alzheimer disease was seen (data not shown). DNP-H reaction and consequent DNP adduction to the walls of large vessels may not be surprising because extracellular matrix proteins abundant in vessels accumulate oxidative modifications, probably owing to their long half-life (
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The specificity of the method to detect free carbonyls was demonstrated by chemical and immunochemical controls. In the former case, reduction of free carbonyls with NaBH4 blocked DNP-H binding (Figure 1D), whereas reduction of Schiff bases with NaCNBH3 was again without effect (not shown). Immunochemical validation was demonstrated by performing all of the following: omitting DNP-H treatment, omitting the antibody to DNP, or absorbing the antibody to DNP with the DNP-H derivative of pyruvate (Figure 1E). These immunochemical controls were critical to ensure the use of an antibody detection technique that is highly sensitive and specific, e.g., we found several different rabbit antisera to DNP that showed such high background staining for neurons that it was often difficult to note specific staining, whereas the rat monoclonal antibody showed no background staining.
Tissue was fixed in the non-crosslinking fixative Methacarn (methanol:chloroform:acetic acid 60:30:10), but we had similar results using unfixed or samples fixed in 80% ethanol. In contrast, tissue fixed in formaldehyde-based fixatives, as well as sections from tissue fixed in Methacarn but later treated with formaldehyde, displayed nonspecific DNP-H adduction throughout the section, making them unsuitable for study.
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Discussion |
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2,4-Dinitrophenylhydrazine (DNP-H), when coupled to immunochemical enhancement, can detect oxidative damage produced by a pathological condition, Alzheimer disease. It is of interest that the adduction of DNP-H was limited to cell bodies of neurons. This is essentially the same localization noted for adducts of the highly reactive lipid peroxidation product hydroxynonenal (
Absence of oxidative damage in the senile plaque is surprising in light of reports that senile plaques are the source of oxygen radicals in Alzheimer disease. First, ß-amyloid, the primary component of senile plaques, is suggested to produce free radicals through a novel mechanism (
Our finding of increased neuronal DNP-H adduction is consistent with biochemical studies demonstrating increased DNP-H-derived adducts by assay of tissue homogenates from normal aging and Alzheimer disease (
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Another approach is to use the enhanced DNP-H immunochemical technique to identify specific proteins modified on immunoblots or by isolation (
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Acknowledgments |
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Supported by grants from the National Institutes of Health (AG09287, AG13846, and AG14249), by the Alzheimer's Association, and by the American Health Assistance Foundation.
Received for publication June 4, 1997; accepted January 7, 1998.
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Literature Cited |
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Butterfield DA, Hensley K, Harris M, Mattson M, Carney J (1994) ß-amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer's disease. Biochem Biophys Res Commun 200:710-715[Medline]
Connor JR, Menzies SL, St. Martin SM, Mufson EJ (1992) A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains. J Neurosci Res 31:75-83[Medline]
Davis RE, Miller S, Herrnstadt C, Ghosh SS, Fahy E, Shinobu LA, Galasko D, Thal LJ, Beal MF, Howell N, Parker WD, Jr. (1997) Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer disease. Proc Natl Acad Sci USA 94:4526-4531
GrundkeIqbal I, Fleming J, Tung YC, Lassmann H, Iqbal K, Joshi JG (1990) Ferritin is a component of the neuritic (senile) plaque in Alzheimer disease. Acta Neuropathol (Berl) 81:105-110[Medline]
Jefferies WA, Food MR, Gabathuler R, Rothenberger S, Yamada T, Yasuhara O, McGeer PL (1996) Reactive microglia specifically associated with amyloid plaques in Alzheimer's disease brain tissue express melanotransferrin. Brain Res 712:122-126[Medline]
Ledesma MD, Bonay P, Colaco C, Avila J (1994) Analysis of microtubule-associated protein tau glycation in paired helical filaments. J Biol Chem 269:21614-21619
Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346-357[Medline]
Montine TJ, Huang DY, Valentine WM, Amarnath V, Saunders A, Weisgraber KH, Graham DG, Strittmatter WJ (1996) Crosslinking of apolipoprotein E by products of lipid peroxidation. J Neuropathol Exp Neurol 55:202-210[Medline]
Nakamura A, Goto S (1995) Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis. J Biochem 119:768-774
Salomon RG, Subbanagounder G, ONeil J, Kaur K, Smith MA, Hoff HF, Perry G, Monnier VM (1997) Levuglandin E2-protein adducts in human plasma and vasculature. Chem Res Toxicol 10:536-545[Medline]
Sayre LM, Zelasko DA, Harris PLR, Perry G, Salomon RG, Smith MA (1997) 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J Neurochem 68:2092-2097[Medline]
Shacter E, Williams JA, Lim M, Levine RL (1994) Differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay. Free Radic Biol Med 17:429-437[Medline]
Smith CD, Carney JM, StarkeReed PE, Oliver CN, Stadtman ER, Floyd RA, Marksberry WR (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci USA 88:10540-10543[Abstract]
Smith MA, Harris PLR, Sayre LM, Beckman JS, Perry G (1997) Widespread peroxynitrite-mediated damage in Alzheimer's disease. J Neurosci 17:2653-2657
Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N (1996) Oxidative damage in Alzheimer's. Nature 382:120-121[Medline]
Smith MA, RudnickaNawrot M, Richey PL, Praprotnik D, Mulvihill P, Miller CA, Sayre LM, Perry G (1995b) Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease. J Neurochem 64:2660-2666[Medline]
Smith MA, Sayre LM, Monnier VM, Perry G (1995a) Radical AGEing in Alzheimer's disease. Trends Neurosci 18:172-176[Medline]
Smith MA, Taneda S, Richey PL, Miyata S, Yan S-D, Stern D, Sayre LM, Monnier VM, Perry G (1994) Advanced Maillard reaction end products are associated with Alzheimer disease pathology. Proc Natl Acad Sci USA 91:5710-5714[Abstract]
Stadtman ER (1993) Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem 62:797-821[Medline]
Sternberger LA (1986) Immunocytochemistry. New York, Wiley
Su JH, Anderson AJ, Cummings BJ, Cotman CW (1994) Immunohistochemical evidence for apoptosis in Alzheimer's disease. Neuroreport 5:2529-2533[Medline]
Szweda LI, Uchida K, Tsai L, Stadtman ER (1993) Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal. Selective modification of an active-site lysine. J Biol Chem 268:3342-3347
Tsang SY, Tam SC, Bremner I, Burkitt MJ (1996) Copper-1,10-phenanthroline induces internucleosomal DNA fragmentation in HepG2 cells, resulting from direct oxidation by the hydroxyl radical. Biochem J 317:13-16[Medline]
Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Nagashima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM (1996) RAGE and amyloid-ß peptide neurotoxicity in Alzheimer's disease. Nature 382:685-691[Medline]
Yan S-D, Chen X, Schmidt A-M, Brett J, Godman G, Zou Y-S, Scott CW, Caputo C, Frappier T, Smith MA, Perry G, Yen S-H, Stern D (1994) Glycated tau protein in Alzheimer disease: a mechanism for induction of oxidant stress. Proc Natl Acad Sci USA 91:7787-7791[Abstract]
Yan SD, Yan SF, Chen X, Fu J, Chen M, Kuppusamy P, Smith MA, Perry G, Godman GC, Nawroth P, Zweier JL, Stern D (1995) Non-enzymatically glycated tau in Alzheimer's disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloid ß-peptide. Nature Med 1:693-699[Medline]
Young HK, Floyd RA, Maidt ML, Dynlacht JR (1996) Evaluation of nitrone spin-trapping agents as radioprotectors. Radiat Res 146:227-231[Medline]