Histidine and lysine as targets of oxidative modification

Summary. Histidine and lysine are two representative targets of oxidative modifications. Histidine is extremely sensitive to a metal-catalyzed oxidation, generating 2-oxo-histidine and its ring-ruptured products, whereas the oxidation of lysine generates carbonyl products, such as aminoadipic semialdehyde. On the other hand, both histidine and lysine are nucleophilic amino acids and therefore vulnerable to modification by lipid peroxidation-derived electrophiles, such as 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes, derived from lipid peroxidation. Histidine shows specific reactivity toward 2-alkenals and 4-hydroxy-2-alkenals, whereas lysine is a ubiquitous target of aldehydes, generating various types of adducts. Covalent binding of reactive aldehydes to histidine and lysine is associated with the appearance of carbonyl reactivity and antigenecity of proteins.     

Formation of Nepsilon-(succinyl)lysine in vivo: a novel marker for docosahexaenoic acid-derived protein modification

Free radical-catalyzed peroxidation of docosahexaenoic acid (DHA, C22:6/omega-3) generates various lipid peroxidation products that covalently modify biomolecules such as proteins. Under a free radical-generating system, DHA significantly modified lysine residues in bovine serum albumin. Upon incubation of oxidized DHA with an amino-compound pyridoxamine or a lysine-containing peptide, N-propanoyl and N-succinyl adducts were determined to be the major modification products. The hydroperoxide levels in the oxidized DHA closely reflected the formation of the N(epsilon)-(succinyl)lysine (SUL) upon reaction with the peptide, indicating that the hydroperoxides of DHA represent a potential pathway for the formation of SUL. To detect the DHA-derived protein modification in vivo, we developed a monoclonal antibody (mAb2B12) specific to SUL and found that the antibody specifically reacts with the SUL moiety. The formation of SUL was then immunochemically demonstrated in the liver of mice fed with DHA followed by intraperitoneal injection of carbon tetrachloride (CCl(4)), a hepatic lipid peroxidation model. Immunoreactive materials with mAb2B12 were observed in the DHA + CCl(4) group, but were not significant in the control, DHA-alone, and CCl(4)-alone groups. These data suggest that the formation of DHA-derived adducts such as SUL may be implicated in the oxidative damage observed in DHA-enriched tissues.     

N(epsilon)-(3-methylpyridinium)lysine, a major antigenic adduct generated in acrolein-modified protein

Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N(alpha)-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N(epsilon)-(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.     

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.     

Immunochemical detection of a novel lysine adduct using an antibody to linoleic acid hydroperoxide-modified protein

We have previously prepared the polyclonal antibody to the 13-hydroperoxyoctadecadienoic acid-modified protein (13Ab) (Kato et al. 1997. J. Lipid Res. 38: 1334-1346), however, the epitopes have not yet been structurally identified. In this study, we identified a novel amide-type adduct as one of the major epitopes of 13Ab and characterized the endogenous formation. Upon incubation of the lysine derivative with peroxidized linoleic acid, the formation of N epsilon -(azelayl)lysine (AZL) was confirmed using liquid chromatography-mass spectrometry. The chemically synthesized azelayl protein was significantly recognized by 13Ab. The peroxidation products of different polyunsaturated fatty acids also generated several analogous carboxyalkylamide-type adducts to AZL by the reaction with the lysine derivative, whereas 13Ab specifically recognized AZL, suggesting that the AZL moiety may be one of the major epitopes of 13Ab. The immunoreactive materials of 13Ab were immunohistochemically detected in atherosclerotic lesions from hypercholesterolemic rabbits. More strikingly, the immunoreactivity was significantly enhanced when the sections were treated with alkali or phospholipase A2 for hydrolyzing the ester bonds prior to the staining. These results suggest that the lipid hydroperoxide-derived carboxylic adducts, such as AZL, and their esters linked with phospholipids may be generated in vivo and involved in the pathogenesis of atherosclerosis associated with oxidative stress.     

Carbonyl stress and detoxification ability in the male genital tract and testis of rats

Carbonyl compounds, which are naturally produced and augmented under oxidative stress, have deleterious effects on the reproductive system. The aldo-keto reductase (AKR) family of enzymes catalyze the reductive detoxification of various carbonyl compounds in an NADPH-dependent manner. To elucidate involvement of AKR in detoxification of endogenously produced carbonyls in the male reproductive system, we investigated the differential expression and tissue localization of aldehyde reductase (ALR) and protein adducts produced by reaction with lipid peroxidation products. A strong immunoreactivity to an anti-ALR antibody was observed in the epithelia of the epididymis, vas deferens, seminal vesicle, and prostate gland. Virtually the same cells were stained with a monoclonal antibody (mAb) 5F6, raised against an acrolein-modified protein. In the testis, however, mAb5F6 specifically stained the nuclei of somatic cells and less differentiated spermatogenic cells. While acrolein inactivated glutathione reductase, an enzyme involved in recycling oxidized glutathione, AKR activity was affected at the high concentration only. The colocalization of lipid peroxidation products and AKR in the epithelia of the male genital tract indicates that these tissues are exposed to oxidative stress and possess a protective system coordinately.     

Formation of acrolein-derived 2'-deoxyadenosine adduct in an iron-induced carcinogenesis model

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acrolein-modified 2' -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N6-propano-2' -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2' -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2' -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.     

Site-specific proteomic analysis of lipoxidation adducts in cardiac mitochondria reveals chemical diversity of 2-alkenal adduction

The modification of proteins by lipid peroxidation products has been linked to numerous diseases and age-related disorders. Here we report on the identification of endogenous protein targets of electrophilic 2-alkenals in cardiac mitochondria. An aldehyde/keto-specific chemical labeling and affinity strategy in combination with LC-MS/MS resulted in 39 unique lipoxidation sites on 27 proteins. Several of the target sites were modified by a variety of 2-alkenal products including acrolein, β-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Many of the adduction sites are implicated in the catalytic function of key mitochondrial enzymes suggesting potential impact on pathways and overall mitochondrial function.     

Thiolation of protein-bound carcinogenic aldehyde. An electrophilic acrolein-lysine adduct that covalently binds to thiols

Acrolein, a representative carcinogenic aldehyde that could be ubiquitously generated in biological systems under oxidative stress, shows facile reactivity with the epsilon-amino group of lysine to form N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product (Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) J. Biol. Chem. 273, 16058-16066). In the present study, we determined the electrophilic potential of FDP-lysine and established a novel mechanism of protein thiolation in which the FDP-lysine generated in the acrolein-modified protein reacts with sulfhydryl groups to form thioether adducts. When a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, was incubated with acrolein-modified bovine serum albumin in sodium phosphate buffer (pH 7.2) at 37 degrees C, a significant loss of sulfhydryl groups, which was accompanied by the loss of enzyme activity and the formation of high molecular mass protein species (>200 kDa), was observed. The FDP-lysine adduct generated in the acrolein-modified protein was suggested to represent a thiol-reactive electrophile based on the following observations. (i) N(alpha)-acetyl-FDP-lysine, prepared from the reaction of N(alpha)-acetyl lysine with acrolein, was covalently bound to glyceraldehyde-3-phosphate dehydrogenase. (ii) The FDP-lysine derivative reacted with glutathione to form a GSH conjugate. (iii) The acrolein-modified bovine serum albumin significantly reacted with GSH to form a glutathiolated protein. Furthermore, the observation that the glutathiolated acrolein-modified protein showed decreased immunoreactivity with an anti-FDP-lysine monoclonal antibody suggested that the FDP-lysine residues in the acrolein-modified protein served as the binding site of GSH. These data suggest that thiolation of the protein-bound acrolein may be involved in redox alteration under oxidative stress, whereby oxidative stress generates the increased production of acrolein and its protein adducts that further potentiate oxidative stress via the depletion of GSH in the cells.     

4-Hydroxynonenal-protein adducts: A reliable biomarker of lipid oxidation in liver diseases

The aldehyde 4-hydroxynonenal (HNE) is a major end-product of peroxidation of membrane n-6-polyunsaturated fatty acids. Primary reactants for HNE are the amino acids cysteine, histidine and lysine, and quantitatively, proteins and peptides represent the most important group of HNE-targeted biomolecules. HNE-protein adducts actually elude the metabolism of the aldehyde, particularly active in the liver, so that they can be easily detected in the hepatic tissue itself and in peripheral blood, and quantified by using immunoassays. Since consistently detectable in various liver disease processes and well related to the intensity of necro-inflammation, HNE-protein adducts may be considered a particularly good marker of lipid oxidation during liver injury. In addition, the demonstrated adduction reaction of HNE with important signalling proteins strongly suggests a pathogenetic role for this lipid aldehyde in the progression of liver diseases.     

Acrolein impairs ATP binding cassette transporter A1-dependent cholesterol export from cells through site-specific modification of apolipoprotein A-I

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, but the factors that control its reactions with nucleophilic groups on proteins remain poorly understood. Lipid peroxidation and threonine oxidation by myeloperoxidase are potential sources of acrolein during inflammation. Because both pathways are implicated in atherogenesis and high density lipoprotein (HDL) is anti-atherogenic, we investigated the possibility that acrolein might target the major protein of HDL, apolipoprotein A-I (apoA-I), for modification. Tandem mass spectrometric analysis demonstrated that lysine 226, located near the center of helix 10 in apoA-I, was the major site modified by acrolein. Importantly, this region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid. Indeed, we found that conversion of Lys-226 to N(epsilon)-(3-methylpyridinium)lysine by acrolein associated quantitatively with decreased cholesterol efflux from cells via the ATP-binding cassette transporter A1 pathway. In the crystal structure of truncated apoA-I, Glu-234 lies adjacent to Lys-226, suggesting that negatively charged residues might direct the modification of specific lysine residues in proteins. Finally, immunohistochemical studies with a monoclonal antibody revealed co-localization of apoA-I with acrolein adducts in human atherosclerotic lesions. Our observations suggest that acrolein might interfere with normal reverse cholesterol transport by HDL by modifying specific sites in apoA-I. Thus, acrolein might contribute to atherogenesis by impairing cholesterol removal from the artery wall.     

Metal-catalyzed oxidation of 2-alkenals generates genotoxic 4-oxo-2-alkenals during lipid peroxidation

Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent adducts, which are associated with aging and disease processes. Since lipid peroxidation is a complex process and occurs in multiple stages, there might be yet unknown reaction pathways. Here, we analyzed comprehensively 2′-deoxyguanosine (dG) adducts with oxidized arachidonic acid using liquid chromatography–tandem mass spectrometry and found the formation of 7-(2-oxo-hexyl)-etheno-dG as one of the major unidentified adducts. The formation of this adduct was reproduced in the reaction of dG with 2-octenal and predominantly with 4-oxo-2-octenal (OOE). We also found that other 2-alkenals (with five or more carbons) generate corresponding 4-oxo-2-alkenal-type adducts. Importantly, it was found that transition metals enhanced the oxidation of C4-position of 2-octenal, leading to the formation of OOE-dG adduct. These findings demonstrated a new pathway for the formation of 4-oxo-2-alkenals during lipid peroxidation and might provide a mechanism for metal-catalyzed genotoxicity.     

4-Hydroperoxy-2-nonenal is not just an intermediate but a reactive molecule that covalently modifies proteins to generate unique intramolecular oxidation products

α,β-Unsaturated aldehydes generated during lipid peroxidation, such as 4-oxoalkenals and 4-hydroxyalkenals, can give rise to protein degeneration in a variety of pathological states. Although the covalent modification of proteins by these end products has been well studied, the reactivity of unstable intermediates possessing a hydroperoxy group, such as 4-hydroperoxy-2-nonenal (HPNE), with protein has received little attention. We have now established a unique protein modification in which the 4-hydroperoxy group of HPNE is involved in the formation of structurally unusual lysine adducts. In addition, we showed that one of the HPNE-specific lysine adducts constitutes the epitope of a monoclonal antibody raised against the HPNE-modified protein. Upon incubation with bovine serum albumin, HPNE preferentially reacted with the lysine residues. By employing N(α)-benzoylglycyl-lysine, we detected two major products containing one HPNE molecule per peptide. Based on the chemical and spectroscopic evidence, the products were identified to be the N(α)-benzoylglycyl derivatives of N(ε)-4-hydroxynonanoic acid-lysine and N(ε)-4-hydroxy-(2Z)-nonenoyllysine, both of which are suggested to be formed through mechanisms in which the initial HPNE-lysine adducts undergo Baeyer-Villiger-like reactions proceeding through an intramolecular oxidation catalyzed by the hydroperoxy group. On the other hand, using an HPNE-modified protein as the immunogen, we raised a monoclonal antibody against the HPNE-modified protein and identified one of the HPNE-specific lysine adducts, N(ε)-4-hydroxynonanoic acid-lysine, as an intrinsic epitope of the monoclonal antibody. Furthermore, we demonstrated that the HPNE-specific epitopes were produced not only in the oxidized low density lipoprotein in vitro but also in the atherosclerotic lesions. These results indicated that HPNE is not just an intermediate but also a reactive molecule that could covalently modify proteins in biological systems.     

Carbonylation of mitochondrial proteins in Drosophila melanogaster during aging

Age-related changes in carbonylation of mitochondrial proteins were determined in mitochondria from the flight muscles of Drosophila melanogaster. Reactivity with antibodies against (i) adducts of dinitrophenyl hydrazone (DNP), commonly assumed to react broadly with derivatized carbonyl groups, (ii) malondialdehyde (MDA), or (iii) hydroxynonenal (HNE), was compared at five different ages of flies. MDA and HNE are carbonyl-containing products of lipid peroxidation, which can form covalent adducts with proteins. Specific objectives were to address the following inter-related issues: (1) what are the sources of adducts involved in protein carbonylation in mitochondria during aging; (2) is carbonylation by different adducts detectable solely by the DNP antibodies, as assumed widely; (3) can the adducts formed by lipid peroxidation products in vivo, be used as markers for monitoring age-associated changes in oxidative damage to proteins. The total amounts of immunoreactive proteins, detected by all three antibodies, were found to increase with age; however, the immunodensity of individual reactive bands and the magnitude of the increases were variable, and unrelated to the relative abundance of a protein. While some protein bands were strongly immunopositive for all three antibodies, others were quite selective. The amounts of high molecular weight cross-linked proteins (>200kDa) increased with age. In general, the anti-HNE antibody reacted with more protein bands compared to the anti-MDA or -DNP antibody. The results suggest that sources of the carbonyl-containing protein adducts vary and no single antibody reacts with all of them. Overall, the results indicate that HNE shows robust age-associated increases in adductation with mitochondrial proteins, and is a good marker for monitoring protein oxidative damage during aging.     

Aldehyde adducts generated during lipid peroxidation modification of proteins

Abstract

Various lines of evidence indicate that an important part in the pathogenesis of atherosclerosis is the modification of the plasma low-density lipoproteins (LDLs). A large number of pro-inflammatory and pro-atherogenic properties have been ascribed to the oxidatively modified LDLs and their components. There is considerable evidence to support the role of lipid peroxidation products, reactive aldehydes in particular, originating from the oxidized LDL as important signaling molecules in the context of the atherosclerotic lesion. These aldehydes generated during the peroxidation of LDL exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts on the apolipoprotein B-100 particle in LDL. Characterization of the aldehyde adducts generated in the protein is therefore critical in understanding the nature of the oxidized LDL. However, the majority of adducts generated during the oxidative modification of LDL have not yet been chemically characterized. In this review, the current status of aldehyde adducts quantitatively analyzed in the Cu²⁺-oxidized LDL is reviewed.     

The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Crotonaldehyde is a representative alpha,beta-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA-Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA-Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA-Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.     

New developments in the isoprostane pathway: identification of novel highly reactive gamma-ketoaldehydes (isolevuglandins) and characterization of their protein adducts.

The bicyclic endoperoxide prostaglandin (PG) H2 undergoes nonenzymatic rearrangement not only to PGE2 and PGD2, but also to levuglandins (LG) E2 and D2, which are highly reactive gamma-ketoaldehydes. Isoprostanes (IsoPs) are PG-like compounds that are produced by nonenzymatic peroxidation of arachidonic acid. PGH2-like endoperoxides are intermediates in this pathway. Therefore, we explored whether the IsoP endoperoxides also undergo rearrangement to form IsoLGs. Oxidation of arachidonic acid in vitro resulted in the formation of abundant quantities of compounds that were established to be IsoLGs by using mass spectrometric analyses. However, the formation of IsoLGs could not be detected in biological systems subjected to an oxidant stress. We hypothesized that this was due to extremely rapid adduction of IsoLGs to proteins. This notion was supported by the finding that LGE2 adducted to albumin at a rate that exceeded that of 4-hydroxynonenal by several orders of magnitude: >50% of LGE2 had adducted within 20 s. We therefore undertook to characterize the nature of LG adducts. Using liquid chromatography electrospray tandem mass spectrometry, we established that LGs form oxidized pyrrole adducts (lactams and hydroxylactams) with the epsilon-amino group of lysine. Oxidation of low density lipoprotein resulted in readily detectable IsoLG adducts on apolipoprotein B after enzymatic digestion of the protein to individual amino acids. These studies identify a novel class of ketoaldehydes produced by the IsoP pathway that form covalent protein adducts at a rate that greatly exceeds that of other known aldehyde products of lipid peroxidation. Elucidation of the nature of the adducts formed by IsoLGs provides the basis to explore the formation of IsoLGs in vivo and investigate the potential biological ramifications of their formation in settings of oxidant injury.     

Localization of 4-hydroxy-2-nonenal-modified proteins in kidney following iron overload.

Intraperitoneal (IP) injection of ferric nitrilotriacetate (Fe-NTA) to rats and mice results in iron-induced free radical injury and cancer in kidneys. We sought to clarify the exact localization of acute oxidative damage in Fe-NTA-induced nephrotoxicity by performing immunogold light and electron microscopic (EM) techniques using an antibody against 4-hydroxy-2-nonenal (HNE)-modified proteins. Biochemical assays were done to provide complementary quantitative data. Renal accumulation of lipid peroxidation-derived aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-alkenals (4-HDA), increased in parallel with protein carbonyl content, an indicator of protein oxidation, 30 min after administration of Fe-NTA. Immunogold light microscopy showed that HNE-modified proteins increased at 30 min with positivity localized to proximal tubular cells. Immunogold EM demonstrated that HNE-modified proteins were mainly in the mitochondria and nuclei of the proximal tubular epithelium. The intensity of labeling at both the light and EM levels increased together with levels of biochemically measured lipid peroxidation products and protein carbonyl content. Our data suggest that the mechanism of acute nephrotoxicity of Fe-NTA involves mitochondrial and nuclear oxidative damage, findings that may help to define the mechanisms of iron-induced cell injury.     

The reaction of 2-thiobarbituric acid with biologically active alpha,beta-unsaturated aldehydes

The standard assay for lipid peroxidation is the measurement of the pink, 532 n, absorbing chromogen which is formed upon reaction of 2-thiobarbituric acid (TBA) with the lipid peroxidation product malonaldehyde (MDA). The present studies indicate that the toxic lipid peroxidation product trans-4-hydroxynonenal and its dehydration product trans, trans-nonadienal react with TBA to form chromogens which absorb maximally at 530 and 532 nm, respectively. Other biologically active alpha, beta-unsaturated aldehydes, such as acrolein and crotonaldehyde, short-chain homologs of alkenals formed during lipid peroxidation, and trans,trans-muconaldehyde, a novel diene dialdehyde, react with TBA to form products which absorb maximally at 495 nm. The molar extinction coefficients of the aldehyde: TBA chromogens formed were found to vary widely, suggesting that only small contributions to the 532 nm absorption by TBA adducts of reactive aldehydes other than MDA may be encountered during the use of the TBA assay.     

An anti-CD11d integrin antibody reduces cyclooxygenase-2 expression and protein and DNA oxidation after spinal cord injury in rats

Our studies have shown that treatment with a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 after spinal cord injury (SCI) decreases intraspinal inflammation, myeloperoxidase activity, lipid peroxidation and protein nitration, improving neurological function in rats. Using severe clip compression SCI in the rat, immunohistochemistry and western blotting were employed to assess the effects of an anti-CD11d mAb treatment on spinal cord cyclooxygenase-2 (COX-2) expression, formation of 8-hydroxy-2-deoxyguanosine (8-OHdG, a marker of RNA and DNA oxidation) and protein carbonylation (a marker of protein oxidation). We also assessed treatment effects on the expression of apurinic/apyrimidinic endonuclease (redox effector factor-1, APE/Ref-1), a multifunctional enzyme involved in the base excision repair of apurinic/apyrimidinic sites in DNA. The expression of COX-2 and formation of 8-OHdG and protein carbonyl groups were increased after SCI while APE/Ref-1 expression was decreased. Anti-CD11d mAb treatment clearly attenuated COX-2 expression and 8-OHdG and protein carbonyl formation and rescued APE/Ref-1 expression after SCI. This study suggests that anti-CD11d mAb treatment significantly reduces intraspinal free radical formation after SCI, thereby reducing protein and DNA oxidative damage. These effects likely underlie tissue preservation and improved neurological function resulting from the mAb treatment.