Scavenging dicarbonyls with 5'-O-pentyl-pyridoxamine increases HDL net cholesterol efflux capacity and attenuates atherosclerosis and insulin resistance.

OBJECTIVE: Oxidative stress contributes to the development of insulin resistance (IR) and atherosclerosis. Peroxidation of lipids produces reactive dicarbonyls such as Isolevuglandins (IsoLG) and malondialdehyde (MDA) that covalently bind plasma/cellular proteins, phospholipids, and DNA leading to altered function and toxicity. We examined whether scavenging reactive dicarbonyls with 5'-O-pentyl-pyridoxamine (PPM) protects against the development of IR and atherosclerosis in Ldlr-/- mice. METHODS: Male or female Ldlr-/- mice were fed a western diet (WD) for 16 weeks and treated with PPM versus vehicle alone. Plaque extent, dicarbonyl-lysyl adducts, efferocytosis, apoptosis, macrophage inflammation, and necrotic area were measured. Plasma MDA-LDL adducts and the in vivo and in vitro effects of PPM on the ability of HDL to reduce macrophage cholesterol were measured. Blood Ly6Chi monocytes and ex vivo 5-ethynyl-2'-deoxyuridine (EdU) incorporation into bone marrow CD11b+ monocytes and CD34+ hematopoietic stem and progenitor cells (HSPC) were also examined. IR was examined by measuring fasting glucose/insulin levels and tolerance to insulin/glucose challenge. RESULTS: PPM reduced the proximal aortic atherosclerosis by 48% and by 46% in female and male Ldlr-/- mice, respectively. PPM also decreased IR and hepatic fat and inflammation in male Ldlr-/- mice. Importantly, PPM decreased plasma MDA-LDL adducts and prevented the accumulation of plaque MDA- and IsoLG-lysyl adducts in Ldlr-/- mice. In addition, PPM increased the net cholesterol efflux capacity of HDL from Ldlr-/- mice and prevented both the in vitro impairment of HDL net cholesterol efflux capacity and apoAI crosslinking by MPO generated hypochlorous acid. Moreover, PPM decreased features of plaque instability including decreased proinflammatory M1-like macrophages, IL-1ß expression, myeloperoxidase, apoptosis, and necrotic core. In contrast, PPM increased M2-like macrophages, Tregs, fibrous cap thickness, and efferocytosis. Furthermore, PPM reduced inflammatory monocytosis as evidenced by decreased blood Ly6Chi monocytes and proliferation of bone marrow monocytes and HSPC from Ldlr-/- mice. CONCLUSIONS: PPM has pleotropic atheroprotective effects in a murine model of familial hypercholesterolemia, supporting the therapeutic potential of reactive dicarbonyl scavenging in the treatment of IR and atherosclerotic cardiovascular disease.

Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis.

BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.

Mitochondrial Isolevuglandins Contribute to Vascular Oxidative Stress and Mitochondria-Targeted Scavenger of Isolevuglandins Reduces Mitochondrial Dysfunction and Hypertension.

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr-/- mice.

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr-/- mice, a model of FH. Compared to hypercholesterolemic Ldlr-/- mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr-/- mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

Scavenging 4-Oxo-2-nonenal.

4-Oxo-2-nonenal (ONE), a product of cellular lipid oxidation, reacts nonspecifically with the lysine residues of proteins and is generated in increased amounts during degenerative diseases and cancer. We show that pyridoxamine, salicylamine, and related 2-aminomethylphenols react with ONE, to form pyrrolo[2,1-b][1,3]oxazines with the participation of both the amino and the phenolic groups. 2-Aminomethylphenols react with ONE as well as with the Michael adducts of ONE much more rapidly than lysine, suggesting their use for therapeutically scavenging ONE.

Isolevuglandin-type lipid aldehydes induce the inflammatory response of macrophages by modifying phosphatidylethanolamines and activating the receptor for advanced glycation endproducts.

AIMS: Increased lipid peroxidation occurs in many conditions associated with inflammation. Because lipid peroxidation produces lipid aldehydes that can induce inflammatory responses through unknown mechanisms, elucidating these mechanisms may lead to development of better treatments for inflammatory diseases. We recently demonstrated that exposure of cultured cells to lipid aldehydes such as isolevuglandins (IsoLG) results in the modification of phosphatidylethanolamine (PE). We therefore sought to determine (i) whether PE modification by isolevuglandins (IsoLG-PE) occurred in vivo, (ii) whether IsoLG-PE stimulated the inflammatory responses of macrophages, and (iii) the identity of receptors mediating the inflammatory effects of IsoLG-PE. RESULTS: IsoLG-PE levels were elevated in plasma of patients with familial hypercholesterolemia and in the livers of mice fed a high-fat diet to induce obesity and hepatosteatosis. IsoLG-PE potently stimulated nuclear factor kappa B (NFκB) activation and expression of inflammatory cytokines in macrophages. The effects of IsoLG-PE were blocked by the soluble form of the receptor for advanced glycation endproducts (sRAGE) and by RAGE antagonists. Furthermore, macrophages derived from the bone marrow of Ager null mice failed to express inflammatory cytokines in response to IsoLG-PE to the same extent as macrophages from wild-type mice. INNOVATION: These studies are the first to identify IsoLG-PE as a mediator of macrophage activation and a specific receptor, RAGE, which mediates its biological effects. CONCLUSION: PE modification by IsoLG forms RAGE ligands that activate macrophages, so that the increased IsoLG-PE generated by high circulating cholesterol levels or high-fat diet may play a role in the inflammation associated with these conditions.

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

DC isoketal-modified proteins activate T cells and promote hypertension.

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1ß, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.

Levuglandin forms adducts with histone h4 in a cyclooxygenase-2-dependent manner, altering its interaction with DNA.

Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has long been linked with the development of cancer, although little is known about any epigenetic effects of COX-2. A product of COX-2 activation, levuglandin (LG) quickly forms covalent bonds with nearby primary amines, such as those in lysine, which leads to LG-protein adducts. Here, we demonstrate that COX-2 activity causes LG-histone adducts in cultured cells and liver tissue, detectable through LC-MS, with the highest incidence in histone H4. Adduction is blocked by a γ-ketoaldehyde scavenger, which has no effect on COX-2 activity as measured by PGE2 production. Formation of the LG-histone adduct is associated with an increased histone solubility in NaCl, indicating destabilization of the nucleosome structure; this is also reversed with scavenger treatment. These data demonstrate that COX-2 activity can cause histone adduction and loosening of the nucleosome complex, which could lead to altered transcription and contribute to carcinogenesis.

A liquid chromatography-tandem mass spectrometry method for measurement of N-modified phosphatidylethanolamines.

N-Acyl phosphatidylethanolamines (NAPEs) are synthesised in response to stress in a variety of organisms from bacteria to humans. More recently, nonenzymatic modification of the ethanolamine headgroup of phosphatidylethanolamine (PE) by various aldehydes, including levuglandins/isoketals (which are gamma-ketoaldehydes [gammaKAs] derived from arachidonic acid), has also been demonstrated. The levels of these various N-modified PEs formed during stress and their biological significance remain to be fully characterized. Such studies require an accurate, facile, and cost-effective method for quantifying N-modified PEs. Previously, NAPE and some of the nonenzymatically N-modified PE species have been quantified by mass spectrometry after hydrolysis to their constituent N-acylethanolamine by enzymatic hydrolysis, most typically with Streptomyces chromofuscus phospholipase D. However, enzymatic hydrolysis is not cost-effective for routine analysis of a large number of samples, and hydrolytic efficiency may vary for different N-modified PEs, making quantitation more difficult. Therefore, we sought a robust and inexpensive chemical hydrolysis approach. Methylamine (CH(3)NH(2))-mediated deacylation has previously been used in headgroup analysis of phosphatidylinositol phosphates. Therefore, we developed an accurate assay for NAPEs and gammaKA-PEs using CH(3)NH(2)-mediated deacylation and quantitation of the resulting glycerophospho-N-modified ethanolamines by liquid chromatography-tandem mass spectrometry.

Characterization of scavengers of gamma-ketoaldehydes that do not inhibit prostaglandin biosynthesis.

Expression of cyclooxygenase-2 (COX-2) is associated with the development of many pathologic conditions. The product of COX-2, prostaglandin H(2) (PGH(2)), can spontaneously rearrange to form reactive gamma-ketoaldehydes called levuglandins (LGs). This gamma-ketoaldehyde structure confers a high degree of reactivity on the LGs, which rapidly form covalent adducts with primary amines of protein residues. Formation of LG adducts of proteins has been demonstrated in pathologic conditions (e.g., increased levels in the hippocampus in Alzheimer's disease) and during physiologic function (platelet activation). On the basis of knowledge that lipid modification of proteins is known to cause their translocation and to alter their function, we hypothesize that modification of proteins by LG could have functional consequences. Testing this hypothesis requires an experimental approach that discriminates between the effects of protein modification by LG and the effects of cyclooxygenase-derived prostanoids acting through their G-protein coupled receptors. To achieve this goal, we have synthesized and evaluated a series of scavengers that react with LG with a potency more than 2 orders of magnitude greater than that with the epsilon-amine of lysine. A subset of these scavengers are shown to block the formation of LG adducts of proteins in cells without inhibiting the catalytic activity of the cyclooxygenases. Ten of these selective scavengers did not produce cytotoxicity. These results demonstrate that small molecules can scavenge LGs in cells without interfering with the formation of prostaglandins. They also provide a working hypothesis for the development of pharmacologic agents that could be used in experimental animals in vivo to assess the pathophysiological contribution of levuglandins in diseases associated with cyclooxygenase up-regulation.

Characterization of covalent adducts of nucleosides and DNA formed by reaction with levuglandin.

Enhanced expression of cyclooxygenase-2 (COX-2) is associated with development of several cancers. The product of COX-2, prostaglandin H(2) (PGH(2)), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form the highly reactive levuglandin E(2) (LGE(2)) or D(2) (LGD(2)). Incubation with LGE(2) causes DNA-protein cross-linking in cultured cells, suggesting that levuglandins can directly react with DNA. We report the identification by liquid chromatography-tandem mass spectrometry of a stable levuglandin-deoxycytidine (LG-dC) adduct that forms upon reaction of levuglandin with DNA. We found that LGE(2) reacted with deoxycytidine, deoxyadenosine, or deoxyguanosine in vitro to form covalent adducts with a dihydroxypyrrolidine structure, as deduced from selective ion fragmentation. For LG-deoxycytidine adducts, the initial dihydroxypyrrolidine structure converted to a pyrrole structure over time. Reaction of LG with DNA yielded a stable LG-dC adduct with a pyrrole structure. These results describe the first structure of levuglandinyl-DNA adducts and provide the tools with which to evaluate the potential for LG-DNA adduct formation in vivo.

Low concentrations of reactive gamma-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation.

Oxidative stress has been strongly implicated in pathological processes. Isoketals are highly reactive gamma-ketoaldehydes of the isoprostanes pathway of free radical-induced peroxidation of arachidonic acid that are analogous to cyclooxygenase-derived levuglandins. Because aldehydes, that are much less reactive than isoketals, have been shown to trigger platelet activation, we investigated the effect of one isoketal (E(2)-IsoK) on platelet aggregation. Isoketal potentiated aggregation and the formation of thromboxane B(2) in platelets challenged with collagen at a concentration as low as 1 nM. Moreover, the potentiating effect of 1 nM isoketal on collagen-induced platelet aggregation was prevented by pyridoxamine, an effective scavenger of gamma-ketoaldehydes. Furthermore, we provide evidence for the involvement of p38 mitogen-activated protein kinase in isoketal-mediated platelet priming, suggesting that isoketals may act upstream the activation of collagen-induced cytosolic phospholipase A(2). Additionally, the incubation of platelets with 1 nM isoketal led to the phosphorylation of cytosolic phospholipase A(2). The cytosolic phopholipase A(2) inhibitors AACOCF3 and MAFP both fully prevented the increase in isoketal-mediated platelet aggregation challenged with collagen. These results indicate that isoketals could play an important role in platelet hyperfunction observed in pathological states such as atherosclerosis and thrombosis through the activation of the endogenous arachidonic acid cascade.

Globin s-propyl cysteine and urinary N-acetyl-S-propylcysteine as internal biomarkers of 1-bromopropane exposure.

1-Bromopropane (1-BP), an alternative to ozone-depleting solvents, is a neuro and reproductive toxicant in animals and humans. In this study, the dose responses for urinary AcPrCys and S-propylcysteine (PrCys) adducts on globin and neurofilaments were determined as a function of 1-BP exposure level and duration in the rat; and globin PrCys adducts and urinary AcPrCys were quantified in samples obtained from workers in a 1-BP production facility. Rats were exposed to 1-BP by inhalation for 2 weeks at 0, 50, 200, or 800 ppm and to 1-BP at 0 or 50 ppm for 4 weeks. After the 4-week exposures ended, half of the animals were euthanized immediately and half euthanized 8 days later. Urinary AcPrCys was measured using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and gas chromatograph-mass spectrometry (GC/MS); and PrCys adducts were determined on globin and neurofilaments using LC/MS/MS. In rats, PrCys adduct and urinary AcPrCys levels demonstrated a linear dose response relative to exposure level. PrCys globin adducts demonstrated a linear cumulative dose response over the 4-week exposure period. Elimination of AcPrCys appeared biphasic with detectable levels still present in urine up to 8 days postexposure. A significant increase in globin PrCys adducts was observed in the 1-BP workers relative to control workers; and urinary AcPrCys increased with increasing 1-BP ambient exposure levels. The results of these studies demonstrate the ability of 1-BP to covalently modify proteins in vivo and support the potential of urinary AcPrCys and globin PrCys adducts to serve as biomarkers of 1-BP exposure in humans.

Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity.

Isoketals and levuglandins are highly reactive gamma-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge alpha-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive gamma-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogues of pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogues, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogues to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.

Dietary copper enhances the peripheral myelinopathy produced by oral pyrrolidine dithiocarbamate.

The neurotoxic hazard of a dithiocarbamate is influenced by route of exposure and acid stability of the dithiocarbamate. As an example, oral administration of the acid labile dithiocarbamate N,N-diethyldithiocarbamate (DEDC) causes a central-peripheral axonopathy thought to result from acid-promoted decomposition to CS2 in the stomach. In contrast, parenteral administration of DEDC, which bypasses the acidic environment of the stomach, causes a primary demyelination that is thought to be mediated through the intact parent dithiocarbamate. The relative acid stability of pyrrolidine dithiocarbamate (PDTC) suggests that a significant portion of a dose can be absorbed intact following oral exposure with the potential to produce a primary myelin injury. The present study was performed to characterize the neurotoxicity of PDTC and evaluate the possible role of copper in dithiocarbamate-mediated demyelination. Male Sprague Dawley rats were administered PDTC in drinking water and given either a normal- or high-copper diet for 18, 47, or 58 weeks. Examination of peripheral nerve by light microscopy and electron microscopy at the end of exposures revealed primary myelin lesions and axonal degeneration in the PDTC groups, with a significant increase in the severity of several lesions observed for the PDTC, high-copper group relative to the PDTC normal-copper diet. ICP-AES metal analysis determined that the PDTC groups had significantly increased brain copper, and at 58 weeks a significant increase in copper was seen in the sciatic nerve of PDTC high-copper animals relative to PDTC normal-copper diet animals. Although RP-HPLC analysis could not detect globin alkylaminocarbonyl cysteine modifications analogous to those seen with parenteral DEDC, LC/MS/MS identified (pyrrolidin-1-yl carbonyl)cysteine adducts on PDTC-exposed rat globin. These findings are consistent with previous studies supporting the ability of acid-stable dithiocarbamates to mediate myelin injury following oral exposure. The greater severity of lesions associated with dietary copper supplementation and elevated copper levels in nerve also suggests that perturbation of copper homeostasis may contribute to the development of myelin lesions.

Localization of isoketal adducts in vivo using a single-chain antibody.

Isoketals are highly reactive gamma-ketoaldehydes formed by the oxidation of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2alpha). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the number of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo.

Induction of apoptosis in colorectal carcinoma cells treated with 4-hydroxy-2-nonenal and structurally related aldehydic products of lipid peroxidation.

The oxidation of polyunsaturated fatty acids during oxidative stress gives rise to a series of toxic alpha,beta-unsaturated aldehydes, including the electrophile 4-hydroxy-2-nonenal (4-HNE) and the related aldehydes, 4-hydroperoxy-2-nonenal (4-HPNE) and 4-oxo-2-nonenal (4-ONE). We synthesized these compounds, as well as the resolved enantiomers of 4-HNE, and compared their toxicities and apoptotic responses in the human colorectal cancer cell line RKO. All of these molecules execute similar death responses at comparable doses over almost identical time frames in RKO cells. The apoptotic response induced by 4-HPNE, 4-ONE, and 4-HNE enantiomers involves activation of caspases, proteolysis of downstream caspase targets, and nucleosomal DNA fragmentation. The results presented herein suggest that these molecules commonly activate certain signaling pathways that control cell death irrespective of their reactive properties.

Mitochondrial oxidation of 4-hydroxy-2-nonenal in rat cerebral cortex.

4-hydroxy-trans-2-nonenal (HNE) is a neurotoxic product of lipid peroxidation whose levels are elevated in multiple neurodegenerative diseases and CNS trauma. The detoxification of HNE may take the route of glutathione conjugation to the C3 carbon and the oxidation or reduction of the C1 aldehyde. In this work, we examined whether the oxidation of HNE to its corresponding carboxylic acid, 4-hydroxy-trans-2-nonenoate (HNEAcid) was detoxifying event, if it occurred in rat cerebral cortex, and in which subcellular compartments. Our results show that HNEAcid did not form protein adducts and was non-toxic to Neuro 2A cells. HNEAcid formation occurred in rat cerebral cortex slices following exposure to HNE in a time-dependent and dose-dependent fashion. Homogenate studies indicated that HNEAcid formation was NAD+ dependent. Subcellular fractionation demonstrated that mitochondria had the highest specific activity for HNEAcid formation with a KM of 21 micro m HNE. These data indicate that oxidation of HNE to its corresponding acid is a major detoxification pathway of HNE in the CNS and that mitochondria play a role in this process.