Alcohol Metabolic Inefficiency: Structural Characterization of Polymorphism-Induced ALDH2 Dysfunctionality and Allosteric Site Identification for Design of Potential Wildtype Reactivators

Liver mitochondrial aldehyde dehydrogenase 2 (ALDH2) enzyme is responsible for the rapid conversion of acetaldehyde to acetic acid. ALDH2 (E487K) polymorphism results in an inactive allele (ALDH2*2) which cause dysfunctional acetaldehyde metabolism. The 3D structure of an enzyme is crucial to its functionality and a disruption in its structural integrity could result in its metabolic inefficiency and dysfunctionality. Allosteric targeting of polymorphs could facilitate the restoration of wildtype functionalities in ALDH2 polymorphs and serve as an advancement in the treatment of associated diseases. Therefore, structural insights into ALDH2*2 polymorph could reveal the varying degree of alterations which occur at its critical domains and accounts for enzymatic dysfunctionality. In this study, we report the structural characterization of ALDH2*2 polymorph and its critical domains using computational tools. Our findings revealed that the polymorph exhibited significant alterations in stability and flexibility at the catalytic and co-enzyme-binding domain. Moreover, there was an increase in the solvent-exposed surface residues and this indicates structural perturbations. Analysis of the interaction network at ALDH2*2 catalytic domain revealed residual displacement and interaction loss when compared to the wildtype thereby providing insight into the catalytic inefficiency of the polymorph. Interestingly, perturbations induced by ALDH2 polymorphism involves the re-orientation of surface residues, which resulted in the formation of surface exposed pockets. These identified pockets could be potential sites for allosteric targeting. The findings from this study will aid the design of novel site-specific small molecule reactivators with the propensity of restoring wildtype activities for treatment of polymorphic ALDH2 related diseases.     

Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal

4-Oxonon-2-enal (4ONE) was demonstrated to be a product of lipid peroxidation, and previous studies found that it was highly reactive toward DNA and protein. The present study sought to determine whether carbonyl reductase (CR) catalyzes reduction of 4ONE, representing a potential pathway for metabolism of the lipid peroxidation product. Recombinant CR was cloned from a human liver cDNA library, expressed in Escherichia coli, and purified by metal chelate chromatography. Both 4ONE and its glutathione conjugate were found to be substrates for CR, and kinetic parameters were calculated. TLC analysis of reaction products revealed the presence of three compounds, two of which were identified as 4-hydroxynon-2-enal (4HNE) and 1-hydroxynon-2-en-4-one (1HNO). GC/MS analysis confirmed 4HNE and 1HNO and identified the unknown reaction product as 4-oxononanal (4ONA). Analysis of oxime derivatives of the reaction products via LC/MS confirmed the unknown as 4ONA. The time course for CR-mediated, NADPH-dependent 4ONE reduction and appearance of 4HNE and 1HNO was determined using HPLC, demonstrating 4HNE to be a major product and 1HNO and 4ONA to be minor products. Simulated structures of 4ONE in the active site of CR/NADPH calculated via docking experiments predict the ketone positioned as primary hydride acceptor. Results of the present study demonstrate that 4ONE is a substrate for CR/NADPH and the enzyme may represent a pathway for biotransformation of the lipid. Furthermore, these findings reveal that CR catalyzes hydride transfer selectively to the ketone but also to the aldehyde and C=C of 4ONE, resulting in 4HNE, 1HNO, and 4ONA, respectively.     

Mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites are in vivo markers of oxidative stress

Oxidative stress-induced lipid peroxidation leads to the formation of cytotoxic and genotoxic 2-alkenals, such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). Lipid-derived reactive aldehydes are subject to phase-2 metabolism and are predominantly found as mercapturic acid (MA) conjugates in urine. This study shows evidence for the in vivo formation of ONE and its phase-1 metabolites, 4-oxo-2-nonen-1-ol (ONO) and 4-oxo-2-nonenoic acid (ONA). We have detected the MA conjugates of HNE, 1,4-dihydroxy-2-nonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), the lactone of HNA, ONE, ONO, and ONA in rat urine by liquid chromatography-tandem mass spectrometry comparison with synthetic standards prepared in our laboratory. CCl(4) treatment of rats, a widely accepted animal model of acute oxidative stress, resulted in a significant increase in the urinary levels of DHN-MA, HNA-MA lactone, ONE-MA, and ONA-MA. Our data suggest that conjugates of HNE and ONE metabolites have value as markers of in vivo oxidative stress and lipid peroxidation.     

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.     

Polymorphism of aldehyde dehydrogenase and alcohol sensitivity

The metabolism of acetaldehyde has received considerable attention in the past years owing to its acute and chronic toxic effects in humans. Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of acetaldehyde in liver and other organs. Two major isozymes of hepatic ALDH (ALDH I or E2 and ALDH II or E1), which differ in their structural and functional properties, have been characterized in humans. The ALDH I with a low Km for acetaldehyde is predominantly of mitochondrial origin and ALDH II which has a relatively higher Km is of cytosolic origin. An inherited deficiency of ALDH I isozyme has been found among Japanese and Chinese which is primarily responsible for producing acute alcohol sensitivity symptoms (flushing response) after drinking mild doses of alcohol. Biochemical, immunochemical and molecular genetics data indicate a structural mutation in the ALDH I isozyme gene responsible for the loss in catalytic activity. Population genetic studies indicate a wide prevalence of this ALDH polymorphism among individuals of Mongoloid race. Flushing response to alcohol shows familial resemblances and preliminary family data from Japan, China and Korea hint to an autosomal codominant inheritance for ALDH I isozyme deficiency. The ALDH polymorphism is apparently responsible for the low incidence of alcoholism in Japanese, Chinese and Koreans. Alcohol-induced sensitivity due to ALDH isozyme deficiency may act as an inhibitory factor against excessive alcohol drinking thereby imparting a protection against alcoholism.     

The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase

Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE.     

Aldehyde dehydrogenase 2 inhibition potentiates 4-hydroxy-2-nonenal induced decrease in angiogenesis of coronary endothelial cells

Coronary endothelial cell (EC) dysfunction including defective angiogenesis is reported in cardiac diseases. 4-Hydroxynonenal (4HNE) is a lipid peroxidation product, which is increased in cardiac diseases and implicated in cellular toxicity. Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial enzyme that metabolizes 4HNE and reduces 4HNE-mediated cytotoxicity. Thus, we hypothesize that ALDH2 inhibition potentiates 4HNE-mediated decrease in coronary EC angiogenesis in vitro. To test our hypothesis, first, we treated the cultured mouse coronary EC (MCEC) lines with 4HNE (25, 50, and 75 μM) for 2 and 4 hours. Next, we pharmacologically inhibited ALDH2 by disulfiram (DSF) (2.5 μM) before challenging the cells with 4HNE. In this study, we found that 4HNE attenuated tube formation which indicates decreased angiogenesis. Next, we found that 4HNE has significantly downregulated the expressions of vascular endothelial growth factor receptor (VEGFR) 2 (P < .05 for mRNA and P = .005 for protein), Sirtuin 1 (SIRT 1) (P < 0.0005 for mRNA), and Ets-related gene (ERG) (P < 0.0001 for mRNA and P < 0.005 for protein) in MCECs compared with control. ALDH 2 inhibition by DSF potentiated 4HNE-induced decrease in angiogenesis (P < 0.05 vs 4HNE at 2 h and P < 0.0005 vs 4HNE at 4 h) by decreasing the expressions of VEGFR2 (P < 0.005 for both mRNA and protein), SIRT 1 (P < 0.05), and ERG (P < 0.005) relative to 4HNE alone. Thus, we conclude that ALDH2 acts as a proangiogenic signaling molecule by alleviating the antiangiogenic effects of 4HNE in MCECs.     

Inactivation of cytosolic aldehyde dehydrogenase via S-nitrosylation in ethanol-exposed rat liver

Aldehyde dehydrogenase (ALDH) isozymes are critically important in the metabolism of acetaldehyde, thus preventing its accumulation after ethanol-exposure. We previously reported that mitochondrial ALDH2 could be inactivated via S-nitrosylation in ethanol-exposed rats. This study was aimed at investigating whether cytosolic ALDH1, with a relatively-low-Km value (11-18 microM) for acetaldehyde, could be also inhibited in ethanol-exposed rats. Chronic or binge ethanol-exposure significantly decreased ALDH1 activity, which was restored by addition of dithiothreitol. Immunoblot analysis with the anti-S-nitroso-Cys antibody showed one immunoreactive band in the immunoprecipitated ALDH1 only from ethanol-exposed rats, but not from pair-fed controls, suggesting S-nitrosylation of ALDH1. Therefore inactivation of ALDH1 via S-nitrosylation can result in accumulation of acetaldehyde upon ethanol-exposure.     

Aldehyde dehydrogenase activity as the rate-limiting factor for acetaldehyde metabolism in rat liver

The velocity of acetaldehyde metabolism in rat liver may be governed either by the rate of regeneration of NAD from NADH through the electron transport system or by the activity of aldehyde dehydrogenase (ALDH). Measurements of oxygen consumption revealed that the electron transport system was capable of reoxidizing ALDH-generated NADH much faster than it was produced and hence was not rate-limiting for aldehyde metabolism. To confirm that ALDH activity was the rate-limiting factor, low-Km ALDH in slices or intact mitochondria was partially inhibited by treatment with cyanamide and the rate of acetaldehyde metabolism measured. Any inhibition of low-Km ALDH resulted in a decreased rate of acetaldehyde metabolism, indicating that no excess of low-Km ALDH existed. Approximately 40% of the metabolism of 200 microM acetaldehyde in slices was not catalyzed by low-Km ALDH. Fifteen of this 40% was catalyzed by high-Km ALDH. A possible contribution by aldehyde oxidase was ruled out through the use of a competitive inhibitor, quinacrine. Acetaldehyde binding to cytosolic proteins may account for the remainder. By measuring acetaldehyde accumulation during ethanol metabolism, it was also established that low-Km ALDH activity was rate-limiting for acetaldehyde oxidation during concomitant ethanol oxidation.     

Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.     

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure–activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde ≥C2=C3 double bond>>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six–nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.     

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure-activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde >/=C2=C3 double bond>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six-nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.     

Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.     

Reactivity of atropaldehyde,a felbamate metabolite in human liver tissue in vitro

Antiepileptic therapy with a broad spectrum drug felbamate (FBM) has been limited due to reports of hepatotoxicity and aplastic anemia associated with its use. It was proposed that a bioactivation of FBM leading to formation of alpha,beta-unsaturated aldehyde, atropaldehyde (ATPAL) could be responsible for toxicities associated with the parent drug. Other members of this class of compounds, acrolein and 4-hydroxynonenal (HNE), are known for their reactivity and toxicity. It has been proposed that the bioactivation of FBM to ATPAL proceeds though a more stable cyclized product, 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one (CCMF) whose formation has been shown recently. Aldehyde dehydrogenase (ALDH) and glutathione transferase (GST) are detoxifying enzymes and targets for reactive aldehydes. This study examined effects of ATPAL and its precursor, CCMF on ALDH, GST and cell viability in liver, the target tissue for its metabolism and toxicity. A known toxin, HNE, which is also a substrate for ALDH and GST, was used for comparison. Interspecies difference in metabolism of FBM is well documented, therefore, human tissue was deemed most relevant and used for these studies. ATPAL inhibited ALDH and GST activities and led to a loss of hepatocyte viability. Several fold greater concentrations of CCMF were necessary to demonstrate a similar degree of ALDH inhibition or cytotoxicity as observed with ATPAL. This is consistent with CCMF requiring prior conversion to the more proximate toxin, ATPAL. GSH was shown to protect against ALDH inhibition by ATPAL. In this context, ALDH and GST are detoxifying pathways and their inhibition would lead to an accumulation of reactive species from FBM metabolism and/or metabolism of other endogenous or exogenous compounds and predisposing to or causing toxicity. Therefore, mechanisms of reactive aldehydes toxicity could include direct interaction with critical cellular macromolecules or indirect interference with cellular detoxification mechanisms.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

Metabolism of 4-hydroxynonenal by the rat hepatoma cell line MH1C1

The metabolism of the toxic lipid peroxidation product 4-hydroxynonenal was investigated in the well-differentiated rat heptoma cell line MH1C1. When exposed to 0.1 mM 4-hydroxynonenal (HNE), MH1C1 cells consumed it in a time-dependent manner. There was a linear relationship between the amount of aldehyde consumed and cell number in the range 0.5 - 4 X 10(6) cells ml-1. This process was unaffected by pyrazole, suggesting that alcohol dehydrogenase is not involved. The whole homogenate of MH1C1 cells consumed added HNE at a rate similar to that in intact cells. Fractionation of the homogenate showed that the highest HNE-metabolizing activity is in the cytosol. The dialysed cytosol had almost no capacity to metabolize HNE, but this was restored by supplementation with NAD, NADH, NADP and NADPH. The metabolism of HNE in MH1C1 cells is thus different from that in hepatocytes, which were shown to utilize cytosolic alcohol dehydrogenase for this process. Both reductive and oxidative pathways could be implicated in the metabolic activity of MH1C1 cells towards HNE as well as binding by glutathione.     

Quantitation of mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites in a smoking cessation study

The breakdown of polyunsaturated fatty acids (PUFAs) under conditions of oxidative stress results in the formation of lipid peroxidation (LPO) products. These LPO products such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) can contribute to the development of cardiovascular and neurodegenerative diseases and cancer. Conjugation with glutathione, followed by further metabolism to mercapturic acid (MA) conjugates, can mitigate the effects of these LPO products in disease development by facilitating their excretion from the body. We have developed a quantitative method to simultaneously assess levels of 4-oxo-2-nonen-1-ol (ONO)-MA, HNE-MA, and 1,4-dihydroxy-2-nonene (DHN)-MA in human urine samples utilizing isotope-dilution mass spectrometry. We are also able to detect 4-hydroxy-2-nonenoic acid (HNA)-MA, 4-hydroxy-2-nonenoic acid lactone (HNAL)-MA, and 4-oxo-2-nonenoic acid (ONA)-MA with this method. The detection of ONO-MA and ONA-MA in humans is significant because it demonstrates that HNE/ONE branching occurs in the breakdown of PUFAs and suggests that ONO may contribute to the harmful effects currently associated with HNE. We were able to show significant decreases in HNE-MA, DHN-MA, and total LPO-MA in a group of seven smokers upon smoking cessation. These data demonstrate the value of HNE and ONE metabolites as in vivo markers of oxidative stress.     

Trans-4-hydroxy-2-hexenal is a neurotoxic product of docosahexaenoic (22:6; n-3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n-3) acid (DHA) is elevated in the CNS in patients with Alzheimer's disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans -4-hydroxy-2-hexenal (HHE), a six carbon analog of the n-6 fatty acid derived trans -4-hydroxy-2-nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD₅₀'s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration- and time-dependent manners. The time-dependent formation of HHE differed from that of F₄-neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5-fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.     

Dietary regulation of catabolic disposal of 4-hydroxynonenal analogs in rat liver

Our previous work in perfused rat livers has demonstrated that 4-hydroxynonenal (HNE) is catabolized predominantly via β oxidation. Therefore, we hypothesized that perturbations in β oxidation, such as diet-altered fatty acid oxidation activity, could lead to changes in HNE levels. To test our hypothesis, we (i) developed a simple and sensitive GC/MS method combined with mass isotopomer analysis to measure HNE and HNE analogs, 4-oxononenal (ONE) and 1,4-dihydroxynonene (DHN), and (ii) investigated the effects of four diets (standard, low-fat, ketogenic, and high-fat mix) on HNE, ONE, and DHN concentrations in rat livers. Our results showed that livers from rats fed the ketogenic diet or high-fat mix diet had high ω-6 polyunsaturated fatty acid concentrations and markers of oxidative stress. However, high concentrations of HNE (1.6 ± 0.5 nmol/g) and ONE (0.9 ± 0.2 nmol/g) were found only in livers from rats fed the high-fat mix diet. Livers from rats fed the ketogenic diet had low HNE (0.8 ± 0.1 nmol/g) and ONE (0.4 ± 0.07 nmol/g), similar to rats fed the standard diet. A possible explanation is that the predominant pathway of HNE catabolism (i.e., β oxidation) is activated in the liver by the ketogenic diet. This is consistent with a 10-fold decrease in malonyl-CoA in livers from rats fed a ketogenic diet compared to a standard diet. The accelerated catabolism of HNE lowers HNE and HNE analog concentrations in livers from rats fed the ketogenic diet. On the other hand, rats fed the high-fat mix diet had high rates of lipid synthesis and low rates of fatty acid oxidation, resulting in the slowing down of the catabolic disposal of HNE and HNE analogs. Thus, decreased HNE catabolism from a high-fat mix diet induces high concentrations of HNE and HNE analogs. The results of this work suggest a potential causal relationship to metabolic syndrome induced by Western diets (i.e., high-fat mix), as well as the effects of a ketogenic diet on the catabolism of lipid peroxidation products in liver.     

Mass spectrometric evidence for long-lived protein adducts of 4-oxo-2-nonenal

Substantial work has been carried out to elucidate the nature of protein modification by 4-hydroxy-2-nonenal (HNE) and its relatives. Its keto cousin, 4-oxo-2-nonenal (ONE), which arises from linoleic acid oxidation independently of HNE, was previously reported to form Michael adducts with His and Cys that can subsequently, in part, condense with Lys residues to give imidazolylpyrrole cross-links. Despite mass spectrometric evidence also for ONE-Lys Michael adducts, the latter do not accumulate in solution. A long-lived adduct that has the same mass as the ONE Lys Michael adduct is suggested instead to be the isomeric 4-ketoamide that arises, along with other adducts, from the reversibly-formed ONE Lys Schiff base. The Lys-ketoamide and His-Lys imidazolylpyrrole cross-links appear to be unusually prominent markers of stable protein modification by ONE.