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.     

Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy-trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 microM). Acrolein potently inhibited SSADH activity (IC(50)=15 microM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC(50) of 110 microM. Trans-2-hexenal (HEX) and crotonaldehyde (100 microM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.     

Human aldo-keto reductase AKR7A2 protects against the cytotoxicity and mutagenicity of reactive aldehydes and lowers intracellular reactive oxygen species in hamster V79-4 cells

Aldo-keto reductase (AKR) enzymes are critical for the detoxication of endogenous and exogenous aldehydes. Previous studies have shown that the AKR7A2 enzyme is catalytically active toward aldehydes arising from lipid peroxidation, suggesting a potential role against the consequences of oxidative stress, and representing an important detoxication route in mammalian cells. The aim of this study was to determine the ability of AKR7A2 to protect cells against aldehyde cytotoxicity and genotoxicity and elucidate its potential role in providing resistance to oxidative stress. A transgenic mammalian cell model was developed in which AKR7A2 was overexpressed in V79-4 cells and used to evaluate the ability of AKR7A2 to provide resistance against toxic aldehydes. Results show that AKR7A2 provides increased resistance to the cytotoxicity of 4-hydroxynonenal (HNE) and modest resistance to the cytotoxicity of trans, trans-muconaldehyde (MUC) and methyglyoxal, but provided no protection against crotonaldehyde and acrolein. Cells expressing AKR7A2 were also found to be less susceptible to DNA damage, showing a decrease in mutation rate cause by 4-HNE compared to control cells. Furthermore, the role of the AKR7A2 enzyme on the cellular capability to cope with oxidative stress was assessed. V79 cells expressing AKR7A2 were more resistant to the redox-cycler menadione and were able to lower menadione-induced ROS levels in both a time and dose dependent manner. In addition, AKR7A2 was able to maintain intracellular GSH levels in the presence of menadione. Together these findings indicate that AKR7A2 is involved in cellular detoxication pathways and may play a defensive role against oxidative stress in vivo.     

Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro.

Using a filter-binding assay based on precipitation of pUC13 plasmid DNA bound to calf-thymus histones, we have determined the efficiency of formation of DNA-protein crosslink formation induced by several aldehyde compounds in vitro. Formaldehyde, glutaraldehyde and acrolein were the most potent, causing 1 crosslink per 2.7 kbp of DNA at 1.5, 8 and 150 microM, respectively. All other compounds tested gave 1 crosslink per plasmid molecule in the mM concentration range as follows: acetaldehyde, 115 mM; propionaldehyde, 295 mM; butyraldehyde, 360 mM; crotonaldehyde, 8.5 mM; trans-2-pentenal, 6.3 mM. Significant decreases in the efficiency of DPXL formation were observed with monofunctional aldehydes of higher carbon chain length. For example, the concentration of formaldehyde needed to give 1 crosslink per molecule was almost 10(5) times less than that of acetaldehyde. Acetaldehyde differs from formaldehyde only by one saturated carbon. The presence of an unsaturated bond between the 2-3 carbons improved the potential for crosslink formation. For example, acrolein was over 500-fold more potent than propionaldehyde. Glutaraldehyde was almost as potent as formaldehyde, indicating that the bifunctional nature of this 5-carbon saturated aldehyde may be crucial to its high efficiency of DNA-protein crosslinking.     

Regulation of constitutive neutrophil apoptosis by the alpha,beta-unsaturated aldehydes acrolein and 4-hydroxynonenal

Reactive alpha,beta-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the alpha,beta-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative alpha,beta-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 microM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that alpha,beta-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.     

The role of aldehyde dehydrogenases in the malonic dialdehyde metabolism in the rat liver

The enzymes catalyzing the NAD-dependent oxidation of malonic dialdehyde (MDA) were isolated from rat liver extracts. Upon 5'-AMP-Sepharose chromatography MDA dehydrogenase was separated into two isoforms, I and II. Isoform I was eluted from the affinity carrier with a 0.1 M phosphate buffer pH 8.0. This isoform had a broad substrate specificity towards aliphatic and aromatic aldehydes. Kinetic studies showed that short- and medium-chain aliphatic aldehydes (C2-C6) were characterized by the lowest Km values and the highest Vmax values. The Km' values for MDA and acetaldehyde were 2.8 microM and 0.69 microM, respectively. Isoform II was eluted with a 0.1 M phosphate buffer pH 8.0 containing 0.5 mM NAD, was the most active with medium- and long-chain aliphatic aldehydes (C6-C11) and had Km values for MDA and acetaldehyde equal to 37 microM and 52 microM, respectively. Isoform I was much more sensitive towards disulfiram inhibition than isoform II. Both isoforms had an identical molecular mass (93 kD) upon gel filtration. It is concluded that MDA dehydrogenase isoform I is identical to mitochondrial aldehyde dehydrogenase having a low Km for acetaldehyde, whereas isoform II may be localized in liver cytosol. The role of aldehyde dehydrogenases in the metabolism of aldehydes derived from lipid peroxidation is discussed.     

Chemotactic activity of the lipid peroxidation product 4-hydroxynonenal and homologous hydroxyalkenals

The effect of the lipid peroxidation product 4-hydroxynonenal and homologous aldehydes (4-hydroxyoctenal, 4-hydroxyundecenal, 4-hydroxytetradecenal and 4-hydroxypentadecenal) on migration and polarization of rat neutrophils was examined. The most effective aldehydes were 4-hydroxyoctenal and 4-hydroxypentadecenal, which stimulated oriented migration at ED50 = 1.4 X 10(-12) M and 1.3 X 10(-12) M, resp., whereas the other aldehydes had ED50 between 1 X 10(-7) and 6 X 10(-11) M. The peptides fMet-Phe and fMet-Leu-Phe used as positive controls had ED50 values of 4.2 X 10(-7) M and 4.5 X 10(-10) M resp. The 4-hydroxyalkenals induced only a small increase of the percentage of polarized cell and did not enhance the random migration. The effects of 4-hydroxyalkenals were only observed when the incubation buffer contained bovine serum albumin (BSA), in the absence of BSA neither the aldehydes nor the peptides exhibited chemotactic properties. Since the aldehydes easily react with the sulfhydryl groups of the BSA to form the S-alkylated BSA in an equilibrium reaction, the chemotactic substance could either be the free aldehyde or the BSA-aldehyde adduct. The adduct prepared from BSA and 4-hydroxynonenal was chemotactic at doses of 0.65 to 0.0065 mg/ml, when tested in the presence of unmodified BSA. Since the adduct released free 4-hydroxyalkenal during the assay in the reverse reaction, it can not be decided whether the active principle is the aldehyde itself or the aldehyde attached to the BSA. From the effective doses of the aldehydes (10(-7) to 10(-12)M) and the BSA-aldehyde adduct it appears very unlikely that the BSA itself gained chemotactic properties through the alkylation of its sulfhydryl groups by the aldehyde.     

Lipoxin A4 inhibits phosphoinositide hydrolysis in human neutrophils

Lipoxins (LX) are trihydroxytetraene metabolites derived from arachidonic acid via an interaction between the 5- and 15-lipoxygenases. Preincubation of [3H] myo-inositol labeled PMN with 10-7M and 10-5M LXA4 for 1 minute at 37 degrees C resulted in a concentration dependent inhibition of the generation of [3H] IP3 and [3H] IP in cells subsequently stimulated by increasing doses of LTB4 or FMLP for 1 minute at 37 degrees C. Preincubation of PMN with LXA4 did not inhibit specific binding of [3H] LTB4 to PMN. These results indicate that LXA4 inhibits chemotactic factor-induced phosphoinositide hydrolysis at a post-receptor level.     

Human aldo-keto reductases 1B1 and 1B10: a comparative study on their enzyme activity toward electrophilic carbonyl compounds

Aldo-keto reductase family 1 member B1 (AKR1B1, 1B1 in brief) and aldo-keto reductase family 1 member B10 (AKR1B10, 1B10 in brief) are two proteins with high similarities in their amino acid sequences, stereo structures, and substrate specificity. However, these two proteins exhibit distinct tissue distributions; 1B10 is primarily expressed in the gastrointestinal tract and adrenal gland, whereas 1B1 is ubiquitously present in all tissues/organs, suggesting their difference in biological functions. This study evaluated in parallel the enzyme activity of 1B1 and 1B10 toward alpha, beta-unsaturated carbonyl compounds with cellular and dietary origins, including acrolein, crotonaldehyde, 4-hydroxynonenal, trans-2-hexenal, and trans-2,4-hexadienal. Our results showed that 1B10 had much better enzyme activity and turnover rates toward these chemicals than 1B1. By detecting the enzymatic products using high-performance liquid chromatography, we measured their activity to carbonyl compounds at low concentrations. Our data showed that 1B10 efficiently reduced the tested carbonyl compounds at physiological levels, but 1B1 was less effective. Ectopically expressed 1B10 in 293T cells effectively eliminated 4-hydroxynonenal at 5 μM by reducing to 1,4-dihydroxynonene, whereas endogenously expressed 1B1 did not. The 1B1 and 1B10 both showed enzyme activity to glutathione-conjugated carbonyl compounds, but 1B1 appeared more active in general. Together our data suggests that 1B10 is more effectual in eliminating free electrophilic carbonyl compounds, but 1B1 seems more important in the further detoxification of glutathione-conjugated carbonyl compounds.     

DNA damage caused by lipid peroxidation products

Lipid peroxidation is a process involving the oxidation of polyunsaturated fatty acids (PUFAs), which are basic components of biological membranes. Reactive electrophilic compounds are formed during lipid peroxidation, mainly alpha, beta-unsaturated aldehydes. These compounds yield a number of adducts with DNA. Among them, propeno and substituted propano adducts of deoxyguanosine with malondialdehyde (MDA), acrolein, crotonaldehyde and etheno adducts, resulting from the reactions of DNA bases with epoxy aldehydes, are a very important group of adducts. The epoxy aldehydes are more reactive towards DNA than the parent unsaturated aldehydes. The compounds resulting from lipid peroxidation mostly react with DNA showing both genotoxic and mutagenic action; among them, 4-hydroxynonenal is the most genotoxic, while MDA is the most mutagenic. DNA damage caused by the adducts of lipid peroxidation products with DNA can be removed by the repairing action of glycosylases. The formed adducts have been hitherto analyzed using the IPPA (Imunopurification-(32)P-postlabelling assay) method and via gas chromatography/electron capture negtive chemical ionization/mass spectrometry (GC/EC NCI/MS). A combination of liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used in recent years.     

Structural characteristics of a lipid peroxidation product, trans-2-nonenal, that favour inhibition of membrane-associated phosphotyrosine phosphatase activity

Protein-tyrosine phosphatases (PTPs) are very susceptible to oxidation by reactive oxygen species (ROS), which induce the oxidation of catalytic cysteines, thereby inactivating these PTPs. PTPs are also inactivated by treatment with different aldehydes (such as trans-2-nonenal), produced after tissue damage by ROS. However, the molecular mechanisms behind such aldehyde-due inactivation remain unknown. Using commercially available compounds, we examined the structural characteristics of trans-2-nonenal that allow the inhibition of platelet membrane-associated PTP activity, as well as how these compounds affect the dynamics of SH-, CO- and NH2- protein groups on the membranes. PTP was effectively inhibited by physiological amounts of trans-2-nonenal (1-10 microM). Incubation with trans-2-nonene (10 microM) also decreased PTP activity, although to a lower extent. Treatment with nonyl aldehyde almost eliminated PTP inhibition. Decreases in protein thiols were visible after trans-2-nonenal and trans-2-nonene treatments. Both the latter compounds also increased protein carbonyls (although trans-2-nonenal was more effective) and decreased protein amino groups to an equal extent. Collectively, our data indicate that alpha,beta unsaturation (and not a double bond in another position) is the most important structural determinant for PTP inhibition, the alkenal with 9-carbon atoms being the most effective in eliciting such inhibition. The data allow us to predict the modification of sulfhydryls and/or the formation of addition products with lysyl or histidyl residues, and hence the kind of specific antibodies that it would be necessary to generate in order to test such modifications directly.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.     

1,N2-deoxyguanosine adducts of acrolein,crotonaldehyde, and trans-4-hydroxynonenal cross-link to peptides via Schiff base linkage

DNA-protein cross-links (DPCs) are formed upon exposure to a variety of chemical and physical agents and pose a threat to genomic integrity. In particular, acrolein and related aldehydes produce DPCs, although the chemical linkages for such cross-links have not been identified. Here, we report that oligodeoxynucleotides containing 1,N(2)-deoxyguanosine adducts of acrolein, crotonaldehyde, and trans-4-hydroxynonenal can form cross-links with the tetrapeptide Lys-Trp-Lys-Lys. We concluded that complex formation is mediated by a Schiff base linkage because DNA-peptide complexes were covalently trapped following reduction with sodium cyanoborohydride, and pre-reduction of adducted DNAs inhibited complex formation. A previous NMR study demonstrated that duplex DNA catalyzes ring opening for the acrolein-derived gamma-hydroxy-1,N(2)-propanodeoxyguanosine adduct to yield an aldehydic function (de los Santos, C., Zaliznyak, T., and Johnson, F. (2001) J. Biol. Chem. 276, 9077-9082). Consistent with this earlier observation, the adducts under investigation were more reactive in duplex DNA than in single-stranded DNA, and we concluded that the ring-open aldehydic moiety is the induced tautomer in duplex DNA for adducts exhibiting high relative reactivity. Adducted DNA cross-linked to Arg-Trp-Arg-Arg and Lys-Trp-Lys-Lys with comparable efficiency, and N(alpha)-acetylation of peptides dramatically inhibited trapping; thus, the reactive nucleophile is located at the N-terminal alpha-amine of the peptide. These data suggest that Schiff base chemistry can mediate DPC formation in vivo following the formation of stable aldehyde-derived DNA adducts.     

Action of 2-nonenal and 4-hydroxynonenal on phosphoinositide-specific phosopholipase C in undifferentiated and DMSO-differentiated HL-60 cells

The promyelocytic cell line HL-60 has been used as an in vitro model to study the mechanism of action of two chemotactic aldehydes, 2-nonenal and 4-hydroxynonenal. Increasing aldehyde concentrations have been added to undifferentiated and DMSO-differentiated cells incubated at 37 degrees C and their effect on phosphoinositide-specific phospholipase C has been analysed by using a specific inositol-1,4,5-tris-phosphate assay system. Concentrations of 2-nonenal between 10(-9) and 10(-7) M significantly increased the enzymatic-activity in DMSO-differentiated HL-60 cells, while 10(-9) and 10(-8) M concentrations were active in the undifferentiated cells. 4-Hydroxynonenal was able to activate phospholipase C both in undifferentiated and DMSO-differentiated cells at concentrations ranging from 10(-8) to 10(-6) M. The concentrations of both compounds active on phospholipase C displayed a good correspondence with those which had been reported to be chemotactic towards rat neutrophils. In the case of 4-hydroxynonenal, the present results confirm its ability to activate phospholipase C, which we had previously shown in isolated neutrophil plasma membranes. The comparison of the effects of 2-nonenal and 4-hydroxynonenal on chemotaxis and phospholipase C activation suggests a common mechanism of action for both aldehydes, for which the presence of the double bond seems to be required.     

Kinetics of membrane-bound aldehyde dehydrogenase from Acinetobacter calcoaceticus

In recent investigations we were able to demonstrate that the NADP-dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus is an inducible enzyme localized in intracytoplasmic membranes limiting alkane inclusions. Long-chain aliphatic hydrocarbons and alkanols are inducers of the enzyme. It was purified by us and now kinetically characterized using the enzyme-micelle form, which contains bacterial phospholipids and a detergent (sodium cholate), too. The pH optimum of aldehyde dehydrogenase was determined to be at pH 10. The enzyme showed substrate inhibition (by aldehyde excess). The Ks and Km values of the leading substrate NADP+ were found to be 8.6 X 10(-5) and 10.3 X 10(-5)M independent of the chain-length of the aldehydes. The Km values of the aldehydes decreased depending on increasing chain-length (butanal: 1.6 X 10(-3), decanal: 1.5 X 10(-6)M). The Ki values (for inhibition by aldehyde excess) showed a similar behaviour (butanal: 7.5 X 10(-3), decanal: 3.5 X 10(-5)M) as well as the optimal aldehyde concentrations inducing the "maximal" reaction velocity (butanal: 5mM, decanal: 6 microM). The number of inhibiting aldehyde molecules per enzyme-substrate complex was determined to be n = 1. NADPH showed product inhibition kinetics (Ki(NADPH) = 2.2 X 10(-4)M), fatty acids did not. We were unable to measure a reverse reaction. The following ions and organic compounds were non-competitive inhibitors of the enzyme: Sn2+, Fe2+, Cu2+, BO3(3-), CN-, EDTA, o-phenanthroline, p-chloromercuri-benzoate, mercaptoethanol, phenylmethylsulfonyl fluoride, and diisopropylfluorophosphate; iodoacetate did not influence enzyme activity. Chloral hydrate was a competitive inhibitor of the aldehydes. Ethyl butyrate activates the enzyme, dependent on the chain-length of the aldehyde substrates.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Substrate specificity of human and yeast aldehyde dehydrogenases

Human aldehyde dehydrogenase (ALDH) family may contribute to metabolism of hydrocarbons, biogenic amines, retinoids, steroids, and lipid peroxidation. We previously reported kinetic properties of human cytosolic ALDH1 and mitochondrial ALDH2 towards oxidation of the straight-chain and branched-chain aliphatic aldehydes with various chain lengths [S.J. Yin, M.F. Wang, C.L. Han, S.L. Wang, Substrate binding pocket structure of human aldehyde dehydrogenases: a substrate specificity approach, Adv. Exp. Med. Biol. 372 (1995) 9-16]. We present here substrate specificities for aromatic and heterocyclic aldehydes with purified human liver ALDH1 and ALDH2, and also with yeast mitochondrial ALDH2 for comparison. Kinetic assay for human ALDHs was performed in 50mM HEPES, pH 7.5 and 25 degrees C, containing 0.5mM NAD(+), 1.7% (v/v) acetonitrile (as a solvent carrier for aldehydes) and varied concentrations of substrate, and for yeast ALDH2 the assay was determined in the same reaction mixture except containing 3mM NAD(+) and addition of 200 mM KCl. With respect to phenylacetaldehyde, 2-phenylpropionaldehyde, benzaldehyde, p-nitrobenzaldehyde, cinnamaldehyde, 2-furaldehyde and indole-3-acetaldehyde, human liver ALDH1 exhibited K(M) ranging from 0.25 to 4.8 microM, V(max) of 0.34-2.4U/mg, and catalytic efficiency, V(max)/K(M), 0.070-3.9U/(mg microM); human ALDH2 exhibited K(M) ranging from less than 0.15-0.74 microM, V(max) of 0.039-0.51 U/mg, and V(max)/K(M), 0.15-1.0U/(mg microM). Human ALDH1 and ALDH2 exhibited substate inhibition constants (K(i)) for phenylacetaldehyde, 95 and 430 microM, respectively. Yeast ALDH2 exhibited K(M) for straight-chain aliphatic aldehydes (C1-C10), 2.3-210 microM, and substrate inhibition constants (C2-C10), 79-2900 microM, with a trend of being smaller K(M) and K(i) for longer chain lengths; and K(M) for cinnamaldehyde, benzaldehyde, and 2-furaldehyde, 5.0, 79, and 1000 microM, respectively. Therefore human ALDH1/ALDH2 and yeast ALDH2 can contribute to detoxification or metabolism of various exogenous/endogenous aliphatic and aromatic aldehydes. The systematic changes in kinetic parameters for oxidation of structurally related aldehydes may reflect subtle functional topographic distinctions of substrate pocket for human and yeast ALDHs.     

Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol pathway enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure-activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with gamma-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation 1, gamma-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with gamma-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.