Characterization of 2'-deoxyadenosine adducts derived from 4-oxo-2-nonenal, a novel product of lipid peroxidation

Analysis of the reaction between 2'-deoxyadenosine and 4-oxo-2-nonenal by liquid chromatography/mass spectrometry revealed the presence of three major products (adducts A(1), A(2), and B). Adducts A(1) and A(2) were isomeric; they interconverted at room temperature, and they each readily dehydrated to form adduct B. The mass spectral characteristics of adduct B obtained by collision-induced dissociation coupled with multiple tandem mass spectrometry were consistent with those expected for a substituted etheno adduct. The structure of adduct B was shown by NMR spectroscopy to be consistent with the substituted etheno-2'-deoxyadenosine adduct 1' '-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]heptane-2' '-one. Unequivocal proof of structure came from the reaction of adducts A(1) and A(2) (precursors of adduct B) with sodium borohydride. Adducts A(1) and A(2) each formed the same reduction product, which contained eight additional hydrogen atoms. The mass spectral characteristics of this reduction product established that the exocyclic amino group (N(6)) of 2'-deoxyadenosine was attached to C-1 of the 4-oxo-2-nonenal. The reaction of 4-oxo-2-nonenal with calf thymus DNA was also shown to result in the formation of substituted ethano adducts A(1) and A(2) and substituted etheno adduct B. Adduct B was formed in amounts almost 2 orders of magnitude greater than those of adducts A(1) and A(2). This was in keeping with the observed stability of the adducts. The study presented here has provided additional evidence which shows that 4-oxo-2-nonenal reacts efficiently with DNA to form substituted etheno adducts.     

Characterization of 4-oxo-2-nonenal as a novel product of lipid peroxidation

Fe(II)-mediated decomposition of 13-[S-(Z,E)]-9, 11-hydroperoxyoctadecadienoic (hydroperoxylinoleic) acid resulted in the formation of three alpha,beta-unsaturated aldehydes. At low Fe(II) concentrations or at early time points after the addition of Fe(II), two major products were observed. The least polar product had chromatographic properties that were identical with those of 4-oxo-2-nonenal. Conversion of this product to its bis-oxime derivative with hydroxylamine hydrochloride resulted in two syn- and two anti-oxime isomers that had chromatographic and mass spectral properties identical with the properties of products derived from an authentic standard of 4-oxo-2-nonenal. This confirmed for the first time that 4-oxo-2-nonenal is a major product of the Fe(II)-mediated breakdown of lipid hydroperoxides. The more polar product had chromatographic properties that were similar to those of 4-hydroperoxy-2-nonenal. LC/MS analysis of its syn- and anti-oxime isomers confirmed this structural assignment. Thus, 4-hydroperoxy-2-nonenal is a previously unrecognized major product of lipid hydroperoxide decomposition. At high Fe(II) concentrations and at longer incubation times, a third more polar product was observed with chromatographic properties that were identical to those of 4-hydroxy-2-nonenal. The syn- and anti-oxime isomers had chromatographic and mass spectral properties identical with the properties of products derived from an authentic standard of 4-hydroxy-2-nonenal. It appears that 4-hydroperoxy-2-nonenal is formed initially and that it is then converted to 4-hydroxy-2-nonenal in the presence of high Fe(II) concentrations or by extended incubations in the presence of low Fe(II) concentrations. It is conceivable that some of the 4-hydroperoxy-2-nonenal is also converted to 4-oxo-2-nonenal. However, we cannot rule out the possibility that it is also formed by a concerted mechanism from a rearrangement product of 13-[S-(Z,E)]-9, 11-hydroperoxyoctadecadienoic acid.     

Covalent modifications to 2'-deoxyguanosine by 4-oxo-2-nonenal, a novel product of lipid peroxidation

Two major products (adducts A and B) from the reaction of 2-deoxyguanosine (dGuo) with 13-hydroperoxylinoleic acid were detected by liquid chromatography/mass spectrometry (LC/MS). Adducts A and B were also the major products formed enzymatically when dGuo was incubated in the presence of linoleic acid and lipoxygenase. The mass spectral fragmentation patterns of adducts A and B suggested that unique modifications to the nucleoside had been introduced. This resulted in the characterization of a novel bifunctional electrophile, 4-oxo-2-nonenal, as the principal breakdown product of linoleic acid hydroperoxide. In subsequent studies, adduct A was found to be a substituted ethano dGuo adduct that was a mixture of three isomers (A(1)-A(3)) that all decomposed to form adduct B. Adduct A(1) was the hemiacetal form of 3-(2-deoxy-beta-D-erythropentafuranosyl)-3,5,6, 7-tetrahydro-6-hydroxy-7-(heptane-2-one)-9H-imidazo[1, 2-alpha]purine-9-one. Adducts A(2) and A(3) were the diastereomers of the open chain ketone form. Adduct B was the substituted etheno dGuo adduct, 3-(2-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(heptane-2 -one)-9-hydroxy[1,2-alpha]purine, the dehydration product of adducts A(1)-A(3). Identical covalent modifications to dGuo were observed when calf-thymus DNA was treated with 4-oxo-2-nonenal. These data illustrate the diversity of reactive electrophiles produced from the peroxidative decomposition of lipids and have implications in fully assessing the role of lipid peroxidation in mutagenesis and carcinogenesis.     

Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal

Lipid peroxidation yields the aldehydes 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). Protein adduction by 4HNE is thought to be involved in the pathogenesis of several diseases. Currently, the reactivity of 4ONE toward proteins is unknown. The purpose of this study was to identify amino acids that react with 4HNE and 4ONE, characterize the chemical structure of the adduct, and determine the preference for amino acid modification. Model peptides containing one or more nucleophilic residues (i.e., Arg, Cys, His, Met, and Lys) were reacted with 4HNE and 4ONE and analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Post-source decay analysis was used to confirm peptide modification. The bimolecular rate constant for adduction of amino acids and peptides by 4HNE and 4ONE was measured. Results of this work indicate that Cys, His, and Lys are modified by 4HNE and 4ONE. In addition, Arg was adducted by 4ONE. The predominant adduct resulting from modification of peptides by 4HNE or 4ONE had a mass of 156 or 154 Da (respectively), indicating that adduction occurs via Michael addition. Reactivity of amino acids toward 4HNE and 4ONE was found to have the following order: Cys > His > Lys (> Arg for 4ONE). The presence of an Arg on a Cys-containing peptide increased the reaction rate with 4HNE and 4ONE by a factor of approximately 5-6 compared to the Cys nucleophile alone. Rate constants determined for the modification of Cys by the lipid aldehydes demonstrated a >100-fold difference in reactivity between 4HNE and 4ONE toward Cys. Results of the present study indicate that both 4HNE and 4ONE modify amino acid nucleophiles; however, the reactivity between these two lipid aldehydes differs both qualitatively and quantitatively.     

Low glutathione level favors formation of DNA adducts to 4-hydroxy-2(E)-nonenal, a major lipid peroxidation product

Lipids are known to be major targets of oxidative stress in cells. In addition to deleterious effects on membranes and various cellular processes, lipid peroxidation has been proposed to be an indirect genotoxic pathway. Indeed, reactive aldehydes produced upon degradation of lipid hydroperoxides may add to DNA bases. In the present work, we investigated the DNA damaging properties of exogenously added 4-hydroxy-2(E)-nonenal (HNE) in human THP1 monocytes. To provide quantitative data on the possible role of HNE in oxidative genotoxicity, we applied an accurate HPLC-MS approach to the quantification of HNE adducts to DNA and of HNE conjugates to glutathione (HNE-GSH), the product of the major detoxification pathway of HNE in cells. We confirmed that GSH was more reactive than DNA toward HNE in cells, with a ratio of 25000 between the amounts of HNE-GSH and DNA adducts. In addition, we found that the conjugate of HNE to cysteine was produced in much lower yield than HNE-GSH, while that of N-acetylcysteine could not be detected. We also observed that a decrease in the GSH content resulted in the favored formation of DNA lesions. If our data based on an intense and short exposure to HNE can be extended to an in vivo situation where low concentrations of HNE are produced on a long time scale, the present results suggest that although the amount of DNA adducts is low upon treatment by exogenous HNE, their formation could be favored upon oxidative stress. Indeed, this last process leads to concomitant consumption of GSH by oxidation and induction of lipid peroxidation.     

Rat aldose reductase-like protein (AKR1B14) efficiently reduces the lipid peroxidation product 4-oxo-2-nonenal

In this study, we examined the substrate specificity, inhibitor sensitivity and kinetic mechanism of a rat aldose reductase-like protein, which is named AKR1B14 in the aldo-keto reductase (AKR) superfamily. AKR1B14 catalyzed the nicotinamide adenine dinucleotide phosphate reduced form (NADPH)-dependent reduction of carbonyl compounds (derived from lipid peroxidation and glycation), xenobiotic aromatic aldehydes and some aromatic ketones. 4-Oxo-2-nonenal, the best substrate showing a K(m) value of 0.16 μM, was reduced into less reactive 4-oxo-2-nonenol, and its cytotoxicity was attenuated by the overexpression of the enzyme in cultured cells. The enzyme also showed low K(m) values (0.9-10 μM) for medium-chain aliphatic aldehydes (such as 4-hydroxynonenal, 1-hexenal and farnesal) and 3-deoxyglucosone, although the K(m) values for short-chain substrates (such as isocaproaldehyde, acrolein and methylglyoxal) were high (16-600 μM). In the reverse reaction, aliphatic and aromatic alcohols were oxidized by AKR1B14 at low rates. AKR1B14 was inhibited by aldose reductase inhibitors such as tolrestat and epalrestat, and their inhibition patterns were noncompetitive versus the aldehyde substrate and competitive with respect to the alcohol substrate. Kinetic analyses of the oxidoreduction and dead-end inhibition suggest that the reaction follows an ordered sequential mechanism.     

Mass spectrometric characterization of modifications to angiotensin II by lipid peroxidation products, 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal

The octapeptide angiotensin II (Ang II; Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) is the primary active hormone of the renin/angiotensin system (RAS) and has been implicated in various cardiovascular diseases. Numerous structure-activity relationship studies have identified Asp(1), Arg(2), and His(6) of Ang II to be critical for its biological activity and receptor binding. From the reactions of Ang II with lipid peroxidation-derived aldehydes, 4-oxo-2(E)-nonenal (ONE) or 4-hydroxy-2(E)-nonenal (HNE), we have identified the major modifications to the N-terminus, Asp(1), Arg(2), and His(6) of Ang II by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization-time-of-flight/MS (MALDI-TOF/MS). The identities of ONE- and HNE-modified Ang II were confirmed by tandem mass spectrometry (MS/MS) and postsource decay (PSD)-TOF/MS before and after the reaction with sodium borohydride. In the reaction with ONE, a pyruvamide-Ang II that formed via oxidative decarboxylation of N-terminal Asp was detected as the most abundant product after 48 h of incubation. It was followed by Arg-modified [Arg(2)(ONE-H(2)O)]-Ang II and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II. The Michael addition products of [His(6)(HNE)]-Ang II were the most abundant products in the beginning of the reaction with HNE, followed by the dehydrated Michael addition products of [His(6)(HNE-H(2)O)]-Ang II. [His(6)(HNE)]-Ang II was dehydrated to [His(6)(HNE-H(2)O)]-Ang II during the prolonged incubation, and [His(6)(HNE-H(2)O)]-Ang II became the major products after 7 days. The model reactions of N(α)-tert-butoxycarbonyl (tBoc)-Arg with ONE and tBoc-His with HNE were performed and compared with the Ang II reaction. tBoc-Arg readily reacted with ONE to produce a compound analogous to [Arg(2)(ONE-H(2)O)]-Ang II, which confirmed Arg as one of the important target nucleophiles of ONE. However, tBoc-His exclusively formed a Michael addition product upon the reaction with HNE. The unexpected formation of [His(6)(HNE-H(2)O)]-Ang II can be explained by the proximity of His(6) to C-terminal carboxylate in the specific conformation of Ang II, which facilitates the dehydration of Michael addition products. Therefore, our results suggest a possible discrepancy in the adduction chemistry of ONE and HNE for model amino acids and endogenous bioactive peptides, which is governed by the microenvironment of peptides, such as the specific amino acid sequence and conformation. Such stable ONE- and HNE-derived modifications to Ang II could potentially modulate its functions in vivo by disrupting the interaction with Ang II type 1 (AT(1)) receptor and/or inhibiting the enzyme activity of aminopeptidase A (APA), which cleaves the N-terminal Asp residue of Ang II to generate Ang III.     

Disposition in rat of [2-3H]-trans-4-hydroxy-2,3-nonenal,a product of lipid peroxidation

1. 4-Hydroxy-2,3-nonenal (HNE) is an end product of lipid peroxidation (LPO) and a well known cytotoxic aldehyde that exhibits a variety of biological effects. In this study the in vivo disposition and covalent binding of i.p. administered [2-³H]HNE was examined in the rat.

2. It was found that several metabolites of [2-³H]HNE are excreted in urine among which at least four mercapturic acids. 1,4-Dihydroxynonane mercapturic acid (DHN-MA) appeared to be the most abundant mercapturic acid excreted in urine (3·5% of the dose) and the excretion of the other three mercapturic acids amounted to 2% of the dose.

3. Within 48 h following i.p. administration of 5 or 25 μmol/kg bodyweight [2-³H]HNE (specific activity 4 μCi/μmol) about 25% of the radioactivity was excreted in urine, whereas 18% of the radioactivity appeared in the faeces.

4. After 48 h, 7% of the radioactivity was still present in the liver and 0·2% in other organs, but this radioactivity appeared not to be covalently bound to cellular macro-molecules. It was found that only 0·13% of the radioactivity was covalently bound in the liver and even less in the other organs.     

Identification of 4-oxo-2-hexenal and other direct mutagens formed in model lipid peroxidation reactions as dGuo adducts

We searched for mutagens that react with 2'-deoxyguanosine (dGuo) in model systems of lipid peroxidation. To autoxidation systems of methyl linoleate (model of omega-6 fat), methyl alpha-linolenate (MLN) (model of omega-3 fat), and commercial salad oil, dGuo was added. The reaction mixtures were analyzed by HPLC. Six adducts were detected, and their structures were determined by 1H and 13C NMR, UV, and mass spectra and by comparison with synthetic authentic samples. The mutagens that reacted with dGuo to form these adducts were proposed as glyoxal, glyoxylic acid, ethylglyoxal, and 4-oxo-2-hexenal (4-OHE). The formation of 8-hydroxy-dGuo, an oxidized product of dGuo, was also detected in the model reaction mixtures. Among them, glyoxal and glyoxylic acid are known mutagens, while ethylglyoxal and 4-OHE, produced from MLN, have not been reported as mutagens thus far. We confirmed the mutagenic activity of 4-OHE with Salmonella strains, TA100 and TA104, without S9 mix. These compounds may be involved in lipid peroxide-related cancers.     

A study on ceftriaxone-induced lipid peroxidation using 4-hydroxy-2-nonenal as model marker

4-Hydroxy-2-nonenal, an indicator of lipid peroxidation process. was used as model marker to explore ceftriaxone-induced lipid peroxidation in goat liver homogenate. Ceftriaxone was found to induce lipid peroxidation significantly. It was further found that ascorbic acid could significantly arrest ceftriaxone-induced lipid peroxidation. The results corroborate the earlier findings with malondialdehyde as model marker.     

Formation of a substituted 1,N(6)-etheno-2'-deoxyadenosine adduct by lipid hydroperoxide-mediated generation of 4-oxo-2-nonenal

Analysis of the reaction between 2'-deoxyadenosine and 13-hydroperoxylinoleic acid by liquid chromatography/constant neutral loss mass spectrometry revealed the presence of two major products (adducts A and B). Adduct A was shown to be a mixture of two isomers (A(1) and A(2)) that each decomposed with the loss of water to form adduct B. The mass spectral characteristics of adduct B were consistent with the substituted 1, N(6)-etheno-2'-deoxyadensoine adduct 1' '-[3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]heptan-2' '-one. Adducts A(1), A(2), and B were formed when 2'-deoxyadenosine was treated with synthetic 4-oxo-2-nonenal, which suggested that it was formed by the breakdown of 13-hydroperoxylinoleic acid. A substantial increase in the rate of formation of adducts A(1), A(2), and B was observed when 13-hydroperoxylinoleic acid and 2'-deoxyadenosine were incubated in the presence of Fe(II). Thus, 4-oxo-2-nonenal was most likely formed by a homolytic process. Although adducts A(1), A(2), and B were formed in the reaction between 4-hydroxy-2-nonenal and 2'-deoxyadenosine, a number of additional products were observed. This suggested that 4-hydroxy-2-nonenal was not a precursor in the formation of 4-oxo-2-nonenal from 13-hydroperoxylinoleic acid. This study has provided additional evidence which shows that 4-oxo-2-nonenal is a major product of lipid peroxidation and that it reacts efficiently with DNA to form substituted etheno adducts.     

Glutathione depletion enhances the formation of endogenous cyclic DNA adducts derived from t-4-hydroxy-2-nonenal in rat liver

Earlier, we detected the cyclic adducts of deoxyguanosine (dG) derived from t-4-hydroxy-2-nonenal (HNE), a long chain alpha,beta-unsaturated aldehyde (enal) product from oxidation of omega-6 polyunsaturated fatty acids, in tissue DNA of rats and humans as endogenous DNA damage. Recent evidence implicates the cyclic HNE adducts in human liver carcinogenesis. Because glutathione (GSH) protects against oxidative stress, we undertook a study to examine the effect of GSH depletion on the HNE-derived cyclic adducts in vivo. Four F344 rats were administered L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at 10 mM in drinking water for 2 weeks. Rats in the control group were given water only. Livers were harvested, and each liver was divided into portions for GSH and DNA adduct analyses. The BSO treatment depleted hepatic GSH by 77%; the GSH levels were reduced from 6.3 +/- 0.3 in the control rats to 1.5 +/- 0.1 micromol/g tissues in the treated group. The formation of HNE-dG adducts, analyzed by an HPLC-based 32P-postlabeling assay, was increased by 4-fold, from 6.2 +/- 2.2 nmol/mol dG in liver DNA of control rats to 28.5 +/- 16.1 nmol/mol dG in the rats treated with BSO (p <0.05). The formation of 8-oxodG in liver DNA was also increased as a result of BSO treatment, although the increase was not statistically significant. These results further support the endogenous origin of HNE-dG adducts and, more importantly, indicate a critical role that GSH plays in protecting against in vivo formation of the promutagenic cyclic DNA adducts derived from HNE.     

Novel 1,N(6)-etheno-2'-deoxyadenosine adducts from lipid peroxidation products

trans,trans-2,4-Decadienal (DDE) is a widespread alpha, beta-unsaturated aldehyde found, for example, in food, water, and environmental pollutants. DDE is also endogenously generated as a breakdown product of lipid peroxidation in cell membranes. In the work presented here, the reaction of DDE with 2'-deoxyadenosine (dAdo) was investigated in an effort to assess its possible DNA damage potential. Besides 1,N(6)-etheno-2'-deoxyadenosine and two products, namely, 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1,2,3-octanetriol (adduct I) and 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1,2-heptanediol (adduct II), previously described by our group, two novel etheno adducts were identified. Thus, 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1-hexanol (adduct III) and 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-2,3-epoxy-1-octanol (adduct IV) were isolated by reverse-phase high-performance liquid chromatography and characterized on the basis of extensive spectroscopic measurements. The formation of the adducts is likely to involve initial DDE oxidation followed by generation of reactive intermediates such as diepoxides, epoxides, and/or hydroperoxides. The subsequent reaction of the latter oxidation products with dAdo will give rise to the four described adducts. We also demonstrated here that upon oxidation, DDE reacts with calf thymus DNA, producing the four dAdo adducts. Interestingly, two of them are the expected products arising from the reaction of dAdo with 4-hydroxy-trans-2-nonenal (HNE) and trans-2-octenal, two other important breakdown lipid peroxidation products. The reactivity of DDE with DNA is lower than that of the latter aldehydes. However, DDE produced a wider variety of adducts. The characterization of the different DNA-etheno adducts and the determination of the mechanism of formation are of great importance for a better understanding of the deleterious biological effects associated with this class of compounds.     

Aldose reductase catalyzes reduction of the lipid peroxidation product 4-oxonon-2-enal

Recent studies found that 4-oxonon-2-enal (4ONE) is a highly reactive product of lipid peroxidation that can modify peptides and protein sulfhydryls. Because aldose reductase (AR) was shown earlier to catalyze reduction of an alpha,beta-unsaturated lipid aldehyde, 4-hydroxynon-2-enal (4HNE), it was systematically investigated if this enzyme could represent a pathway for 4ONE metabolism as well. 4ONE, the glutathione (GSH) conjugate of 4ONE (GS-4ONE), and 1-hydroxynonen-4-one (1HNO), the predicted initial metabolite of 4ONE reduction, were incubated with AR and NADPH, and kinetic constants were measured. The initial product of AR-mediated 4ONE reduction was identified as 1HNO, which could be further reduced to DHN, catalyzed by AR. This result indicates that the order of 4ONE carbonyl reduction is aldehyde and then ketone. 1HNO was found to be an electrophile toward GSH with reactivity approximately 55-fold less than 4ONE but approximately 2-fold higher than that of 4HNE. The enzyme had activity toward GS-4ONE, exhibiting a approximately 4-fold higher k(cat)/K(M) for GS-4ONE as compared to 4ONE. In the presence of NADPH, 4ONE did not inactivate AR, whereas in the absence of the cofactor, approximately 60% of the enzyme activity was lost. The orientation of 4ONE in the AR active site was predicted using molecular modeling to explain the reactivity of 4ONE toward the enzyme. These simulations revealed that concurrent with NADPH binding to AR, Cys 298 is oriented such that the thiol group will not interact with 4ONE. Results of the present study are the first to demonstrate that AR may represent a pathway for metabolism of 4ONE and GS-4ONE.     

Model studies on protein side chain modification by 4-oxo-2-nonenal

trans-4-Oxo-2-nonenal (ONE) has recently been demonstrated to be a direct product of lipid peroxidation. In earlier studies to elucidate the structure of the trans-4-hydroxy-2-nonenal (HNE)-derived fluorescent Lys-Lys cross-link, we showed that ONE was capable of both oxidative and nonoxidative cross-linking of amines. A more comprehensive study on nonoxidative modification of protein nucleophiles by ONE is described here, focusing on the initial Michael addition of imidazole, thiol, and amine groups to C2 or C3 to give 4-keto aldehydes that can then condense with amines to form nucleophile-substituted pyrroles. 2,3-Substituted pyrroles (major) and 2,4-substituted pyrroles (minor) were distinguished by 2D NMR techniques, and N(tau)-substitution is preferred over N(pi)-substitution in the Michael addition of histidine. Mechanisms of both nonoxidative and oxidative side chain reactions of ONE are discussed, as is the relative propensity (ONE > HNE) to induce cross-linking of the model proteins ribonuclease A and beta-lactoglobulin.     

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal,a lipid peroxidation end product

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress.     

Structural and functional changes in human insulin induced by the lipid peroxidation byproducts 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Lipid peroxidation produces many reactive byproducts including 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) derived from the peroxidation of n-3 and n-6 polyunsaturated fatty acids, respectively. HNE and HHE can modify circulating biomolecules through the formation of covalent adducts. It remains, however, unknown whether HHE and HNE could induce functional and structural changes in the insulin molecule, which may in turn be pivotal in the development of insulin resistance and diabetes. Recombinant human insulin was incubated in the presence of HHE or HNE, and the formation of covalent adducts on insulin was analyzed by mass spectrometry analysis. Insulin tolerance test in mice and stimulation of glucose uptake by 3T3 adipocytes and L6 muscle cells were used to evaluate the biological efficiency of adducted insulin compared with the native one. One to 5 adducts were formed on insulin through Michael adduction, involving histidine residues. Glucose uptake in 3T3-L1 and L6C5 cells as well as the hypoglycemic effect in mice was significantly reduced after treatment with adducted insulin compared to native insulin. The formation of HNE- and HHE-Michael adducts significantly disrupts the biological activity of insulin. These structural and functional abnormalities of the insulin molecule might contribute to the pathogenesis of insulin resistance.     

Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation, in rat precision-cut liver slices

4-Hydroxy-2-nonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological and cytotoxic effects. The in vitro metabolism of [4-(3)H]-HNE by rat precision-cut liver slices was investigated. Liver slices rapidly metabolize HNE - about 85% of 0.1 microM [4-(3)H]-HNE was degraded within 5 min of incubation. The main metabolites of HNE identified were 4-hydroxynonenoic acid (HNA), glutathione-HNE-conjugate (HNE-GSH), glutathione-1,4-dihydroxynonene-conjugate (DHN-GSH) and cysteine-HNE-conjugate (HNE-CYS). Whereas glutathione conjugation demonstrated saturation kinetics (K(m)=412.2+/-152.7 microM and V(max)=12.3+/-2.5 nmol h(-1) per milligram protein), HNA formation was linear up to 500 microM HNE in liver slices. In contrast to previous reports, no trace of the corresponding alcohol of the HNE, 1,4-dihydroxynon-2-ene was detected in the present study. Furthermore, the beta-oxidation of HNA including the formation of tritiated water was demonstrated. The identification of 4-hydroxy-9-carboxy-2-nonenoic acid and 4,9-dihydroxynonanoic acid demonstrated that omega-oxidation significantly contributes to the biotransformation of HNE in liver slices.     

Mass spectroscopic characterization of protein modification by 4-hydroxy-2-(E)-nonenal and 4-oxo-2-(E)-nonenal

The modification of proteins by 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) was investigated using mass spectroscopic approaches. Electrospray ionization MS analysis of HNE- and ONE-treated myoglobin and apomyoglobin revealed that the latter more "open" protein structure resulted in more extensive modification. Reductive methylation of Lys residues halved the extent of modification, implicating the importance of adduction of HNE and ONE to both His and Lys residues. HPLC-MS/MS analysis of tryptic and chymotryptic peptides of HNE- or ONE-adducted apomyoglobin was aided by the knowledge of structures previously elucidated through model reactions. In the case of HNE, the adducts detected were the HNE-His Michael adduct (on H24, H36, H64, and H113), its dehydrated form (on H36), and the HNE-Lys pyrrole adduct (on K16, K42, K45, K145, and K147). In the case of the more reactive ONE, the adducts detected were the ONE-His Michael adduct (on H24), the ONE-Lys pyrrolinone adduct (on K16 and K145), and the ONE-His-Lys pyrrole cross-link (linking K16 to H24 in the C(5) peptide). Although previous analyses of tryptic peptides yielded findings about the nature of His modification, the current chymotryptic peptide analysis produced the first structural characterization of Lys modification on intact proteins by HNE and ONE using mass spectrometry.