Lipid Peroxidation Product Acrolein Research Articles

Metabolic Investigation on the Interaction Mechanism between Dietary Dihydrochalcone Intake and Lipid Peroxidation Product Acrolein Reduction.

Acrolein (ACR), a lipid peroxidation product, pathologically participates in various chronic diseases. In vitro evidence suggestes that dietary dihydrochalcones (DHCs) potentiate safe and alternative therapeutics to synthetic pharmaceuticals for ACR scavenging. Here, to investigate whether ingested DHCs could trap ACR and thereof result in reductions in endogenous ACR in mice is aimed. Three doses of phloretin (25, 100, and 400mgkg-1 ), a major dietary DHC, are orally administrated to mice and 24h urine and fecal samples are collected, respectively. High-resolution MS-based targeted metabolomics reveal for the first time that phloretin and its oxidized metabolite are able to trap endogenous ACR via formation of ACR conjugates. Quantification further demonstrate that a) more than 13% of ingested phloretin can dose-dependently trap 0.77-9.92nmol of ACR within 24h; b) phloretin ingestion leads to marked reductions in both free ACR and ACR metabolites in mouse urine compared to control; and c) trapping reactions by phloretin can account for up to 20.1% of the total decreases in endogenous ACR, depending on the administration doses. Findings from this study indicate that regular consumption of DHCs-rich diets holds great promise to alleviate the development of ACR-associated chronic diseases.

The Onset of Systemic Oxidative Stress Associated with the Accumulation of Lipid Peroxidation Product Acrolein in the Skin of Patients with Small-Vessel Vasculitis.

Small-vessel vasculitis (SVV) is the inflammation of the vessel wall that can result in hemorrhage and/or ischemia. Among the histological findings in SVV are increased infiltrating neutrophils, which, due to their oxidative burst and myeloperoxidase activity, release excessive reactive oxygen species, triggering a chain reaction of lipid peroxidation and yielding reactive aldehydes such as acrolein. The implication of oxidative stress in the pathogenesis of SVV was studied, focusing on acrolein immunohistochemistry in the affected skin vessels and systemic stress response. Samples from SVV patients and healthy subjects were collected and analyzed for total serum peroxides, total antioxidant capacity, inflammatory and immunological parameters, as well as for the presence of acrolein–protein adducts in the skin tissue specimens. The obtained data showed that systemic redox homeostasis and iron metabolism are altered in SVV patients. Possible biomarkers in the evaluation of oxidative status, disease activity and prevalence were indicated. Furthermore, a strong correlation between the accumulation of acrolein–protein adducts in the skin and the progression of the disease was revealed. Thus, the results of this study demonstrate that SVV is not only associated with systemic oxidative stress but also with tissue-specific oxidative stress that promotes acrolein formation and protein modification correlating with the severity of cutaneous vasculitis.

Cardiospecific Overexpression of ATPGD1 (Carnosine Synthase) Increases Histidine Dipeptide Levels and Prevents Myocardial Ischemia Reperfusion Injury.

BACKGROUNDMyocardial ischemia reperfusion (I/R) injury is associated with complex pathophysiological changes characterized by pH imbalance, the accumulation of lipid peroxidation products acrolein and 4‐hydroxy trans‐2‐nonenal, and the depletion of ATP levels. Cardioprotective interventions, designed to address individual mediators of I/R injury, have shown limited efficacy. The recently identified enzyme ATPGD1 (Carnosine Synthase), which synthesizes histidyl dipeptides such as carnosine, has the potential to counteract multiple effectors of I/R injury by buffering intracellular pH and quenching lipid peroxidation products and may protect against I/R injury. METHODS AND RESULTSWe report here that β‐alanine and carnosine feeding enhanced myocardial carnosine levels and protected the heart against I/R injury. Cardiospecific overexpression of ATPGD1 increased myocardial histidyl dipeptides levels and protected the heart from I/R injury. Isolated cardiac myocytes from ATPGD1‐transgenic hearts were protected against hypoxia reoxygenation injury. The overexpression of ATPGD1 prevented the accumulation of acrolein and 4‐hydroxy trans‐2‐nonenal–protein adducts in ischemic hearts and delayed acrolein or 4‐hydroxy trans‐2‐nonenal–induced hypercontracture in isolated cardiac myocytes. Changes in the levels of ATP, high‐energy phosphates, intracellular pH, and glycolysis during low‐flow ischemia in the wild‐type mice hearts were attenuated in the ATPGD1‐transgenic hearts. Two natural dipeptide analogs (anserine and balenine) that can either quench aldehydes or buffer intracellular pH, but not both, failed to protect against I/R injury.CONCLUSIONSEither exogenous administration or enhanced endogenous formation of histidyl dipeptides prevents I/R injury by attenuating changes in intracellular pH and preventing the accumulation of lipid peroxidation derived aldehydes.

Identification and Quantitation of the Lipation Product 2-Amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic Acid (MP-Lysine) in Peanuts.

The lipid peroxidation product acrolein was semiquantitated by GC-MS (EI) in unheated and heated peanut oil, respectively, representing a model system for peanut roasting. Depending on the heating time, acrolein levels significantly increased from 0.2 to 10.7 mg/kg oil. As a result of heating N(α)-acetyl-l-lysine and acrolein, the pyridinium derivative 2-acetamido-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-acetyl lysine) was identified. In addition, the lysine derivative 2-amino-6-[5-(hydroxymethyl)-3,6-dihydro-2H-pyridin-1-yl]hexanoic acid was identified after reduction and hydrolysis. After preparation of 2-amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine) as reference material, its amounts were quantitated in acrolein-modified peanut proteins by HPLC-ESI-MS/MS after acid hydrolysis, showing that at low acrolein concentrations, the modification of lysine could be entirely explained by the formation of MP-lysine. Furthermore, for the first time, MP-lysine was quantitated in peanut samples in amounts up to 10.2 mg/kg, showing an increase depending on the roasting time. Thus, MP-lysine might represent a marker to evaluate the extent of food protein lipation by acrolein.

Abstract 1689: Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis for 7-carboxymethylguanine and 7-carboxyethylguanine in human liver DNA

Abstract The carcinogenic nitrosamines N-nitrososarcosine (NSAR) and 3-(methylnitrosamino)propionic acid (MNPA) have been detected in the diet, tobacco products, and human urine. Metabolic activation of NSAR by methyl hydroxylation would produce a DNA carboxymethylating agent while similar metabolism of MNPA would produce a carboxyethylating agent, each of which would be expected to react at the 7 position of guanine to give 7-carboxymethylguanine (7-CMG) and 7-carboxyethylguanine (7-CEG), respectively. Furthermore, acrylic acid, a major industrial product, environmental contaminant and likely endogenous metabolite, and β-propiolactone, are also known to react with DNA to give 7-CEG. We developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS-SRM) method to quantify these adducts in human liver DNA. [15N5]7-CMG and [15N5]7-CEG were synthesized and used as internal standards. After addition of the internal standards, the DNA was hydrolyzed enzymatically and the hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-CMG and 7-CEG was treated with acetyl chloride in methanol to convert them to the corresponding methyl esters. After re-purification by solid-phase extraction, the appropriate fraction was analyzed by LC-ESI-MS/MS-SRM for the transitions m/z 224[M + H]+ → m/z 164[(M + H) - C2H4O2]+ for 7-CMG and m/z 238[M + H]+ → m/z 152 [BH]+ for 7-CEG. Human liver DNA samples had clear peaks co-eluting with [15N5]7-CEG, but no peaks were observed at the retention time of 7-CMG. The on column detection limits were approximately 10 and 5 fmol, respectively, for the methyl esters of 7-CMG and 7-CEG. The detection limit for the methyl ester of 7-CEG in DNA (starting with 1 mg) was approximately 8 fmol. Known addition studies using 7-CEG and calf thymus DNA demonstrated that the method was accurate and precise. Artifactual formation of 7-CEG during DNA isolation and enzyme hydrolysis was excluded. The method was applied for the analysis of 27 human liver samples. 7-CEG, but not 7-CMG, was detected in each sample, mean ± S.D, 408 ± 375 fmol/µmol guanine (82 adducts per 109 nucleotides), range 17 − 1491 fmol/µmol guanine. This is the first report demonstrating the presence of 7-CEG in DNA from humans. While MNPA, β-propiolactone and acrylic acid are possible sources of this DNA adduct, we consider acrylic acid to be the most likely because of its ubiquitous occurrence and probable endogenous formation from the lipid peroxidation product acrolein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1689.

Lipid peroxidation product acrolein as a predictive biomarker of prostate carcinoma relapse after radical surgery

Cancer recurrence after radical surgery might happen even in the case of patients with localized prostate carcinoma treated by radical prostatectomy. Therefore, identifying predictive markers of tumour recurrence is very important, so this study evaluated the presence of lipid peroxidation product acrolein in primary prostate carcinomas, assuming that acrolein could be involved in prostate carcinogenesis as was recently shown for colon cancer. Samples obtained by radical prostatectomy of 70 patients were analysed, out of which 27 patients suffered afterwards from tumour recurrence, while 43 patients were disease free. Immunohistochemistry using genuine monoclonal antibodies against acrolein-protein adducts revealed the association of acrolein with progression of carcinoma. The logistic regression combining clinical parameters together with the biochemical markers of disease and acrolein immunohistochemistry has shown that the relapse might be predicted with 90% accuracy if tumour-positive surgical margins, stage of disease and the intensity of acrolein presence in tumour stroma were taken together.

Structural modification of soy protein by the lipid peroxidation product acrolein

Acrolein was selected as a representative secondary byproduct of lipid peroxidation to investigate the effect of oxidative modification of reactive aldehyde on soy protein structure. Acrolein reacted with histidine, lysine and cysteine residues in soy protein to form covalent adducts, leading to protein carbonylation and degradation of sulfhydryl groups. Circular dichroism spectra showed that soy protein modification by acrolein was related to loss of α-helix and increase of β-sheet structure. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly implied that acrolein induced soy protein aggregation, and results obtained by size-exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of acrolein. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that acrolein caused soy protein cross-linking which non-disulphide covalent bonds were involved in the formation of cross-linking, and subunits of β-conglycinin were more vulnerable to acrolein than that of glycinin.

Screening for DNA adducts by data-dependent constant neutral loss-triple stage mass spectrometry with a linear quadrupole ion trap mass spectrometer.

A two-dimensional linear quadrupole ion trap mass spectrometer (LIT/MS) was employed to simultaneously screen for DNA adducts of environmental, dietary, and endogenous genotoxicants, by data-dependent constant neutral loss scanning followed by triple-stage mass spectrometry (CNL-MS3). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M + H - 116]+) in the MS/MS scan mode triggered the acquisition of MS3 product ion spectra of the aglycone adducts [BH2]+. Five DNA adducts of the tobacco carcinogen 4-aminobiphenyl (4-ABP) were detected in human hepatocytes treated with 4-ABP, and three DNA adducts of the cooked-meat carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were identified in the livers of rats exposed to MeIQx, by the CNL-MS3 scan mode. Buccal cell DNA from tobacco smokers was screened for DNA adducts of various classes of carcinogens in tobacco smoke including 4-ABP, 2-amino-9H-pyrido[2,3-b]indole (AalphaC), and benzo[a]pyrene (BaP); the cooked-meat carcinogens MeIQx, AalphaC, and 2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (PhIP); and the lipid peroxidation products acrolein (AC) and trans-4-hydroxynonenal (HNE). The CNL-MS3 scanning technique can be used to simultaneously screen for multiple DNA adducts derived from different classes of carcinogens, at levels of adduct modification approaching 1 adduct per 108 unmodified DNA bases, when 10 microg of DNA is employed for the assay.

Age‐dependent telomere‐shortening is repressed by phosphorylated α‐tocopherol together with cellular longevity and intracellular oxidative‐stress reduction in human brain microvascular endotheliocytes

Cellular life-span of neonatal human brain microvascular endotheliocytes (HBME) was estimated by population doubling levels (PDLs) for serial subcultivations until spontaneous proliferation stoppage, and was 2.4-fold longer for continuous administration with the 6-O-phosphorylated derivative (TocP) of alpha-tocopherol (Toc), being bio-available owing to its water-solubility, or TocP plus 2-O-phosphorylated ascorbate (Asc2P), and 1.3-fold longer with Asc2P, at a dose of 150 microM, than for the non-administered control. Enlarged cell diameters indicative of cellular aging were repressed for TocP-administered cells as analyzed with a channelizer. Age-dependent shortening of telomeric DNA length (291 bp/PDL) was slowed markedly for TocP (165 bp/PDL) or TocP plus Asc2P, but slightly for Asc2P. Telomerase activity as assessed by the PCR-based TRAP method was detectable slightly at younger ages but no longer at middle ages for the non-administered cells, but, for TocP-administered cells, was intensely detected at younger ages and appreciably until middle ages. Intracellular TocP amounts were not changed age-dependently in contrast to a marked decrease in Toc which accrued from TocP esterolysis. This may be partly attributed to age-dependent changes in the lipid peroxidation product acrolein (ACR), which was abundant at older ages in non-administered cells, but scarcely in TocP-administered cells. Furthermore, intracellular reactive oxygen species (ROS) such as H(2)O(2) and hydroperoxides as detected using the redox indicator CDCFH-DA was less abundant in TocP-administered cells than in non-administered cells. Thus the telomeric-DNA retention, concurrently with retained telomerase activity, was shown to be correlated with cellular longevity, and may be supported by diminished oxidative stress, in hydrophobic microenvironment, which can be achieved by TocP rather than AscP.

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein–protein adducts in 113 human colon tumours. The presence of acrolein–protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.

Cysteine Modification by Lipid Peroxidation Products Inhibits Protein Disulfide Isomerase

A proteomic approach was applied to mitochondrial protein isolated from the livers of rats fed a combination high-fat and ethanol diet to identify proteins modified by 4-hydroxynonenal (4-HNE). Using this approach, the endoplasmic reticulum chaperone, protein disulfide isomerase (PDI), which participates in the maturation of newly synthesized proteins through promoting correct disulfide formation, was consistently found to be modified by 4-HNE. Further mass spectral analysis of PDI isolated from the animals revealed modification of an active site Cys residue thought to be involved in client protein binding. To test the hypothesis that 4-HNE inhibits the chaperone, purified bovine PDI was treated with concentrations of 4-HNE ranging from 20 to 200 microM (10-100-fold molar excess aldehyde), resulting in 14-56% inhibition, respectively. Similar treatments with the lipid peroxidation products acrolein (ACR) and 4-oxononenal (4-ONE) resulted in 60 and 100% inhibition, respectively, suggesting inactivation of the chaperone via Cys modification. Thiol sensitivity was confirmed through concentration-dependent inhibition of PDI by the Cys modifier N-ethylmaleimide (NEM). While some degree of sensitivity to these lipid aldehydes is suggested by the data, when compared to inactivation of other proteins by 4-HNE, PDI has demonstrated a relative resistance. It was also observed that physiologic (e.g., 4 mM) concentrations of GSH were capable of removing the 4-HNE adducts, likely serving as a protective mechanism against inactivation by 4-HNE and other lipid peroxidation products. However, because an active site Cys was found to be modified by 4-HNE on PDI in vivo, it is possible that the protective effect of GSH on the chaperone decreases under conditions of sustained oxidative stress, such as during chronic alcohol consumption, as GSH is depleted. The data presented here thus suggest potential impairment of an important molecular chaperone during oxidative stress.

Reactivity of hydrazinophthalazine drugs with the lipid peroxidation products acrolein and crotonaldehyde.

The nucleophilic drug hydralazine strongly inhibits cell toxicity mediated by acrolein, a short chain 2-alkenal formed during lipid peroxidation. We here report the chemistry of acrolein-trapping by hydralazine, and show that together with its structural analogue dihydralazine, it also readily traps crotonaldehyde. Isolable reaction products included (1E)-acrylaldehyde phthalazin-1-ylhydrazone (E-APH), (1Z)-acrylaldehyde phthalazin-1-ylhydrazone (Z-APH), (1E,2E)-but-2-enal phthalazin-1-ylhydrazone (E-BPH) and (1Z,2E)-but-2-enal phthalazin-1-ylhydrazone (Z-BPH). Concentration-dependent formation of (1E)-acrylaldehyde phthalazin-1-ylhydrazone was observed in the culture media of cells co-exposed to hydralazine and the acrolein precursor allyl alcohol. These aldehyde-sequestering properties of hydrazinophthalazine drugs may contribute to the protection they provide against 2-alkenal-mediated toxicity.

Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells.

A hallmark of several neurodegenerative disorders, including Alzheimer's disease and tauopathies, is the hyperphosphorylation of the microtubule-associated protein tau. Tau phosphorylation by proline-directed and non-proline-directed protein kinases has been tested using antibodies PHF1 and 12E8, respectively. The effect of the lipid peroxidation product acrolein on these modes of phosphorylation has been assayed. We have found that acrolein, a peroxidation product from arachidonic acid, increases the phosphorylation of tau at the site recognized by PHF-1 both in human neuroblastoma cells and in primary cultures of mouse embryo cortical neurons. Whereas the basal phosphorylation of tau protein at the PHF1 site seems to be largely mediated by glycogen synthase kinase-3 (which is also activated in response to Abeta peptide), the acrolein-induced tau hyperphosphorylation at the same site is also due to p38 stress-activated kinase. These results support the view that oxidative stress and subsequent formation of lipid peroxidation products may contribute to tau protein phosphorylation in Alzheimer's disease and tauopathies.

Expression of rat liver glutathione-S-transferase GSTA5 in cell lines provides increased resistance to alkylating agents and toxic aldehydes

The glutathione-S-transferases (GST) are a major contributor to the eukaryotic cell's defences against chemical and oxidative stress. However, the role of individual GST isoenzymes in conferring resistance to xenobiotics has not been fully determined. We have examined the effect of the rat GSTA5 isoenzyme in the detoxication of alkylating agents and aldehydes by constructing a cell line in which it is stably expressed. The hamster fibroblast cell line V79 was transfected with a construct expressing GSTA5 from the CMV promoter. A stable clone (V79-GSTA5) was isolated after selecting for the neomycin phosphotransferase gene present on the introduced DNA. The cell line showed significantly increased levels of resistance towards the alkylating agents chorambucil and melphalan. Levels of resistance were 4–6-fold greater in V79-GSTA5 cells than in control cells. Increased levels of resistance were also observed towards the lipid peroxidation product acrolein (IC 50=80 μM compared with 17 μM in control cells). The V79-GSTA5 cells also showed a 4-fold increase in resistance to trans, trans muconaldehyde (IC 50=4 μM compared with l μM for control cells). GSTA5 did not protect against 4-hydroxynonenal, but it did provide greater levels of protection to hydrogen peroxide, with an IC 50 of 380 μM in V79-GSTA5 compared with 180 μM in control cells. In contrast, V79-GSTA5 cells were more sensitive to methyl glyoxal, suggesting that a methyl glyoxal–glutathione conjugate is more toxic that the parental compound. These data contribute towards the evaluation of the role of GSTA5 in the detoxication of these compounds.