Formation Of HNE Research Articles

Metabolic pathways for removing reactive aldehydes are diminished in the skeletal muscle during heart failure.

Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in theGastrocnemius and soleus muscles were analyzed by LC/MS-MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius musclewere decreased after 14weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5-2fold in the gastrocnemius muscle after 14weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting.

Assessing the Impact of Pomegranate Peel Extract Active Packaging and High Hydrostatic Pressure Processing on Color and Oxidative Stability in Sliced Nitrate/Nitrite-Reduced Iberian Dry-Cured Loins

Our study aimed to assess the impact of active packaging with pomegranate peel extract (0.06 mg gallic acid eq./cm2) and/or high-pressure treatment (600 MPa, 7 min) on the instrumental color, lipid, and protein oxidation of Iberian dry loins formulated with reduced nitrate/nitrite levels (0, 37.5, and 150 mg/kg) during 100-day refrigerated storage (4 °C). CIE L*a*b* coordinates were measured, and malondialdehyde, carbonyls, and free thiol contents served as markers for lipid and protein oxidation. Active packaging lowered CIE L* (35.4 vs. 34.1) and a* (15.5 vs. 14.5) and increased yellowness (15.6 vs. 16.3) and hue (45.2 vs. 48.4), while pressurization increased CIE L* (33.1 vs. 36.3) and diminished a* values (16.1 vs. 13.9). Ongoing nitrate/nitrite amounts significantly influenced lipid peroxidation, protein carbonyl formation, and free thiol loss. Active packaging and high-pressure processing had varying effects on carbonyl and thiol contents. Neither pressurization nor active packaging impacted malondialdehyde formation. Pressurization enhanced the formation of 4-HNE (503 vs. 697 pg/g). Protein oxidation proved more sensitive to changes, with active packaging offering protection against protein carbonylation (15.4 vs. 14.7 nmol carbonyls/mg protein), while pressurization induced thiol loss (34.3 vs. 28.0 nmol Cys eq./mg protein). This comprehensive understanding provides essential insights for the meat industry, emphasizing the necessity for customized processing conditions to enhance color stability, lipid preservation, and protein integrity in dry-cured loin slices.

Pharmacological inhibition of catalase induces peroxisome leakage and suppression of LPS induced inflammatory response in Raw 264.7 cell.

Peroxisomes are metabolically active organelles which are known to exert anti-inflammatory effects especially associated with the synthesis of mediators of inflammation resolution. However, the role of catalase and effects of peroxisome derived reactive oxygen species (ROS) caused by lipid peroxidation through 4-hydroxy-2-nonenal (4-HNE) on lipopolysaccharide (LPS) mediated inflammatory pathway are largely unknown. Here, we show that inhibition of catalase by 3-aminotriazole (3-AT) results in the generation of peroxisomal ROS, which contribute to leaky peroxisomes in RAW264.7 cells. Leaky peroxisomes cause the release of matrix proteins to the cytosol, which are degraded by ubiquitin proteasome system. Furthermore, 3-AT promotes the formation of 4HNE-IκBα adduct which directly interferes with LPS induced NF-κB activation. Even though, a selective degradation of peroxisome matrix proteins and formation of 4HNE- IκBα adduct are not directly related with each other, both of them are could be the consequences of lipid peroxidation occurring at the peroxisome membrane.

Digestive n-6 Lipid Oxidation, a Key Trigger of Vascular Dysfunction and Atherosclerosis in the Western Diet: Protective Effects of Apple Polyphenols.

A main risk factor of atherosclerosis is a Western diet (WD) rich in n-6 polyunsaturated fatty acids (PUFAs) sensitive to oxidation. Their oxidation can be initiated by heme iron of red meat leading to the formation of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic aldehyde. An increased 4-HNE production is implicated in endothelial dysfunction and atherosclerosis. By contrast, a diet rich in proanthocyanidins reduces oxidative stress and arterial diseases. This study evaluates the effects of a WD on vascular integrity in ApolipoproteinE (ApoE-/- ) mice and the protective capacity of apple extract and puree rich in antioxidant proanthocyanidins. ApoE-/- mice are fed during 12 weeks with a WD with or without n-6 PUFAs. Moreover, two WD + n-6 PUFAs groups are supplemented with apple puree or phenolic extract. An increase in digestive 4-HNE production associated with a rise in plasmatic 4-HNE and oxidized LDL concentrations is reported. Oxidizable n-6 PUFAs consumption is associated with a worsened endothelial dysfunction and atherosclerosis. Interestingly, supplementations with apple polyphenol extract or puree prevented these impairments while reducing oxidative stress. n-6 lipid oxidation during digestion may be a key factor of vascular impairments. Nevertheless, an antioxidant strategy can limit 4-HNE formation during digestion and thus durably protect vascular function.

Heme-Iron-Induced Production of 4-Hydroxynonenal in Intestinal Lumen May Have Extra-Intestinal Consequences through Protein-Adduct Formation.

Some epidemiological studies show that heme iron consumption, in red meat, is associated to the development of several chronic diseases, including cancers and cardio-metabolic diseases. As heme iron intestinal absorption is finely regulated, we hypothesized that heme iron may act indirectly, through the peroxidation of dietary lipids, in food or in the intestinal lumen during digestion. This heme-iron-induced lipid peroxidation provokes the generation of toxic lipid oxidation products that could be absorbed, such as 4-hydroxynonenal (HNE). In a first experiment, heme iron given to rats by oral gavage together with the linoleic-acid-rich safflower oil induced the formation of HNE in the intestinal lumen. The HNE major urinary metabolite was elevated in the urine of the treated rats, indicating that this compound has been absorbed. In a second experiment, we showed that stable isotope-labeled HNE given orally to rats was able to reach non-intestinal tissues as a bioactive form and to make protein-adducts in heart, liver and skeletal muscle tissues. The presence of HNE-protein adducts in those tissues suggests a putative biological role of diet-originating HNE in extra-intestinal organs. This finding could have major consequences on the onset/development of chronic diseases associated with red meat over-consumption, and more largely to peroxidation-prone food consumption.

N-6 Polyunsaturated fatty acid oxidation increase oxidative stress, endothelial dysfunction and atherosclerosis in ApoE mice fed with chronic Western diet. Prevention strategy by apple polyphenols

Atherosclerosis is a leading cause of mortality associated with endothelial dysfunction, oxidative stress and inflammation. One of the main risk factors is a Western diet rich in n-6 polyunsaturated fatty acids (n-6PUFA) which are sensitive to oxidation by heme iron of red meat in the gastrointestinal tract. This oxidation may increase 4-hydroxy-2-nonenal (4-HNE) formation, a cytotoxic aldehyde generated from n-6PUFA. Moreover, coupling of 4-HNE to LDL can lead to endothelial dysfunction and atherosclerosis development. By contrast, a diet rich in proanthocyanidins (PCs) (polyphenols, flavonoid class) is known to reduce oxidative stress as well as coronary artery disease. Evaluate chronic in Western diet effects and the potential protective capacity of PCs on vascular function and atherosclerosis development in an ApoE-/- mouse model. ApoE-/- mice were fed with: high-fat/red meat (HF-RM); HF-RM + n-6PUFA; HF-RM + n-6PUFA supplemented with apple puree or a phenolic extract (0.06% w/w) for 12 weeks. We reported an increase in fecal 4-HNE formation associated with a rise in plasmatic 4-HNE and oxLDL concentrations in n-6PUFA group. Moreover, adding oxidizable n-6PUFA to high fat diet was associated with ROS production (anion superoxide), worsened endothelial dysfunction, blood pressure and atheromatous plaque formation (136 vs. 166mm 2 in HF-RM). However, necrotic core size, fibrosis and lipid accumulation in atheromatous plaque were not affected. Interestingly, both polyphenol supplementations prevented the exacerbation of oxidative stress, endothelial dysfunction and atherosclerosis impairments as well as an increase in eNOS activity ( Table 1 ). Apple polyphenols limit the formation of absorbable 4-HNE during digestion and consequently protect vascular integrity against the damages of oxidative stress. Consumption of PC-rich fruits should thus be encouraged within the Western diet.

4‐HNE Unfavorably Alters Muscle Mitochondrial Bioenergetics and Cell Viability

Linoleic acid consumption has increased by orders of magnitude over the past century, eliciting an increasing awareness of and appreciation for its metabolites, particularly 4‐hydroxynonenal (4‐HNE). Elevated 4‐HNE formation is causally implicated in myriad neurodegenerative, cardiovascular, autoimmune, and metabolic disorders. The purpose of this project was to identify the effect of 4‐HNE on muscle cell mitochondrial bioenergetics. Our primary observation was that physiological levels of 4‐HNE compromised muscle cell mitochondrial oxygen consumption, leading to reduced oxygen flux despite a substantial production of reactive oxygen species (ROS). Indeed, when viewed in light of the reduced oxygen consumption, the relative production of ROS with 4‐HNE was even more pronounced. These findings could explain the significant reduction of cell viability demonstrated. In conclusion, this work joins the growing body of evidence suggesting a need to scrutinize dietary linoleic acid consumption.

Retardation of 4-Hydroxy-2-Transnonenal (HNE), a Toxic Aldehyde Formation by Antioxidants in Heat-Treated Corn Oil at Frying Temperature

The antioxidative properties of four antioxidants such as rosemary extracts (RE), tert-butylhydroquinone (TBHQ), ascorbyl palmitate (AP), citric acid (CA) and their mixtures were investigated on the formation of 4-hydroxy-2-transnonenal (HNE) in commercial corn oil heated at 185°C for up to 6 hours. Among the antioxidants 100 ppm RE and a mixture of 200 ppm tertiary butylated hydroquinone (TBHQ) + 100 ppm ascorbyl palmitate (AP) + 50 ppm citric acid (CA) exhibited excellent antioxidative activity, as determined by the thiobarbituric acid reaction (TBARS) assay, measuring the formations of the secondary lipid oxidation products and by high-performance liquid chromatography (HPLC), measuring the formation of the toxic α, β-unsaturated hydroxyaldehyde HNE after heat treatment of corn oil at 185°C up to 6 hours. TBHQ, AP and CA alone did not show much protective properties. The synergistic effects of TBHQ + AP + CA mixture shown to reduce the formation of HNE after 6 hours heat-treated corn oil by 27%. RE 100 ppm was also found to be a very effective antioxidant, reducing the formation of HNE after 6 hours heat-treated corn oil in the same condition by 29%.

Limited Antioxidant Effect of Rosemary in Lipid Oxidation of Pan-Fried Salmon.

Consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) rich fatty fish is known to provide an array of health benefits. However, high temperature in food preparation, such as pan-frying, potentially degrades eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of the n-3 PUFAs by heat oxidation. The addition of antioxidant condiments, and herbs in particular, may retard PUFA peroxidation and preserve EPA and DHA during pan-frying. In this study, different types of antioxidant condiments (sage, rosemary, black peppercorn, thyme, basil, and garlic) were tested for antioxidant capacity, and the condiment with the highest capacity was selected for its effect on lipid oxidation of salmon. The changes in fatty acids and lipid peroxidation of salmon, during pan-frying with the selected condiment (olive oil infused with rosemary, RO(infused)), were compared with salmon prepared in extra virgin olive oil, olive oil, or without oil. The total saturated fatty acid was found to be less in pan fried salmon with RO(infused). None of the oil type conserved EPA- and DHA-content in salmon. However, RO(infused) lowered lipid peroxidation by lessening hydroperoxide and 4-HNE formation, but not the other related products (HDHA, HETE, isoprostanes). Our observation indicates that the antioxidant capacity of RO(infused), when it is incorporated with food, becomes limited.

Apple polyphenols decrease endothelial dysfunction and atherosclerosis in ApoE mice fed with chronic Western diet

Atherosclerosis is a leading cause of mortality in industrialized countries and is associated with endothelial dysfunction, oxidative stress and inflammation. One of the main risk factors is a pro-oxidant Western diet rich in ω-6 polyunsaturated fatty acids (ω-6 PUFA) which are sensitive to oxidation by heme iron of red meat in the gastrointestinal tract. This oxidation may increase 4-hydroxy-2-nonenal (4-HNE) formation, a cytotoxic aldehyde generated from ω-6 PUFA. Moreover, coupling of 4-HNE to LDL is a key step in atherosclerosis development. By contrast, a diet rich in proanthocyanidins (PCs) (polyphenols, flavonoid class) is known to reduce oxidative stress as well as coronary artery disease. Evaluate chronic pro-oxidant Western diet effects and the potential protective capacity of PCs on vascular function and atherosclerosis development in an ApoE-/- mouse model. ApoE-/- mice were fed with: high-fat/red meat (HF-RM); HF-RM + ω-6 PUFA; HF-RM + ω-6 PUFA supplemented with apple puree or a phenolic extract (0.06% w/w) for 12 weeks. We reported an increase in fecal 4-HNE formation associated with a rise in plasmatic 4-HNE and LDLox concentrations in ω-6 PUFA group. This increase was prevented by apple polyphenols supplementation. Moreover, adding oxidizable ω-6 PUFA to high fat diet worsens endothelial dysfunction, blood pressure and atheromatous plaque formation (136 vs. 166mm 2 in HF-RM). However, necrotic core size, fibrosis and lipid accumulation in atheromatous plaque were not affected. Interestingly, both polyphenol supplementations prevented the exacerbation of endothelial dysfunction and atherosclerosis impairments which was associated with an increase in eNOS activity. Apple polyphenols limit the formation of absorbable lipid oxidation products during digestion (4-HNE) and consequently protect vascular integrity. Consumption of PC-rich fruits should thus be encouraged within the Western diet.

The role of lipid peroxidation during the progression of human hepatocellular carcinoma (HCC)

Hepatocellular Carcinoma (HCC) is ranked 5th among the most diagnosed cancer and the 3rd most common cause of cancer-related death worldwide. Emerging evidence suggests that HCC metastasis through portal vein tumor thrombus (PVTT) is the leading cause for poor prognosis in HCC patients. However, the mechanisms underlying initiation and progression of HCC remain to be defined. Altered redox status in cancer cells has been linked to lipid peroxidation induced by reactive oxygen species (ROS) and subsequent formation of reactive lipid electrophiles, especially 4-hydroxy-nonenal (4-HNE). Emerging evidence suggests that cancer cells manipulate redox status to acquire anti-apoptotic phenotype. Cardiolipin (CL), a mitochondria-specific inner membrane phospholipid, is critical for maintaining mitochondrial function. Paradoxically, liver tissues contain tetralinoleoyl cardiolipin (TLCL) as the major CL in mitochondria yet emerging evidence suggests that ROS generated in mitochondria may lead to CL peroxidation and activation of intrinsic apoptosis. It remains unclear how CL oxidation leads to apoptosis and its relevance to the pathogenesis of hepatocellular carcinoma (HCC). We employed a mass spectrometry-based lipidomic approach to profile lipids in human tissues of HCC and found that CL was gradually decreased in tumor comparing to peripheral non-cancerous tissues, accompanied by a concomitant decrease of oxidized CL and its oxidation product, 4-HNE. Incubation of liver cancer cells with TLCL significantly restored apoptotic sensitivity accompanied by an increase of CL and its oxidation products when treated with staurosporine (STS) or Sorafenib (the standard treatment for late stage HCC patients). Our studies uncovered a novel mechanism by which cancer cells adopt to evade apoptosis, highlighting the importance of mitochondrial control of apoptosis through modulation of CL oxidation and subsequent 4-HNE formation in HCC. Thus, manipulation of mitochondrial CL oxidation and lipid electrophile formation may have potential therapeutic value for diseases linked to oxidative stress and mitochondrial dysfunctions.

Inhibition by microbial metabolites of Chinese dark tea of age-related neurodegenerative disorders in senescence-accelerated mouse prone 8 (SAMP8) mice.

Dark tea has a significant effect on the prevention and treatment of age-related degenerative diseases. At present, further exploration of its functional mechanisms is delayed because of the complexity of post-fermentation microbial metabolites during the production phase. In this study, new isolated microbial metabolites extracted from dark tea were used to explore the neuroprotective effects, and they also helped allow further exploration of the mechanism of dark tea. Taking senescence-accelerated mouse prone 8 (SAMP8) mice as a biological model, we examined the protective effect on brain neurons of post-fermentation microbial metabolites which were extracted from dark tea. The 4-month-old mice were given treatments of the same concentration (10 mg kg-1 d-1) which were l-theanine, 3,3'-azanediylbis(4-hydroxybenzoic acid) (CDT-1) and one of the 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols (CDT-2) by gavage for 14 weeks. Relative measurements such as RT-PCR, ELISA, western blotting, and section staining (HE, Nissl and myelin) were carried out. The results showed that l-theanine, CDT-1 and CDT-2 could inhibit the decrease in body weight, and down-regulate the formation of 4-HNE and ubiquitinated protein aggregates and the Aβ metabolic pathway. They could also increase endogenous antioxidant capacity, relieve cell hypoxia, and reduce the rate of neuronal apoptosis. This means that their protective activity regarding SAMP8 neurons was excellent and the activity of CDT-2 was the most significant.

Mitochondrial control of apoptosis through modulation of cardiolipin oxidation in hepatocellular carcinoma: A novel link between oxidative stress and cancer

Altered redox status in cancer cells has been linked to lipid peroxidation induced by reactive oxygen species (ROS) and subsequent formation of reactive lipid electrophiles, especially 4-hydroxy-nonenal (4-HNE). Emerging evidence suggests that cancer cells manipulate redox status to acquire anti-apoptotic phenotype but the underlying mechanisms are poorly understood. Cardiolipin (CL), a mitochondria-specific inner membrane phospholipid, is critical for maintaining mitochondrial function. Paradoxically, liver tissues contain tetralinoleoyl cardiolipin (TLCL) as the major CL in mitochondria yet emerging evidence suggests that ROS generated in mitochondria may lead to CL peroxidation and activation of intrinsic apoptosis. It remains unclear how CL oxidation leads to apoptosis and its relevance to the pathogenesis of hepatocellular carcinoma (HCC). We employed a mass spectrometry-based lipidomic approach to profile lipids in human tissues of HCC and found that CL was gradually decreased in tumor comparing to peripheral non-cancerous tissues, accompanied by a concomitant decrease of oxidized CL and its oxidation product, 4-HNE. Incubation of liver cancer cells with TLCL significantly restored apoptotic sensitivity accompanied by an increase of CL and its oxidation products when treated with staurosporine (STS) or Sorafenib (the standard treatment for late stage HCC patients). Our studies uncovered a novel mechanism by which cancer cells adopt to evade apoptosis, highlighting the importance of mitochondrial control of apoptosis through modulation of CL oxidation and subsequent 4-HNE formation in HCC. Thus manipulation of mitochondrial CL oxidation and lipid electrophile formation may have potential therapeutic value for diseases linked to oxidative stress and mitochondrial dysfunctions.

4-Hydroxy-nonenal-A Bioactive Lipid Peroxidation Product.

This review on recent research advances of the lipid peroxidation product 4-hydroxy-nonenal (HNE) has four major topics: I. the formation of HNE in various organs and tissues, II. the diverse biochemical reactions with Michael adduct formation as the most prominent one, III. the endogenous targets of HNE, primarily peptides and proteins (here the mechanisms of covalent adduct formation are described and the (patho-) physiological consequences discussed), and IV. the metabolism of HNE leading to a great number of degradation products, some of which are excreted in urine and may serve as non-invasive biomarkers of oxidative stress.

4-Hydroxynonenal: A Superior Oxidative Biomarker Compared to Malondialdehyde and Carbonyl Content Induced by Carbon Tetrachloride in Rats

Carbon tetrachloride (CCl4), a halogenated substance that generates free radical species during metabolism in vivo, induces hepatotoxicity, produces oxidative DNA damage, and increased levels of protein carbonyl, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In this study, Sprague-Dawley rats received single or repeated ip injections of carbon tetrachloride (CCl4), and formation and persistence of carbonyls, MDA, and 4-HNE in plasma were measured using gas chromatography–mass spectrometry. After a single injection of 500 mg/kg CCl4 the in vivo half-lives of MDA and carbonyl content were 1.5 d and 2 d, respectively, while that of 4-HNE was approximately 10 d. Treatment with CCl4 (50, 100, 500, or 1000 mg/kg) dose-dependently increased these oxidative biomarkers in blood. However, formation of protein carbonyls and MDA was less sensitive than 4-HNE to CCl4. Levels of serum glutamic oxaloacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT) (hepatotoxicity markers) rose with CCl4 doses. After a single injection (500 mg/kg), the peak level of SGOT was observed after 8 h but SGPT after 24 h. Overall, 4-HNE was more dose-sensitive and showed greater formation subchronically than other biomarkers. Multiple ip treatments with 300 mg CCl4 /kg (d 1, 3, 6, 10, 14, and 21) demonstrated that 4-HNE formation was highest (18-fold, peak/control) and subchronic up to d 21 (last treatment day), unlike other biomarkers. Data suggest that 4-HNE, MDA, and carbonyl content may be useful oxidative biomarkers for exposure to free radical generating halogenated compounds. However, 4-HNE appears to be a more sensitive and sustainable biomarker for toxicological and risk assessments.

Changes in secondary structure of α-synuclein during oligomerization induced by reactive aldehydes

The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of α-synuclein oligomers in vitro. However, the changes in secondary structure of α-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric α-synuclein peak with a molecular weight of about ∼2000 kDa, which coincided with a decreasing ∼50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a ∼2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced α-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30–160 kDa) and high molecular weight oligomers (≥260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced α-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (≥300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to β-sheet. However, ONE-induced α-synuclein oligomers exhibited a slightly higher degree of β-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to β-sheet structure that coincides with the formation of α-synuclein oligomers; albeit through different kinetic pathways depending on the degree of cross-linking.

4-Hydroxy-2-nonenal (4-HNE) and Its Lipation Product 2-Pentylpyrrole Lysine (2-PPL) in Peanuts.

After synthesis of a deuterated 4-hydroxy-2-nonenal (4-HNE) standard, the formation of 4-HNE during heating of peanut oil and whole peanuts, respectively, was measured by GC-MS. Whereas a significant increase in 4-HNE levels was observed for peanut oil, the amount of 4-HNE decreased when whole peanuts were roasted due to lipation reactions with amino acid side chains of the proteins. The ε-amino group of lysine was identified as the favored reaction partner of 4-HNE. After heating N(α)-acetyl-l-lysine and 4-HNE, a Schiff base, a novel pyridinium derivative, a 2-pentylpyrrol derivative and, following reduction and hydrolysis, a reduced, cyclized Michael adduct were identified. 2-Amino-6-(2-pentyl-1H-pyrrol-1-yl)hexanoic acid (2-PPL) was synthesized and quantitated in peanut proteins, which had been incubated with various amounts of 4-HNE by HPLC-ESI-MS/MS after enzymatic hydrolysis. At low 4-HNE concentrations the modification of lysine could be entirely explained by the formation of 2-PPL. Additionally, 2-PPL was quantified for the first time in peanut samples, and an increase depending on the roasting time was observed. 2-PPL represents a suitable marker to evaluate the extent of food protein lipation by 4-HNE.

Ozonated oils as functional dermatological matrices: Effects on the wound healing process using SKH1 mice

Wound tissue repair is a complex and dynamic process of restoring cellular structures and tissue layers. Improvement of this process is crucial for several pathologies characterized by chronic delayed wound closure such as diabetes, and the investigation of new approaches aimed to ameliorate the wound healing process is under continuous evolution. Recently, the usage of vegetable matrices in the form of ozonated oils has been proposed and several researchers have shown a positive effect in the wound, based on their bactericidal, antiviral, and antifungal properties. The present study was undertaken to compare the effect that different ozonated oils (olive, sesame and linseed) with the same level of ozonation have on wound healing rate in SKH1 mice. Several histological parameters and the level of key proteins such as VEGF and PCNA have been analyzed. Only treatment with ozonated sesame oil shows a faster wound closure in the first 7 days. This effect paralleled with the increased VEGF and PCNA levels, NFκB nuclear translocation and 4-HNE formation. The present study shows that not only the ozonation grade is of importance for the improvement of wound healing process but also the typical composition of the oil.

Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection by-products in drinking water. 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is the most extensively studied endogenous lipid hydroperoxide. Although it is well known that the decomposition of 13-HPODE can be catalyzed by transition metal ions, it is not clear whether halogenated quinones could enhance its decomposition independent of metal ions and, if so, what the unique characteristics and similarities are. Here we show that 2,5-dichloro-1,4-benzoquinone (DCBQ) could markedly enhance the decomposition of 13-HPODE and formation of reactive lipid alkyl radicals such as pentyl and 7-carboxyheptyl radicals, and the genotoxic 4-hydroxy-2-nonenal (HNE), through the complementary application of ESR spin trapping, HPLC–MS, and GC–MS methods. Interestingly, two chloroquinone–lipid alkoxyl conjugates were also detected and identified from the reaction between DCBQ and 13-HPODE. Analogous results were observed with other halogenated quinones. This represents the first report that halogenated quinoid carcinogens can enhance the decomposition of the endogenous lipid hydroperoxide 13-HPODE and formation of reactive lipid alkyl radicals and genotoxic HNE via a novel metal-independent nucleophilic substitution coupled with homolytic decomposition mechanism, which may partly explain their potential genotoxicity and carcinogenicity.

Inhibition of NF-κB Activation by 4-Hydroxynonenal Contributes to Liver Injury in a Mouse Model of Alcoholic Liver Disease

Long-term alcohol exposure sensitizes hepatocytes to tumor necrosis factor-α (TNF) cytotoxicity. 4-Hydroxynonenal (4-HNE) is one of the most abundant and reactive lipid peroxides. Increased hepatic 4-HNE contents present in both human alcoholics and alcohol-fed animals. In the present study, we investigated the effects of intracellular 4-HNE accumulation on TNF-induced hepatotoxicity and its potential implication in the pathogenesis of alcoholic liver disease. Male C57BL/6 mice were fed an ethanol-containing or a control diet for 5 weeks. Long-term alcohol exposure increased hepatic 4-HNE and TNF levels. Cell culture studies revealed that 4-HNE, at nontoxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-κB transactivity. Further investigation demonstrated that 4-HNE prevented TNF-induced inhibitor of κBα phosphorylation without affecting upstream IκB kinase activity. An immunoprecipitation assay revealed that increased 4-HNE content was associated with increased formation of 4-HNE-inhibitor of κBα adduction in both 4-HNE-treated hepatocytes and in the livers of alcohol-fed mice. Prevention of intracellular 4-HNE accumulation by bezafibrate, a peroxisome proliferator-activated receptor-α agonist, protected hepatocytes from TNF killing via NF-κB activation. Supplementation of N-acetylcysteine, a glutathione precursor, conferred a protective effect on alcohol-induced liver injury in mice, was associated with decreased hepatic 4-HNE formation, and improved hepatic NF-κB activity. In conclusion, increased 4-HNE accumulation represents a potent and clinically relevant sensitizer to TNF-induced hepatotoxicity. These data support the notion that removal of intracellular 4-HNE can serve as a potential therapeutic option for alcoholic liver disease.