HNE Concentration Research Articles

Myofibrillar protein lipoxidation in fish induced by linoleic acid and 4-hydroxy-2-nonenal: Insights from LC-MS/MS analysis

The oxidation of fish lipids and proteins is interconnected. The LOX (lipoxygenase)-catalyzed LA (linoleic acid) oxidation system on MPs (myofibrillar proteins) was established in vitro, to investigate the impact of lipoxidation on the physicochemical properties of fish MPs. By detecting HNE (4-hydroxy-2-nonenal) concentration during LA oxidation, the HNE treatment system was established to investigate the role of HNE in this process. In addition, the site specificity of modification on MPs was detected utilizing LC-MS/MS. Both treatments could induce sidechain modification, increase particle size, and cause loss of nutritional value through the reduction in amino acid content of MPs. The HNE group is more likely to alter the MPs' surface hydrophobicity compared to the LA group. By increasing the exposure of modification sites in MPs, the HNE group has more types and number of modifications compared to the LA group. LA group mainly induced the modification of single oxygen addition on MPs instead, which accounted for over 50 % of all modifications. The LA group induced a more pronounced reduction in the solubility of MPs as compared to the HNE group. In conclusion, HNE binding had a high susceptibility to Lys on MPs. Protein aggregation, peptide chain fragmentation, and decreased solubility occurred in the LA group mainly induced by peroxide generated during lipid oxidation or the unreacted LA instead of HNE. This study fills in the mechanism of lipoxidation on protein oxidation in fish and sheds light on the HNE modification sites of MPs, paving the way for the development of oxidation control technology.

4-Hydroxynonenal (HNE), a Toxic Aldehyde in Fried Chicken from Fast Food Restaurants and a Grocery Store

The lipid oxidation product, the toxic 4-hydroxynonenal, was measured for fried chicken in commercial samples from two fast food restaurants and one commercial establishment. The fried chicken samples were breasts, thighs, chicken nuggets and popcorn chicken. Fried chicken samples were separated to breast skin and meat, and thigh skin and meat. Chicken nuggets and popcorn chicken were not separated from the coating materials and the meat was analyzed together. Samples were analyzed for total fat, fatty acid distribution, reactivity with thiobarbituric acid (TBAR), measuring the secondary lipid peroxidation products such as aldehydes, ketones and related carbonyl compounds. Samples were analyzed for HNE, a toxic aldehyde using high performance liquid chromatography (HPLC). Fatty acid distribution showed in breast and thigh meats, a higher level of palmitic acid, compared to the breast and thigh skins. TBAR values were significantly higher in all breast meats compared to skins. TBAR values were significantly higher in thigh meat than in skin samples. HNE concentrations (μg HNE/g fat) were significantly very low in breast skins compared to breast meat. HNE concentration was generally higher in thigh meat than skin but not in every sample. In chicken nuggets, both the TBAR value and HNE concentration were much higher from one establishment than from the two others. Chicken nuggets TBAR and HNE concentrations resembled one of the chicken nugget samples. The average toxic HNE concentration for 100 g fried chicken breast (skins + meat) was 12.55 μg and for thighs (skin + meat) was 26.76 μg. The average total HNE concentration was 2.1 times higher in the fried chicken thighs than in the breasts. It is clear that HNE is produced during the heating process of oils and incorporated into the fried meat and skin samples during the frying process. If HNE, a toxic aldehyde, is consumed with the food, over long periods of time it could be related to a number of pathological conditions.

Effect of 4-hydroxy-2-nonenal treatment on the IgE binding capacity and structure of shrimp (Metapenaeus ensis) tropomyosin

Lipid peroxidation can react with free amines of proteins and induce modification of structural and functional properties. This study presents the IgE binding capacity and structural changes of shrimp tropomyosin (TM) under oxidative stress with 4-hydroxy-2-nonenal (HNE). IgE binding capacity was evaluated with the dot-blot assay and inhibition enzyme-linked immunosorbent assay. A decrease in IgE binding capacity of TM was found with 0.01mM HNE treatment, which was more significant when the HNE concentration was increased to 0.5mM. The conformational changes of TM, as characterized by fluorescence spectroscopy, circular dichroism spectroscopy and ultraviolet absorption spectroscopy, correlated well with IgE binding capacity changes. Further LC–ESI-MS/MS analyses showed that the side-chain groups of alanine, leucine, lysine and histidine had been modified by HNE. These results suggested that the HNE-induced conformational changes of TM significantly influenced its allergenicity and that these changes were caused by the modification of specific amino acids residues.

Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions

The influence of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the amoeba Acanthamoeba castellanii uncoupling protein (AcUCP) in isolated phosphorylating mitochondria was studied. Under phosphorylating conditions, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. The HNE-induced proton leak decreased the yield of oxidative phosphorylation in an HNE concentration-dependent manner. The present study describes how the contributions of ATP synthase and HNE-induced AcUCP in phosphorylating respiration vary when the rate of succinate oxidation is decreased by limiting succinate uptake or inhibiting complex III activity within the range of a constant membrane potential. In phosphorylating mitochondria, at a given HNE concentration (100 μM), the efficiency of AcUCP in mitochondrial uncoupling increased as the respiratory rate decreased because the AcUCP contribution remained constant while the ATP synthase contribution decreased with the respiratory rate. HNE-induced uncoupling can be inhibited by GTP only when ubiquinone is sufficiently oxidized, indicating that in phosphorylating A. castellanii mitochondria, the sensitivity of AcUCP activity to GTP depends on the redox state of the membranous ubiquinone.

Influence of Polydimethylsiloxane on the Formation of 4‐Hydroxynonenal in Soybean Oil at Frying Temperature

AbstractSoybean oils treated with 25 or 100 ppb polydimethylsiloxane (PDMS) and a control with no PDMS were heated at 180 °C for 48 h. The decomposition of linoleate and tocopherols was monitored, as well as the changes in concentration of 4‐hydroxynonenal (HNE). HNE was rapidly formed at the beginning of the heating period in the control and in the oil containing 25 ppb PDMS. HNE reached a maximum concentration of 0.033 μmol/g oil and then began to slowly decline. The maximum HNE concentration was attained at 18 h in the control oil compared with 28 h in the oil with 25‐ppb PDMS. In the oil with 100‐ppb PDMS the HNE increased very slowly during the first 32 h after which the HNE increased faster, reaching 0.033 μmol/g oil at 46 h and showed no decline during the 48 h heating period. Thus, PDMS had a beneficial effect in preventing the formation of the toxic HNE.

Formation of 4‐Hydroxy‐2‐(E)‐Nonenal in a Corn–Soy Oil Blend: a Controlled Heating Study Using a French Fried Potato Model

AbstractThe formation of 4‐hydroxy‐2‐(E)‐nonenal (HNE) in a corn–soy oil blend during frying was investigated. Frozen shoestring potatoes were fried once per hour at 180 °C for 8 h/day over a 4‐day period. As a control, oil was also heated under identical conditions, except that no product was fried. HNE was quantified by GC–MS using a stable isotope dilution assay with pentaflurobenzyl hydroxylamine hydrochloride (PFBHA) and trimethylsilyl 2,2,2‐trifluoro‐n‐(trimethylsilyl)acetimidate (BSTFA) derivatization. The HNE concentration in the potato fryers increased throughout the first day of frying. On subsequent days the daily maximum HNE concentration was reached after fresh oil was added and the fryer was brought to the frying temperature. The potato fryer oil reached a maximum concentration of 5.6 ppm during the second day of frying. Similarly, the HNE concentration of the oil in the control fryer increased throughout the first day of heating. On subsequent days the daily maximum HNE concentration varied throughout the experimental period. The control fryers reached a maximum concentration of 6.3 ppm at the end of the second day of heating. Throughout the experimental period there was a tendency for the oil in the control fryer to have a greater concentration of HNE than the oil in the potato fryer. Overall time of the experiment and heating with food versus heating without frying food and their interaction were significant in terms of HNE formation.

Temperature Dependence of HNE Formation in Vegetable Oils and Butter Oil

AbstractThe temperature dependence of the formation of toxic 4‐hydroxy‐2‐trans‐nonenal (HNE) was investigated in high and low linoleic acid (LA) containing oils such as corn, soybean and butter oils. These oils contain about 60, 54 and 3–4% of LA for corn, soybean and butter oils, respectively. The oils were heated for 0, 0.5, 1, 2, and 3 h at 190 °C and for 0, 5, 15 and 30 min at 218 °C. HNE concentrations in the oils were analyzed by high performance liquid chromatography (HPLC). The maximum HNE concentrations in heated (190 °C) corn, soybean and butter oils were 5.46, 3.73 and 1.85 μg HNE/g oil, respectively. The concentration of HNE at 218 °C increased continuously for all the three oils, although they were heated for much shorter periods compared to the lower temperature of heating (190 °C). HNE concentration at 30 min reached the maximum of 15.48, 10.72 and 6.71 μg HNE/g oil for corn, soybean and butter oils, respectively. HNE concentration at higher temperature (218 °C) was 4.9, 3.7, and 8.7 times higher than at the lower temperature (190 °C) and 30 min of heating for corn, soybean and butter oils, respectively. It was found that HNE formation was temperature dependant in the tested oils.

Accumulation of 4-hydroxynonenal protein adducts and Bax protein in rat hearts during aging

No. 29 Accumulation of 4-hydroxynonenal protein adducts and Bax protein in rat hearts during aging Peter Kaplan⁎, Zuzana Tatarkova, Monika Sivonova, Jan Lehotsky, Dusan Dobrota. Department of Biochemistry, Jessenius Faculty of Medicine, Martin, Slovakia ⁎ Corresponding author. Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Mala Hora 4, SK 036 01 Martin, Slovakia. Tel.: +421 043 4131565; fax: +421 043 4136770. E-mail address: kaplan@jfmed.uniba.sk The accumulation of oxidatively damagedmolecules has been implicated to be a major cause of cellular dysfunction that occurs during the aging process. 4-hydroxynonenal (HNE), amajor lipid peroxidation (LPO) product, exerts various cytotoxic and signalling effects. The aim of this work was to investigate formation of HNE and HNE-protein adducts in hearts of adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. HNE concentration was markedly elevated in old (134.6±4.5% vs. adult group, pb0.01) and senescent group (173.5±1.2%, pb 0.001). To assess potential modification of proteins by HNE we performed Western blot analysis with an antibody recognizing HNE-protein Michael adducts. The results showed enhanced formation of HNE adducts with few proteins of heart homogenates from old and senescent rats. The formation of protein adducts with LPO end-products was also confirmed by spectrofluorimetric methods. In 15and 26-month old rats the fluorescence intensity of lysine residues modified by LPO endproducts increased by 29.0±3.3% (pb0.001) and 79.2±8.3% (pb0.001), respectively, as compared to 6-month old animals. There is increasing evidence that apoptosis plays an important role inmyocardial aging. Because LPOproducts can be apoptotisinducing signals we also evaluated protein levels of pro-apoptotic regulator Bax. Cardiac Bax levels were significantly higher in 15and 26-month old rats when compared to 6-month old animals. These results suggest that HNE is involved in oxidative modification of proteins during aging; however, further studies are required to determine its role in apoptosis during myocardial aging. This work was supported by grants VEGA SR 1/0027/08 and APVV 51-027404.

4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

Introduction4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.MethodsMarkers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.ResultsOur data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.ConclusionOur study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.

The influence of 4‐hydroxy‐2‐nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells

The product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE) is known to cause cell death at high concentrations, while at lower concentrations it can influence cell proliferation and differentiation. In our experiments we used human osteosarcoma cells (HOS), to test the influence of HNE on cell proliferation, differentiation and induction of apoptosis. Apoptosis induction was estimated by TiterTACS TUNEL test. The cells were in parallel counted and the DAPI staining method was used to distinguish between apoptotic and necrotic cells as well as to define the proportion of cells in mitosis. To test the influence of HNE on HOS cell differentiation, cells were treated every second day with HNE. After 10 days, the cells were stained for alkaline phosphatase, a marker for osteoblast differentiation. Cell growth inhibition was caused by supraphysiological concentrations of 10 or 100 microM HNE, while apoptosis was induced with supraphysiological as well as by the physiological amount of the aldehyde (1 microM). Necrosis appeared when cells were treated with 10 or 100 microM, but not with 1 microM HNE. The proportion of cells in mitosis gradually declined with increased HNE concentration. Multiple exposures of HOS cells to 10 microM HNE prevented HOS cell differentiation. These results indicated that HNE inhibits proliferation and differentiation of HOS cells in the same concentration dependent manner as it causes apoptosis. We thus assume that HNE might be one of the important signaling molecules regulating the growth of the human osteosarcoma cells.

Biliary lipid peroxidation products in gallbladder cancer: increased peroxidation or biliary stasis?

Gallbladder cancer is a common malignancy among women in India. The aetiology of gallbladder carcinoma is still obscure and although numerous factors have been implicated, none has stood the test of time. This study was carried out to evaluate the role of lipid peroxidation product 4-hydroxynonenal (HNE) in gallbladder carcinogenesis. High-performance liquid chromatography (HPLC) analysis was carried out in 30 bile samples obtained during laparotomy from patients with gallbladder cancer (n = 10), cholelithiasis (n = 10) and age- and sex-matched controls (n = 10). A significantly high concentration of HNE and lower concentration of biliary pH and electrolyte was observed in patients with carcinoma of the gallbladder compared with controls and patients with cholelithiasis. A significant correlation was also observed between biliary Na+ and K+ (r = 0.72), biliary Ca2+ and Na+ (r = 0.69) among controls. However, there was no correlation between HNE, electrolytes and pH among cancer patients. An increased concentration of biliary HNE and decreased concentration of electrolytes suggest biliary stasis rather then increased peroxidation in cancer patients.

Inhibition of glucose-induced insulin secretion by 4-hydroxy-2-nonenal and other lipid peroxidation products.

Lipid peroxidation due to oxidative stress is accelerated under hyperglycemic conditions such as diabetes mellitus. The effect of 4-hydroxy-2-nonenal (HNE) and other lipid peroxidation products on the ability of isolated rat pancreatic islets to secrete insulin was examined in this study. HNE concentration- and time-dependently deteriorated glucose-induced insulin secretion: insulin secretion was decreased by 50% when measured after incubation of islets with 100 microM HNE for 1 h. Other lipid peroxidation products, e.g. 2-hexenal and 2-butenal, also inhibited glucose-induced insulin secretion. HNE at 100 microM lowered alpha-ketoisocaproate-induced insulin secretion, whereas leucine-induced insulin secretion was stimulated. Insulin secretion induced by 10 mM glyceraldehyde was slightly decreased by HNE. On the other hand, HNE severely decreased insulin secretion induced by 10 mM glyceraldehyde and 2.8 mM glucose. Glucose utilization and glucose oxidation were significantly lowered in islets treated with HNE. The amounts of fructose 1,6-bisphosphate and dihydroxyacetone phosphate in islets were decreased by treatment with HNE, whereas the amount of fructose 6-phosphate was increased. Our study indicates that HNE and other lipid peroxidation products impair insulin secretion induced by glucose probably through affecting both the glycolytic pathway and the citric acid cycle.

Aldehydic products of lipid peroxidation inactivate cytochrome P-450

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.

Raised concentrations of aldehyde lipid peroxidation products in premature infants with chronic lung disease

AIMTo indicate the extent of lipid peroxidation induced by oxidative stress, by measuring aldehyde end products in biological samples.METHODSA highly specific gas chromatography and mass spectrometry (GC/MS) method was used...

(E)-4-Hydroxy-2-Nonenal May be Involved in the Pathogenesis of Parkinson’s Disease

( E)-4-Hydroxy-2-nonenal (HNE) is a toxic end-product of the free radical-stimulated peroxidation of phospholoipid-bound arachidonic acid in cell membranes. There is a growing body of evidence to suggest that free radicals may play an important role in the pathology of Parkinson’s disease. HNE is highly electrophilic and is conjugated to reduced glutathione (GSH) by glutathione S-transferase. The depletion of GSH in the substantia nigra of Parkinson’s patients and in the brainstem of mice treated with the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prompted this study on the concentrations of HNE in the cerebrospinal fluid (CSF) and plasma of Parkinson’s patients and the brainstem of mice treated with MPTP. HNE was identified and quantitated by a highly specific and sensitive method based on the gas chromatography-negative-ion chemical ionisation mass spectrometry of the O-pentafluorobenzyl oxime derivative using 9 D 3-4-hydroxy-2-nonenal as an internal standard. The mean concentration of HNE in the CSF of patients with Parkinson’s disease was 1.47 ± 0.76 μM (mean ± SD, n = 10), while the concentration in the CSF of a group of control patients was 0.38 ± 0.14 μM ( n = 10; p < .01). The mean concentration of HNE in the plasma of Parkinson’s patients was 0.68 ± 0.15 μM ( n = 20) and the concentration in the control group was 0.47 ± 0 12 μM ( n = 20; p < .05). The mean peak concentration of HNE in the brainstem of mice after a single s.c. dose of MPTP (40 mg/kg) was 3.62 ± 0.36 nM/g wet wt. ( n = 17) at 12 h while the control value was 0.45 ± 0.05 nM/g wet wt. ( n = 20; p < .05). The GSH concentration in the brainstem of MPTP-treated mice at 24 h. was 0.65 ± 0.03 μM/g wet wt. ( n = 14) and the control value was 1.25 ± 0.03 μM/g wet wt. ( n = 20; p < .01). The corresponding concentration of GSH-HNE-conjugate at 24 h was 0.32 ± 0.09 μM/g wet wt. ( n = 12) compared with a control value of 0.05 ± 0.02 ( n = 16; p < .01). After treatment with α-tocopherol (2.35 g/kg s.c. daily × 3) the mean concentration of HNE 12 hr. after MPTP injection was 0.89 ± 0.06 nM/g wet wt. ( n = 18). The HNE concentration in a group not treated with α-tocopherol prior to MPTP injection was 3.49 ± 0.09 nM/g wet wt. ( n = 14; p < .05). The concentration of GSH in the mice pretreated with α-tocopherol before MPTP injection was 1.14 ± 0.02 μM/g wet wt. ( n = 17) at 24 h compared to 0.61 ± 0.02 μM/g wet wt. ( n = 14) in the untreated mice ( p < .05). The direct injection of HNE (1, 10, 100, 1,000 μM) into the substantia nigra caused a dose dependent depletion of GSH in the brainstem of mice. The mean concentration of GSH 24 hr after the injection of 100 μM of HNE was 0.43 ± 0.22 μM/g wet wt. ( n = 4) compared with a control value of 1.48 ± 0.02 μM/g wet wt. ( n = 8; p < .05). The corresponding concentration of GSH-HNE-conjugate was 0.32 ± 0.12 μM/g wet wt. ( n = 4) while the control value was 0.04 ± 0.02 μM/g wet wt. ( n = 8). These data suggest that HNE may be a causeative neurotoxin in Parkinson’s disease and that HNE may also be involved in MPTP toxicity.

4-Hydroxynonenal Modifies the Effects of Serum Growth Factors on the Expression of the c- fos Proto-Oncogene and the Proliferation of HeLa Carcinoma Cells

In this study, the effect of 4-hydroxynonenal (HNE), a peroxidation product of ω-6-poly-unsaturated fatty acids, on the expression of the c- fos proto-oncogene and growth factor-induced proliferation of HeLa carcinoma cells in vitro was investigated. The Fos protein forms the heterodimer AP-1 with the Jun protein and regulates the cell cycle by inducing cyclin D1. Agents that are able to induce c- fos include serum, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), all of which were used in this study. The proliferation rate was determined by cell counting (viable and dead cells according to trypan blue exclusion) and the BrdU assay. The c- fos mRNA level was monitored by the reverse transcriptase/polymerase chain reaction. In the absence of HNE, serum-deprived cells responded to serum stimulation with a more than 10-fold increase of the c- fos mRNA level as well as with an increased rate of DNA synthesis and cell multiplication. Both EGF and PDGF (applied in combination with insulin) were able to substitute for FCS and induced rapid growth of the tumor cells preincubated in serum-deprived medium. In the absence of growth factors a negative correlation between the HNE concentration (range: 1–250 μM) and the c- fos mRNA level was observed. We suppose that HNE interferes in this case with the basal activity of the c- fos promoter. EGF, when applied after the HNE treatment, induced rapid growth of the tumor cells preincubated in serum-free medium, if HNE was used in a physiological concentration (1 μM). No difference was observed compared to the HNE-free control. c- fos mRNA level was nearly unchanged. In contrast, a cytotoxic concentration of the aldehyde (100 μM) caused a complete inhibition of proliferation, although a twofold increase of the c- fos mRNA level immediately after the aldehyde treatment was observed. A similar effect of HNE in cytotoxic concentration on c- fos expression was observed when cells were grown in presence of PDGF instead of EGF. Hence, in both cases HNE possibly interferes with the signal transduction pathway, which is initiated by external growth factors. The increased c- fos expression might be part of an abortive attempt to overcome the stressful condition raised by a cytotoxic concentration of HNE.

4-Hydroxy-2-Nonenal Hardly Affects Glycolysis

4-Hydroxy-2-nonenal (HNE), one of the major products of lipid peroxidation, inactivated the rate-limiting enzymes (from animal sources) of the glycolytic pathway and the pentose phosphate pathway when incubated at 37°C for 1 h in the absence of glutathione (GSH). The HNE concentration for half-maximal inactivation of 6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase was 3–10 μM; and that value for pyruvate kinase, glucose-6-phosphate dehydrogenase, and hexokinases I and II was 0.15-0.6 mM. In the presence of 5 mM GSH, however, only PFK, irrespective of the source (muscle, liver, or erythrocyte), was inactivated by 40–-50% when incubated with 0.1 mM HNE for 1 h. Even PFK was not inactivated in the presence of both GSH and its substrate, ATP (2 mM). Glycolysis in human erythrocytes was not affected by treatment of cells with 0.1 mM HNE at 37°C for 30 min. The results suggest that HNE, at concentrations observable under physiological and pathological conditions, hardly affects glycolysis in cells.

4-Hydroxynonenal formation during ischemia and reperfusion of rat small intestine

The metabolism and the washout of 4-hydroxynonenal (HNE), a major lipid peroxidation product, was determined in jejunal intestine of rats under normoxic, ischemic and postischemic conditions. The HNE utilization was by several orders higher than the HNE washout. Under ischemic conditions a drastic decrease of HNE utilization was found, compared with normoxic and postischemic utilization rates. The real flux rate of intracellular HNE formation during ischemia and reperfusion of small intestine was for the first time estimated on the basis of tissue HNE concentration, HNE utilization rate at defined HNE tissue concentration and HNE washout by perfusate circulation. Such estimation led to intestinal HNE formation rates of about 20 nmoles/g/min at normoxia, 20 to 40 nmoles/g/min at ischemia, 100 nmoles/g/min at 10 min of reperfusion and 20 nmoles/g/min at 60 min of reperfusion. Therefore, one can conclude that the tissue concentration of HNE only partly represents the real HNE formation rate and that the formation rate is of particular pathophysiological importance.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

Effects of the lipid peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function.

(E)-4-hydroxy-2-nonenal (HNE) is a lipid peroxide end-product which exerts powerful biological effects in a variety of cell and tissue systems. The effects of exogenous HNE on ram spermatozoa were examined in vitro. HNE inhibited the motility of diluted ram spermatozoa in a dose-dependent (100-400 mumol l-1) manner (P < 0.05). The extent of motility loss varied with sperm concentration as well as with HNE concentration, and was manifested as a progressive decrease in mean sperm velocity. The suppressive effect of 250-500 mmol HNE l-1 on the motility of reactivated ram sperm models (P < 0.05) was prevented by the addition of 1 mmol reduced glutathione l-1 to the reactivation medium, suggesting that HNE inhibits ram sperm motility via oxidation of sulfhydryl groups in the axoneme. Oxygen uptake by ram spermatozoa was inhibited (P < 0.05) by the addition of 100 or 200 mumol HNE l-1. Glucose utilization was maintained in the presence of 200 mumol HNE l-1, suggesting that fructolysis was unaffected by HNE. As was the case with motility, the inhibition of oxidative metabolism by HNE was not reversed by washing the spermatozoa. The activity of ram sperm acrosomal enzymes released by cold shock, as measured by hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), was reduced in the presence of 100 mumol HNE l-1 (P < 0.05). No evidence was found of disruption of the acrosomal outer membrane or the sperm plasma membrane as a result of exposure to HNE.(ABSTRACT TRUNCATED AT 250 WORDS)