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.     

Effects of polyunsaturated fatty acids on calcium response and degranulation from RBL-2H3 cells

To investigate the biological activity of various polyunsaturated fatty acids (PUFAs) on the allergic reaction, we examined the effects of six PUFAs and two saturated fatty acids on calcium response and degranulation from rat basophilic leukemia (RBL-2H3) cells. Between 20 and 40 microM of six PUFAs (omega-6 series: arachidonic acid [AA, C20:4], gamma-linolenic acid [gamma-LN, C18:3] and linoleic acid [LA, C18:2]; omega-3 series: alpha-linolenic acids [alpha-LN, C18:3] and eicosapentaenoic acid [EPA, C20:5]; and omega-9 series: oleic acid [OLE, C18:1]), or two saturated fatty acids (stearic acid [STA, C18:0] and arachidic acid [AD, C20:0]) were used to examine the effects on calcium response and degranulation from RBL-2H3 cells. Calcium response was monitored using the fluorescent calcium indicator fura-2, while degranulation was monitored by measuring histamine release from the cells. Three omega-6 PUFAs (AA, alpha-LN and LA) dose-dependently increased the cytosolic free-calcium concentration and histamine release from RBL-2H3 cells. This phenomenon was specific to the omega-6 PUFAs, the omega-3 PUFAs (alpha-LA and EPA), omega-9 PUFA (OLE) and the saturated fatty acids (STA and AD) had no effect. The increase in the cytosolic free-calcium concentration caused by the omega-6 PUFAs depended on the existence of external calcium, cell viability and the cellular IP(3) levels remained unchanged throughout the experiment. These results suggest that omega-6 PUFAs work as direct mediators of calcium signaling pathways in RBL-2H3 cells.     

Fish oil fatty acids as cardiovascular drugs

Starting in the 1970s the hypothesis that the low mortality from coronary heart disease among the Greenland Eskimos was due to their high consumption of n-3 fish oil fatty acids, initiated many studies to find if the n-3 polyunsaturated fatty acids in fish oils (PUFAs) could prevent cardiac atherosclerosis. To date this possibility has not achieved clinical recognition. The recent literature shows an increase of intervention studies to learn if the fish oil fatty acids can reduce mortality from sudden cardiac death, and the mechanism(s) of such a protective effect. Indeed the most definite beneficial cardiac action of these n-3 PUFAs seems now to be their ability in the short term to prevent sudden cardiac death. It is apparent that over long periods of time the n-3 fish oil fatty acids also prevent atherosclerosis. Definition of the fatty acids to which I will be referring in the text: n-6 (omega-6) polyunsaturated fatty acids; linoleic acid (18:2n-6, LA); arachidonic acid (C20:4n-6, AA). n-3 (omega-3) fatty acids; alpha-linolenic acid (18:3n-3, ALA); eicosapentaenoic acid (20:5n-3, EPA); docosahexaenoic acid (C22:6n-3, DHA). The bold, underlined abbreviation will appear in the text to identify the fatty acid being discussed.     

Role of omega-6 and omega-3 fatty acids in fetal programming

Maternal nutrition plays a critical role in fetal development and can influence adult onset of disease. Linoleic acid (LA) and alpha-linolenic acid (ALA) are major omega-6 (n-6) and n-3 polyunsaturated fatty acids (PUFA), respectively, that are essential in our diet. LA and ALA are critical for the development of the fetal neurological and immune systems. However, in recent years, the consumption of n-6 PUFA has increased gradually worldwide, and elevated n-6 PUFA consumption may be harmful to human health. Consumption of diets with high levels of n-6 PUFA before or during pregnancy may have detrimental effects on fetal development and may influence overall health of offspring in adulthood. This review discusses the role of n-6 PUFA in fetal programming, the importance of a balance between n-6 and n-3 PUFAs in the maternal diet, and the need of further animal models and human studies that critically evaluate both n-6 and n-3 PUFA contents in diets.     

Regioselective formation of acrolein-derived cyclic 1,N(2)-propanodeoxyguanosine adducts mediated by amino acids, proteins, and cell lysates

Acrolein (Acr) is a major component in cigarette smoke and a ubiquitous environmental pollutant. It is also formed as a product of lipid peroxidation. Following ring closure via the Michael addition, Acr modifies deoxyguanosine (dG) in DNA by forming cyclic 1,N(2)-propanodeoxyguanosine adducts (OHPdG). The reactions of Acr with dG yield, depending on the direction of ring closure, two regioisomers, α- and γ-OHPdG, in approximately equal amounts. However, previous (32)P-postlabeling studies showed that the γ isomers were detected predominantly in the DNA of rodent and human tissues. Because of the potential differential biological activity of the isomeric OHPdG adducts, it is important to confirm and study the chemical basis of the regioselective formation of γ isomers in vivo. In this study, it is confirmed that γ-OHPdG adducts are indeed the major isomers formed in vivo as evidenced by a LC-MS/MS method specifically developed for Acr-derived dG adducts. Furthermore, we have shown that the formation of γ-isomers is increased in the presence of amino-containing compounds, including amino acids, proteins, and cell lysates. A product of Acr and arginine that appears to mediate the regioselective formation of γ isomers was identified, but its structure was not fully characterized due to its instability. This study demonstrates that intracellular amino-containing compounds may influence the regiochemistry of the formation of OHPdG adducts and reveals a mechanism for the preferential formation of γ-OHPdG by Acr in vivo.     

Dietary n − 6 and n − 3 polyunsaturated fatty acids: From biochemistry to clinical implications in cardiovascular prevention

Linoleic acid (LA) and alpha linolenic acid (ALA) belong to the n − 6 (omega-6) and n − 3 (omega-3) series of polyunsaturated fatty acids (PUFA), respectively. They are defined “essential” fatty acids since they are not synthesized in the human body and are mostly obtained from the diet. Food sources of ALA and LA are most vegetable oils, cereals and walnuts. This review critically revises the most significant epidemiological and interventional studies on the cardioprotective activity of PUFAs, linking their biological functions to biochemistry and metabolism. In fact, a complex series of desaturation and elongation reactions acting in concert transform LA and ALA to their higher unsaturated derivatives: arachidonic acid (AA) from LA, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) from ALA. EPA and DHA are abundantly present in fish and fish oil. AA and EPA are precursors of different classes of pro-inflammatory or anti-inflammatory eicosanoids, respectively, whose biological activities have been evoked to justify risks and benefits of PUFA consumption. The controversial origin and clinical role of the n − 6/n − 3 ratio as a potential risk factor in cardiovascular diseases is also examined. This review highlights the important cardioprotective effect of n − 3 in the secondary prevention of sudden cardiac death due to arrhythmias, but suggests caution to recommend dietary supplementation of PUFAs to the general population, without considering, at the individual level, the intake of total energy and fats.     

Gamma linolenic acid: an antiinflammatory omega-6 fatty acid

Inflammation plays an important role in health and disease. Most of the chronic diseases of modern society, including cancer, diabetes, heart disease, arthritis, Alzheimer's disease, etc. have inflammatory component. At the same time, the link between diet and disease is also being recognized. Amongst dietary constituents, fat has gained most recognition in affecting health. Saturated and trans fatty acids have been implicated in obesity, heart disease, diabetes and cancer while polyunsaturated fatty acids (PUFAs) generally have a positive effect on health. The PUFAs of omega-3 and omega-6 series play a significant role in health and disease by generating potent modulatory molecules for inflammatory responses, including eicosanoids (prostaglandins, and leukotrienes), and cytokines (interleukins) and affecting the gene expression of various bioactive molecules. Gamma linolenic acid (GLA, all cis 6, 9, 12-Octadecatrienoic acid, C18:3, n-6), is produced in the body from linoleic acid (all cis 6,9-octadecadienoic acid), an essential fatty acid of omega-6 series by the enzyme delta-6-desaturase. Preformed GLA is present in trace amounts in green leafy vegetables and in nuts. The most significant source of GLA for infants is breast milk. GLA is further metabolized to dihomogamma linlenic acid (DGLA) which undergoes oxidative metabolism by cyclooxygenases and lipoxygenases to produce anti-inflammatory eicosanoids (prostaglandins of series 1 and leukotrienes of series 3). GLA and its metabolites also affect expression of various genes where by regulating the levels of gene products including matrix proteins. These gene products play a significant role in immune functions and also in cell death (apoptosis). The present review will emphasize the role of GLA in modulating inflammatory response, and hence its potential applications as an anti-inflammatory nutrient or adjuvant.     

Effect of omega-3 fatty acid supplementation on the arachidonic acid:eicosapentaenoic acid ratio

STUDY OBJECTIVES: To determine the baseline arachidonic acid:eicosapentaenoic acid (AA:EPA) ratio in patients with coronary artery disease and healthy subjects, and whether supplementation of omega-3 fatty acids, administered as fish oil capsules, affects this ratio. DESIGN: Prospective, open-label trial. SETTING: University-affiliated cardiology clinic. SUBJECTS: Thirty patients with stable coronary artery disease (CAD) and 30 healthy subjects. INTERVENTION: All participants received omega-3 fatty acids 1.5 g/day for 4 weeks, followed by 3 g/day for an additional 4 weeks. MEASUREMENTS AND MAIN RESULTS: For each participant, a lipid profile was determined at baseline and after 4 weeks of treatment with each dose. Other laboratory results analyzed were serum AA:EPA ratios, high-sensitivity C-reactive protein (hs-CRP) levels, and blood glucose levels. Mean +/- SD baseline AA:EPA ratios were 39.6 +/- 19.0 in healthy subjects and 23.7 +/- 12.5 in patients with CAD. These ratios decreased significantly in both groups after treatment with 1.5 g/day of omega-3 fatty acids: 9.0 +/- 4.2 in healthy subjects and 10.3 +/- 8.8 in patients with CAD. After treatment with 3 g/day, the ratios were further reduced: 5.1 +/- 3.2 in healthy subjects and 4.9 +/- 2.6 in patients with CAD. Supplementation with omega-3 fatty acids did not significantly affect hs-CRP, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, or blood glucose levels. Triglyceride levels were not reduced in patients with CAD but were significantly decreased in healthy subjects (by 20% decrease with omega-3 fatty acids 1.5 g/day and by 32% decrease with 3 g/day). CONCLUSION: Treatment with omega-3 fatty acids significantly reduced AA:EPA ratios in both healthy subjects and in patients with stable CAD. The treatment had no effect on hs-CRP levels in either group, and it reduced triglyceride levels in healthy subjects but not in patients with CAD.     

Omega 3-fatty acids: health benefits and cellular mechanisms of action

Epidemiological evidence has established that ingestion of long-chain polyunsaturated omega-3 fatty acids (omega-3 PUFAs), abundant in fish oils, have profound effects on many human disorders and diseases, including cardiovascular disease and cancer. Here we briefly review the dietary recommendations and the food sources that are naturally enriched by these fatty acids. There are also a number of products including eggs, bread, and cereals available to supplement omega-3 fatty acid dietary intake. Some of these supplements are proposed to aid different pathological conditions. While the beneficial effects of omega-3 fatty acids can no longer be doubted, their molecular mechanism of action remains elusive. Without question, the action of omega-3 fatty acids is complex and involves a number of integrated signaling pathways. This review focuses on one of the possible cellular mechanisms by which the omega-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), may function. Studies with cancer cells suggest that DHA induces cell cycle arrest and apoptosis by activating protein phosphatases, leading to dephosphorylation of retinoblastoma protein (pRB). Protein phosphatases are also involved with the protein Bcl2, which regulates the release of cytochrome c from mitochondria, and eventually, activation of the apoptotic enzyme caspase 3.     

Effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone.

The comparative effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone were investigated. Docosahexaenoic acid (1-255 microM) and eicosapentaenoic acid (31-255 microM) inhibited phenylephrine-induced contractions, (8-63%) and (20-65%), respectively, which were not altered by indomethacin, NDGA, or by removal of the endothelium. Linoleic acid (18:2n6) and arachidonic acid (20:4n6) also induced significant relaxation. Therefore, fatty acid-induced relaxation of the rat aorta is specific to polyunsaturated fatty acids, 20:5n3, 22:6n3, 18:2n6 and 20:4n6.     

Proper balance of omega-3 and omega-6 fatty acid supplements with topical cyclosporine attenuated contact lens-related dry eye syndrome

Essential fatty acids had been applied in the treatment of dry eye syndrome (DES), but the effects of different combinations of fatty acids have not been investigated. 360 long-term contact lens wearers were included in this double-blinded study. Omega-3 and omega-6 fatty acids were combined in different ratios and supplied to the participants that were randomly divided into six groups, and the effects of different essential fatty acids mixture on DES with or without topical cyclosporine were investigated. More than half of long-term contact lens wearers suffered from DES, which were found to be attenuated by oral supplement of properly balanced O3FA and O6FA fatty acid. The topical cyclosporine treatment considerably inhibited the production of cytokines compared to the cyclosporine negative groups, which further relieved DES. Proper balance of omega-3 and omega-6 fatty acid combination significantly alleviated contact lens-related DES.     

The mechanism of action of omega-3 fatty acids in secondary prevention post-myocardial infarction

BACKGROUND: Omega-3 fatty acids from fish and fish oils can protect against coronary heart disease (CHD), which is still the most common cause of death in the Western economies. Evidence from epidemiological and case cohort studies indicate that consumpton of fatty fish and omega-3 fatty acids reduces the risk of cardiovascular mortality. OBJECTIVE:This article briefly reviews the evidence regarding omega-3 fatty acids and CHD and outlines the mechanisms through which omega-3 fatty acids might confer cardiac benefits over and above the standard secondary prevention strategies. CONCLUSION: The conclusion reached is that omega-3 fatty acids play a significant role in secondary prevention post-myocardial infarction. The mechanisms through which two of these omega-3 fatty acids, eicosapentaenoic acid and docosahexanoic acid, exert their action appear to be distinct and adjuvant to the available standard secondary prevention therapies. The role to be played by the administration of a newly licensed 90% concentrate EPA + DHA formulation (1 g/day capsule: Omacor) is explored.     

Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells

Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H2O2, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H2O2-, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H2O2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST), (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD.     

The mechanism of action of omega-3 fatty acids in secondary prevention post-myocardial infarction

ABSTRACT

Background: Omega-3 fatty acids from fish and fish oils can protect against coronary heart disease (CHD), which is still the most common cause of death in the Western economies. Evidence from epidemiological and case cohort studies indicate that consumption of fatty fish and omega-3 fatty acids reduces the risk of cardiovascular mortality.

Objective: This article briefly reviews the evidence regarding omega-3 fatty acids and CHD and outlines the mechanisms through which omega-3 fatty acids might confer cardiac benefits over and above the standard secondary prevention strategies.

Conclusion: The conclusion reached is that omega-3 fatty acids play a significant role in secondary prevention post-myocardial infarction. The mechanisms through which two of these omega-3 fatty acids, eicosapentaenoic acid and docosahexanoic acid, exert their action appear to be distinct and adjuvant to the available standard secondary prevention therapies. The role to be played by the administration of a newly licensed 90% concentrate EPA + DHA formulation (1?g/day capsule: Omacor) is explored.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophils in vitro. Evidence of formation of antiinflammatory compounds.

Enzymatic transformation of the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of other n-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if these n-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 microM) and different concentrations (0-100 microM) of the n-6 fatty acids linoleic acid (LA) and dihomo-gamma-linolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50 = 45 microM) and DGLA (IC50 = 40 microM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50 = 7.5 microM and IC50 = 0.2 microM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50 = 0.75 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of saturated and polyunsaturated fatty acids on the cytochrome P450 3A activity in rat liver microsomes

This study was conducted to investigate the inhibitory effects of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on cytochrome P450 3A (CYP3A). The inhibition of 6beta-hydroxy testosterone formation from testosterone in rat liver microsomes was used as an index of CYP3A activity. In the present study, among the three types of fatty acids, the rank order of inhibitory effects of fatty acids was SFAs相似文献   

Protein adducts generated from products of lipid oxidation: focus on HNE and one

Modification of proteins in conditions of oxidative stress can contribute to protein dysfunction or tissue damage and disease progression. Bifunctional, most often alpha,beta-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily bind to protein nucleophiles. Modification by bifunctional aldehydes can also lead to intramolecular or intermolecular protein crosslinking. Model studies are revealing the structure of adducts that can then be more readily identified in mass spectrometric studies on proteins exposed to the various pure aldehydes or to peroxidized PUFAs. Although simple Michael and Schiff base adducts are often formed initially, only some of these adducts, such as the HNE- and ONE-derived Michael adducts on Cys and His residues, are found to survive the conditions of proteolysis and HPLC-MS analysis. Reversibly formed adducts, such as the HNE-Lys Michael adduct, can be found on proteolytic peptides only if a NaBH4-reduction step is used prior to proteolysis. Initial adducts can evolve by tautomerization, oxidation, cyclization, dehydration, and sometimes condensation with a second aldehyde molecule (the same or different), to give stable advanced lipoxidation end products (ALEs) that can be found by mass spectrometry. These include the HNE-Lys-derived 2-pentylpyrrole, the ONE-Lys-derived 4-ketoamide, the ONE-derived His-Lys pyrrole crosslink, and a Lys-derived 3-formyl-4-pentylpyrrole that results from combined action of ONE and acrolein. Michael adducts of alpha,beta-unsaturated aldehydes such as HNE and ONE can be derivatized by 2,4-dinitrophenylhydrazine (DNPH) and can thus constitute significant DNPH-detectable protein-bound carbonyl activity that serves as a key indicator of oxidative stress in tissues. It appears that lipid oxidation is a more important contributor to such activity than metal-catalyzed oxidation of protein side-chains.     

Protein Adducts Generated from Products of Lipid Oxidation: Focus on HNE and ONE

Modification of proteins in conditions of oxidative stress can contribute to protein dysfunction or tissue damage and disease progression. Bifunctional, most often α,β-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily bind to protein nucleophiles. Modification by bifunctional aldehydes can also lead to intramolecular or intermolecular protein crosslinking. Model studies are revealing the structure of adducts that can then be more readily identified in mass spectrometric studies on proteins exposed to the various pure aldehydes or to peroxidized PUFAs. Although simple Michael and Schiff base adducts are often formed initially, only some of these adducts, such as the HNE- and ONE-derived Michael adducts on Cys and His residues, are found to survive the conditions of proteolysis and HPLC-MS analysis. Reversibly formed adducts, such as the HNE-Lys Michael adduct, can be found on proteolytic peptides only if a NaBH₄-reduction step is used prior to proteolysis. Initial adducts can evolve by tautomerization, oxidation, cyclization, dehydration, and sometimes condensation with a second aldehyde molecule (the same or different), to give stable advanced lipoxidation end products (ALEs) that can be found by mass spectrometry. These include the HNE-Lys-derived 2-pentylpyrrole, the ONE-Lys-derived 4-ketoamide, the ONE-derived His-Lys pyrrole crosslink, and a Lys-derived 3-formyl-4-pentylpyrrole that results from combined action of ONE and acrolein. Michael adducts of α,β-unsaturated aldehydes such as HNE and ONE can be derivatized by 2,4-dinitrophenylhydrazine (DNPH) and can thus constitute significant DNPH-detectable protein-bound carbonyl activity that serves as a key indicator of oxidative stress in tissues. It appears that lipid oxidation is a more important contributor to such activity than metal-catalyzed oxidation of protein side-chains.     

Effects of di-(2-ethylhexyl)-phthalate (DEHP) and its metabolites on fatty acid homeostasis regulating proteins in rat placental HRP-1 trophoblast cells.

Di-(2-ethylhexyl)-phthalate (DEHP) is a widely used plasticizer and ubiquitous environmental contaminant. The potential health hazards, including teratogenicity, from exposure to DEHP may be related to the role of DEHP or its metabolites in the trans-activation of peroxisome proliferator-activated receptors (PPARs). Fetal essential fatty acid (EFA) homeostasis is controlled by directional transfer across the placenta through a highly regulated process, including PPAR activation. Using HRP-1 rat trophoblastic cells, the effects of DEHP and two of its metabolites, mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (EHA), on the mRNA and protein expression of the three known PPAR isoforms (alpha, beta, and gamma), fatty acid transport protein 1 (FATP1), plasma membrane fatty acid binding protein (FABPpm), and the heart cytoplasmic fatty acid binding protein (HFABP) were investigated. This study also investigated the functional effects of exposure on the uptake and transport of six long chain fatty acids (LCFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), linoleic acid (LA), alpha-linolenic acid (ALA), oleic acid (OA), and stearic acid (SA). In the presence of DEHP, MEHP, and EHA, the expression of PPARalpha, PPARgamma, FATP1, and HFABP were up-regulated in a dose- and time- dependent manner, while PPARbeta and FABPpm demonstrated variable expression. The uptake rates of EFAs (AA, DHA, LA, ALA) increased significantly upon exposure, and the transport of AA (omega-6) and DHA (omega-3) were directionally induced. These results suggest that DEHP, MEHP, and EHA can influence EFA transfer across HRP-1 cells, implying that these compounds may alter placental EFA homeostasis and potentially result in abnormal fetal development.     

Drug-induced protein free radical formation is attenuated by unsaturated fatty acids by scavenging drug-derived phenyl radical metabolites

Aromatic amine drugs like aminoglutethimide (AG) and related congeners have been shown to produce phenyl radicals through metabolism by myeloperoxidase (MPO)/H(2)O(2), which has been proposed to play a role in drug-induced agranulocytosis. AG has also been shown to induce MPO protein radical formation, but the ultimate fate of these metabolically generated phenyl radicals is still unknown. We tested the reactivity of linoleic acid (LA) and GSH with aniline-based compounds in the presence of horseradish peroxidase (HRP)/H(2)O(2) by measuring oxygen consumption. We found a qualitative correlation between drugs or xenobiotics that formed phenyl radical metabolites with the cooxidation of LA. Most compounds that reacted with LA did not react with GSH. Furthermore, an AG-derived phenyl radical was detected by EPR spin-trapping with MNP (2-methyl-2-nitrosopropane), in a reaction containing AG and HRP/H(2)O(2); these spectra were attenuated in the presence of LA and docosahexaenoic acid (DHA) indicating that phenyl radical scavenging occurred. Since it has been proposed that the phenyl radical metabolite leads to protein radical formation on MPO, we investigated the effect of LA and DHA in immuno-spin trapping experiments with MPO-containing HL-60 cell lysate. Using anti-DMPO, a protein radical was detected on a putative MPO fragment from the reaction of DMPO, AG, and glucose/glucose oxidase. When LA or DHA was included in this reaction, protein radical formation was significantly inhibited. Our results show that certain polyunsaturated fatty acids (PUFAs) act as scavengers of aromatic amine drug-derived phenyl radicals which in turn prevent protein radical formation. However, the interaction of phenyl radical drug metabolites with PUFAs will be dictated by their relative concentrations compared to those of other targets. Most importantly, it is possible to differentiate peroxidase substrates that generate phenyl radical metabolites from N-centered radicals on the basis of their reactivity toward GSH vs PUFAs, and PUFAs are targets for metabolically generated phenyl radicals.