Headspace volatile markers for sensitivity of cocoa (Theobroma cacao L.) somatic embryos to cryopreservation

The mechanisms that reduce the viability of plant somatic embryos following cryopreservation are not known. The objective of the present study was to evaluate the sensitivity of cocoa (Theobroma cacao L.) somatic embryos at different stages of an encapsulation-dehydration protocol using stress-related volatile hydrocarbons as markers of injury and recovery. The plant stress hormone ethylene and volatile hydrocarbons derived from hydroxyl radicals (methane) and lipid peroxidation (ethane) were determined using gas chromatography headspace analysis. Ethylene and methane were the only volatiles detected, with both being produced after each step of the cryogenic protocol. Ethylene production was significantly reduced following exposure to liquid nitrogen, but then increased in parallel with embryo recovery. In contrast, the production of methane was cyclic during recovery, with the first cycle occurring earlier for embryos recovered from liquid nitrogen and desiccation than those recovered from earlier steps in the protocol. These results suggest that loss of somatic embryo viability during cryopreservation may be related to the oxidative status of the tissue, and its capacity to produce ethylene. This study has demonstrated that headspace volatile analysis provides a robust non-destructive analytical approach for assessing the survival and recovery of plant somatic embryos following cryopreservation.

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated With Cu2+ ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu2+ to Cu+ and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu+ oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides. (C) 2007 Elsevier Inc. All rights reserved.

Common variants of apolipoprotein A-IV differ in their ability to inhibit low density lipoprotein oxidation

Apolipoprotein A-IV (apoA-IV) inhibits lipid peroxidation, thus demonstrating potential anti-atherogenic properties. The aim of this study was to investigate how the inhibition of low density lipoprotein (LDL) oxidation was influenced by common apoA-IV isoforms. Recombinant wild type apoA-IV (100 mu g/ml) significantly inhibited the oxidation of LDL (50 mu g protein/ml) by 5 mu M CuSO4 (P < 0.005), but not by 100 mu M CuSO4, suggesting that it may act by binding copper ions. ApoA-IV also inhibited the oxidation of LDL by the water-soluble free-radical generator 2,2'-azobis(amidinopropane) dihydrochloride (AAPH; I mM), as shown by the two-fold increase in the time for half maximal conjugated diene formation (T-1/2; P < 0.05) suggesting it can also scavenge free radicals in the aqueous phase. Compared to wild type apoA-IV, apoA-IV-S347 decreased T-1/2 by 15% (P = 0.036) and apoA-IV-H360 increased T-1/2 by 18% (P = 0.046). All apoA-IV isoforms increased the relative electrophoretic mobility of native LDL, suggesting apoA-IV can bind to LDL and acts as a site-specific antioxidant. The reduced inhibition of LDL oxidation by apoA-IV-S347 compared to wild type apoA-IV may account for the previous association of the APOA4 S347 variant with increased CHD risk and oxidative stress. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages - Activation by oxidatively modified LDL and 4-hydroxynonenal

CD36 is an important scavenger receptor mediating uptake of oxidized low- density lipoproteins ( oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase- 1 ( HO- 1), and peroxiredoxin I ( Prx I). 4- Hydroxy- 2- nonenal ( HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2- deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO- 1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO- 1, and Prx I protein levels. Because PPAR-gamma can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.

Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of M-r below 500 was prepared as a model for the low M-r components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low M-r serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL.jlr These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants.

Effects of lipid emulsions on lipid body formation and eicosanoid production by human peripheral blood mononuclear and polymorphonuclear cells

Background & aims: This study investigated the influence of four commercial lipid emulsions, Ivelip, ClinOleic, Omegaven and SMOFlipid (R), on lipid body formation, fatty acid composition and eicosanoid production by cultured human peripheral blood polymorphonuclear cells (PMN) and mononuclear cells (PBMC). Methods: PMN and PBMC were exposed to emulsions at concentrations ranging from 0.01 to 0.04%. Lipid body formation was assessed by microscopy, fatty acid composition by gas chromatography and eicosanoids by ELISA. Results: Stimulation of inflammatory cells and exposure to lipid emulsions promoted the formation of lipid bodies, but there did not appear to be differential effects of the emulsions tested. In contrast, there were differential effects of lipid emulsions on eicosanoid formation, particularly with regards to LTB4 production by PMN. Omegaven dramatically increased production of eicosanoids compared with the other emulsions in a dose-dependent manner. This effect was associated with a significantly higher level of lipid peroxides in the supernatants of cells exposed to Omegaven. Conclusions: Stimulation of inflammatory cells and exposure to lipid emulsions promotes lipid body formation and eicosanoid production, although the differential effects of different emulsions appear to be largely due to lipid peroxidation of unsaturated fatty acids in some emulsions in this in vitro system. (C) 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Effect of apoE genotype and vitamin E on biomarkers of oxidative stress in cultured neuronal cells and the brain of targeted replacement mice

The aetiology of apoE4 genotype-Alzheimer's disease (AD) association are complex. The current study emphasizes the impact of apoE genotype and potential beneficial effects of vitamin E (VE) in relation to oxidative stress. Agonist induced neuronal cell death was examined 1) in the presence of conditioned media containing equal amounts of apoE3 or apoE4 obtained from stably transfected macrophages, and 2) after pretreatment with alpha- and gamma-tocopherol, and -tocotrienol. ApoE3 and apoE4 transgenic mice were fed a diet poor or rich in VE to study the interplay of both apoE genotype and VE status, on membrane lipid peroxidation, antioxidative enzyme activity and glutathione levels in the brain. Cytotoxicity of hydrogen peroxide and glutamate was higher in neuronal cells cultured with apoE4 than apoE3 conditioned media. VE pre-treatment of neurons counteracted the cytotoxicity of a peroxide challenge but not of nitric oxide. No significant effects of apoE genotype or VE supplementation were observed on lipid peroxidation or antioxidative status in the brain of apoE3 and apoE4 mice. VE protects against oxidative insults in vitro, however, no differences in brain oxidative status were observed in mice. Unlike in cultured cells, apoE4 may not contribute to higher neuronal oxidative stress in the brain of young targeted replacement mice.

Associations between apolipoprotein e genotype and circulating F-2-isoprostane levels in humans

Apolipoprotein E (apoE), an important determinant of plasma lipoprotein metabolism, has three common alleles (ε 2, ε 3, and ε 4). Population studies have shown that the risk of diseases characterized by oxidative damage, such as coronary heart disease and Alzheimer's disease, is significantly higher in ε 4 carriers. We evaluated the association between apoE genotypes and plasma F-2-isoprostane levels, an index of lipid peroxidation, in humans. Two hundred seventy-four healthy subjects (104 males, 170 females; 46.9 &PLUSMN; 13.0 yr; 200 whites, 74 blacks; 81 nonsmokers, 64 passive smokers, and 129 active smokers) recruited for a randomized clinical antioxidant intervention trial were included in this analysis. ApoE genotype was determined by PCR and restriction enzyme digestion. Free plasma F2-isoprostane was measured by GC-MS. Genotype groups were compared using multiple regression analysis with adjustment for sex, age, race, smoking status, body mass index, plasma ascorbic acid, and β-carotene. Subjects with ε 3/ε 4 and ε 4/ε 4 genotype (ε 4-carriers) and with ε 2/ε 3 and ε 3/ε 3 (non-ε 4-carriers) were pooled for analysis. In subjects with high cholesterol levels (total cholesterol above 200 mg/dl), plasma F-2-isoprostane levels were 29% higher in ε 4 carriers than in non-ε 4-carriers (P= 0.0056). High-cholesterol subjects that are ε 4 carriers have significantly higher levels of lipid peroxidation as assessed by circulating F-2-isoprostane levels.

Heme-induced biomarkers associated with red meat promotion of colon cancer are not modulated by the intake of nitrite

Red and processed meat consumption is associated with the risk of colorectal cancer. Three hypotheses are proposed to explain this association, via heme-induced oxidation of fat, heterocyclic amines, or N-nitroso compounds. Rats have often been used to study these hypotheses, but the lack of enterosalivary cycle of nitrate in rats casts doubt on the relevance of this animal model to predict nitroso- and heme-associated human colon carcinogenesis. The present study was thus designed to clarify whether a nitrite intake that mimics the enterosalivary cycle can modulate hemeinduced nitrosation and fat peroxidation. This study shows that, in contrast with the starting hypothesis, drinking water added with nitrite to mimic the salivary nitrite content did not change the effect of hemoglobin on biochemicalmarkers linked to colon carcinogenesis, notably lipid peroxidation and cytotoxic activity in the colon of rat. However, ingested sodium nitrite increased fecal nitrosocompounds level, but their fecal concentration and their nature (iron-nitrosyl) would probably not be associated with an increased risk of cancer.We thus suggest that the rat model could be relevant for study the effect of red meat on colon carcinogenesis, in spite of the lack of nitrite in the saliva of rats.

A central role for heme iron in colon carcinogenesis associated with red meat intake

Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP+MeIQx, 50+25 μg/kg in diet) and NOC (induced by NaNO2+NaNO3 0.17+0.23 g/l of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically-induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc- deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67±39 mm2; 2,5% hemoglobin diet: 114±47 mm2, p=0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation.

Consumption of mate tea (Ilex paraguariensis) decreases the oxidation of unsaturated fatty acids in mouse liver

Mate (Ilex paraguariensis) is rich in polyphenolic compounds, which are thought to contribute to the health benefits of tea. Mate tea was administered orally to mice at a dose of 0.5, 1.0 or 2.0 g/kg for 60 d, and changes both in serum lipid concentration and fatty acid composition of liver and kidney were examined. The effects of mate tea on serum and tissue lipid peroxidation were assessed by the evaluation of thiobarbituric acid-reactive substances (TBARS). In tea-consuming mice, both MUFA (18: 1n-9) and PUFA (18: 2n-6 and 20: 4n-6) were increased (P<0.05) in the liver lipid (approximately 90 and 60%, respectively), whereas only MUFA (approximately 20%) were increased in the kidney lipid. The most altered PUFA class was n-6 PUFA, which increased by approximately 60-75 % (P<0.05). This difference in the fatty acid profile in the liver is reflected in the increased PUFA:SFA ratio. Consistent with these results, mice fed with mate tea had much lower TBARS in the liver. No differences (P>0.05) were found in the levels of serum cholesterol, HDL-cholesterol and TAG under the conditions of the present study. These results suggest that treatment with mate tea was able to protect unsaturated fatty acids from oxidation and may have selective protective effects within the body, especially on the liver.

N-acetylcysteine protects against renal injury following bilateral ureteral obstruction

Background. Obstructive nephropathy decreases renal blood flow (RBF) and glomerular filtration rate (GFR), causing tubular abnormalities, such as urinary concentrating defect, as well as increasing oxidative stress. This study aimed to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on the protein expression of aquaporin 2 (AQP2) and endothelial nitric oxide synthase (eNOS), after the relief of bilateral ureteral obstruction (BUO). Methods. Adult male Wistar rats were divided into four groups: sham (sham operated); sham operated + 440 mg/kg body weight (BW) of NAC daily in drinking water, started 2 days before and maintained until 48 h after the surgery; BUO (24-h BUO only); BUO + NAC-pre (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started 2 days before BUO); and BUO + NAC-post (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started on the day of BUO relief). Experiments were conducted 48 h after BUO relief. Results. Serum levels of thiobarbituric reactive substances, which are markers of lipid peroxidation, were significantly lower in NAC-treated rats than in the BUO group rats. The administration of NAC provided significant protection against post-BUO GFR drops and reductions in RBF. Renal cortices and BUO rats presented decreased eNOS protein expression of eNOS in the renal cortex of BUO group rats, whereas it was partially recovered in BUO + NAC-pre group rats. Urine osmolality was significantly lower in BUO rats than in sham group rats or NAC-treated rats, the last also presenting less interstitial fibrosis. Post-BUO downregulation of AQP2 protein expression was averted in the BUO + NAC-pre group rats. Conclusions. This study demonstrates that NAC administration ameliorates the renal function impairment observed 48 h after the relief of 24-h BUO. Oxidative stress is important for the suppression of GFR, RBF, tissue AQP2 and eNOS in the polyuric phase after the release of BUO.

Oxidative variables and antioxidant enzymes activities in the mdx mouse brain

Dystrophin is a protein found at the plasmatic membrane in muscle and postsynaptic membrane of some neurons, where it plays an important role on synaptic transmission and plasticity. Its absence is associated with Duchenne`s muscular dystrophy (DMD), in which cognitive impairment is found. Oxidative stress appears to be involved in the physiopathology of DMD and its cognitive dysfunction. In this regard, the present study investigated oxidative parameters (lipid and protein peroxidation) and antioxidant enzymes activities (superoxide dismutase and catalase) in prefrontal cortex, cerebellum, hippocampus, striatum and cortex tissues from male dystrophic mdx and normal C57BL10 mice. We observed (I) reduced lipid peroxidation in striatum and protein peroxidation in cerebellum and prefrontal cortex; (2) increased superoxide dismutase activity in cerebellum, prefrontal cortex, hippocampus and striatum: and (3) reduced catalase activity in striatum. It seems by our results, that the superoxide dismutase antioxidant mechanism is playing a protective role against lipid and protein peroxidation in mdx mouse brain. (C) 2009 Elsevier Ltd. All rights reserved.

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Modulation of rat neutrophil function in vitro by cis- and trans-MUFA

In the present study, the effects of trans-MUFA, elaidic acid (EA; 18 : 1-9t) and vaccenic acid (VA; 18 : 1-11t) on rat neutrophil functions were compared with those of cis-monounsaturated oleic acid (OA) (18 : 1-9c) and saturated stearic acid (SA; 18 : 0) (10-150 mu M). Trans-fatty acids enhanced neutrophil phagocytic capacity, superoxide (O(2)(center dot-)) and hydrogen peroxide production, and candidacidal activity. The same effects were observed for OA. Cells treated with trans-MUFA showed reduced production of NO(center dot), whereas those treated with OA showed an increase in production. Treatment with SA did not provoke significant effect on the parameters investigated. The increase in O(2)(center dot-) production induced by MUFA was not observed when diphenyleneiodonium, an NADPH oxidase inhibitor, was added to the medium. This finding suggests that MUFA stimulate neutrophil NADPH oxidase activity. The addition of 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-inclol-3-yl)-1H-pyrrole-2,5-dione, a protein kinase C (PKC) inhibitor, and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor, did not affect O(2)(center dot-) production induced by MUFA. Therefore, the mechanisms by which MUFA stimulate NADPH oxidase are not dependent on PKC and do not seem to involve PI3K. Experiments using Zn(2+), an inhibitor of NADPH oxidase H(+) channel, indicated that MUFA activate the NADPH oxidase complex in rat neutrophil due to opening of H(+) channel.