Kinetic Profile of Inflammation Markers in Human Skin In vivo Following Exposure to Ultraviolet B Indicates Synchronic Release of Cytokines and Prostanoids.

Ultraviolet B (UVB) irradiation affects epidermal cells, which respond via a cascade of inflammation markers. After initial in vitro and ex vivo experiments, this study used cutaneous microdialysis to generate a kinetic profile for 16 cytokines and 4 prostanoids in human skin in vivo. Skin areas 9 cm2 were irradiated with UVB (2× minimal erythematous dose) 16 h after catheter placement in the dermis of the volar forearms of healthy volunteers. Dialysates were collected at 4-h intervals up to 64 h and analysed for 5- and 8-iso-PGF2α, 9α,11α-PGF2α and PGE2 by gas chromatography-mass spectrometry (GC/MS). Dialysates were also analysed for interleukin (IL)-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-α, Fas ligand (FasL), interferon-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), RANTES, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) using a multiplex-based cytometric-bead-array. In conclusion, 3 peaks with synchronic release of T helper (TH) 1-directed inflammatory cytokines and prostanoids could be detected post-UVB: an early phase (4-12 h), an intermediate phase (16-24 h) and a late phase (32-40 h). A TH2-directed cytokine response was detectable at intermediate and late phases.

Effects of Topical Tacrolimus and Polyunsaturated Fatty Acids on In Vivo Release of Eicosanoids in Atopic Dermatitis During Dermal Microdialysis.

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease with release of distinct inflammatory signals. This study investigated the presence of eicosanoids in AD skin and the effect of topical agents with potential to suppress inflammation. Twelve patients with moderate AD received topical treatment on either arm with tacrolimus 0.1% ointment or a lotion containing 12% ω-6 fatty acids (polyunsaturated fatty acids; PUFA) twice daily for 5 consecutive days. Interstitial fluid was collected in vivo via dermal microdialysis from 4 defined skin areas: lesional, non-lesional and topically treated skin (tacrolimus or PUFA). Markers of oxidative stress (F2-isoprostanes; 5- and 8-prostaglandin F2α) and inflammation (9α,11α-prostaglandin F2α; and prostaglandin E2) were determined by gas chromatography-mass spectrometry. All eicosanoid levels were reduced in non-lesional and tacrolimus-treated skin. A significant reduction was observed in total F2-isoprostanes; 9α,11α-prostaglandin F2α; and prostaglandin E2 in non-lesional skin and in 9α,11α-prostaglandin F2α in tacrolimus-treated compared with untreated AD skin. In conclusion, treatment with tacrolimus compared with PUFA appears to suppress eicosanoids more efficiently in AD skin.

Prolonged cold storage using a new histidine-tryptophan-ketoglutarate-based preservation solution in isogeneic cardiac mouse grafts.

AIMS: The aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based cold preservation solution in comparison with traditional HTK solution in a mouse cardiac transplant model and to assess the impact of chloride ions and of iron chelators. METHODS AND RESULTS: After 24 h cold ischaemia, traditional HTK-preserved hearts survived up to 13 days (4.4 ± 1.7 days; n = 8)). Hearts stored in the new solution without iron chelators (N46) showed significantly prolonged survival up to 2 months (N46: 11.9 ± 8.7 days; P < 0.01; n = 7) and with iron chelators (DesfLK) up to 3 months (N46 DesfLK: 12.7 ± 13.7 days; n = 6). Re-beating time was significantly shorter with the new solution (HTK: 14.5 ± 5.9 min, N46: 9.2 ± 2.7 min, N46 DesfLK: 7.1 ± 3.7 min; P < 0.01). The new solution showed significantly decreased release of creatine kinase (HTK: 25998 ± 8471 U/L, N46: 13829 ± 7679 U/L, N46 DesfLK: 3093 ± 597 U/L; P < 0.01 and n = 7 each) and lactate dehydrogenase (HTK: 5391 ± 1062 U/L, N46: 3428 ± 1890 U/L, P < 0.05; N46 DesfLK: 682 ± 344 U/L, P < 0.01) and decreased histological evidence of injury. A chloride-poor variant of the new solution showed inferior graft survival. CONCLUSION: The new solution markedly attenuates myocardial injury and yields better graft survival than traditional HTK solution. The presence of chloride ions is crucial for heart preservation. Some protective effects are obviously caused by iron chelators.

Acute atrial tachyarrhythmia induces angiotensin II type 1 receptor-mediated oxidative stress and microvascular flow abnormalities in the ventricles.

AIMS: Patients with paroxysmal atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non ST-segment elevation myocardial infarction) during an arrhythmic event. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography. Here we explored the potential underlying mechanism of these events. METHODS AND RESULTS: A total of 14 pigs were studied using a closed chest, rapid atrial pacing (RAP) model. In five pigs RAP was performed for 7 h (600 b.p.m.; n = 5), in five animals RAP was performed in the presence of angiotensin-II type-1-receptor (AT(1)-receptor) inhibitor irbesartan (RAP+Irb), and four pigs were instrumented without intervention (Sham). One-factor analysis of variance was performed to assess differences between and within the three groups. Simultaneous measurements of fractional flow reserve (FFR) and coronary flow reserve (CFR) before, during, and after RAP demonstrated unchanged FFR (P = 0.327), but decreased CFR during RAP (RAP: 67.7 +/- 7.2%, sham: 97.2 +/- 2.8%, RAP+Irb: 93.2 +/- 3.3; P = 0.0013) indicating abnormal left ventricular (LV) microcirculation. Alterations in microcirculatory blood flow were accompanied by elevated ventricular expression of NADPH oxidase subunit Nox2 (P = 0.039), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1, P = 0.004), and F(2)-isoprostane levels (P = 0.008) suggesting RAP-related oxidative stress. Plasma concentrations of cardiac troponin-I (cTn-I) increased in RAP (RAP: 613.3 +/- 125.8 pmol/L vs. sham: 82.5 +/- 12.5 pmol/L; P = 0.013), whereas protein levels of eNOS and LV function remained unchanged. RAP+Irb prevented the increase of Nox2, LOX-1, and F(2)-isoprostanes, and abolished the impairment of microvascular blood flow. CONCLUSION: Rapid atrial pacing induces AT(1)-receptor-mediated oxidative stress in LV myocardium that is accompanied by impaired microvascular blood flow and cTn-I release. These findings provide a plausible mechanism for the frequently observed cTn-I elevation accompanied with typical angina pectoris symptoms in patients with paroxysmal AF and normal (non-stenotic) coronary arteries.

Dietary vitamin E, brain redox status and expression of Alzheimer's disease-relevant genes in rats.

Oxidative stress is one of the major pathological features of Alzheimer's disease (AD). Here, we investigated whether dietary vitamin E (VE) depletion may induce adverse effects and supplementation with alpha-tocopherol (alphaT) may result in beneficial effects on redox status and the regulation of genes relevant in the pathogenesis of AD in healthy rats. Three groups of eight male rats each were fed diets with deficient ( < 1 mg alphaT equivalents/kg diet), marginal (9 mg alphaT equivalents/kg diet) or sufficient (18 mg alphaT equivalents/kg diet) concentrations of natural-source VE for 6 months; a fourth group was fed the VE-sufficient diet fortified with alphaT (total VE, 146 mg alphaT equivalents/kg diet). Feeding of the experimental diets dose dependently altered alphaT concentrations in the cortex and plasma. No significant changes in F2-isoprostane concentrations, activities of antioxidative enzymes (total superoxide dismutase, Se-dependent glutathione peroxidase) and concentrations of glutathione or the expression of AD-relevant genes were observed. In this non-AD model, depletion of VE did not induce adverse effects and supplementation of alphaT did not induce positive effects on the parameters related to the progression of AD.

UVB irradiation-induced impairment of keratinocytes and adaptive responses to oxidative stress.

UVB irradiation of human skin is known to induce pathophysiological processes as oxidative stress and inflammation. HaCaT keratinocytes represent a well-established in vitro model system to investigate the influence of UVB irradiation on cell cultures. It was the aim of these investigations to study the effects of moderate UVB doses on cellular and mitochondrial integrity of HaCaT keratinocytes, biomarkers of oxidative stress and antioxidant protection by superoxide dismutases. F(2)-isoprostane concentrations were UVB dose-dependently enhanced reaching a plateau at 50 mJ/cm(2). Cell viability was reduced and apoptosis was enhanced with increasing UVB doses. The activities of the respiratory chain complexes were practically not altered at lower UVB doses, up to 50 mJ/cm(2), whereas remarkable decreases, also for the levels of cardiolipin species, were seen at 100 mJ/cm(2). As an adaptive response to the enhanced oxidative stress, protein levels of MnSOD increased about 3-fold at 50 mJ/cm(2) and decreased at higher doses. From the data it can be concluded that keratinocytes are sufficiently protected at low UVB doses, whereas higher doses lead to irreversible cell damage.

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio.

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils.

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.

Beta-carotene cleavage products induce oxidative stress in vitro by impairing mitochondrial respiration.

Carotenoids are widely used as important micronutrients in food. Furthermore, carotenoid supplementation has been used in the treatment of diseases associated with oxidative stress. However, in some clinical studies harmful effects have been observed, for example, a higher incidence of lung cancer in individuals exposed to extraordinary oxidative stress. The causal mechanisms are still unclear. Carotenoid cleavage products (CCPs), including highly reactive aldehydes and epoxides, are formed during oxidative attacks in the course of antioxidative action. Here, we tested the hypothesis that CCPs may increase oxidative stress by impairing mitochondrial function. We found that CCPs strongly inhibit state 3 respiration of isolated rat liver mitochondria even at concentrations between 0.5 and 20 microM. This was true for retinal, beta-ionone, and mixtures of cleavage products, which were generated in the presence of hypochlorite to mimic their formation in inflammatory regions. The inhibition of mitochondrial respiration was accompanied by a reduction in protein sulfhydryl content, decreasing glutathione levels and redox state, and elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favor functional deterioration of the adenine nucleotide translocator. The findings may reflect a basic mechanism of increasing the risk of cancer induced by CCPs.

Beta-carotene breakdown products may impair mitochondrial functions--potential side effects of high-dose beta-carotene supplementation.

Beta-carotene (BC) and other carotenoids are mainly considered as belonging to the group of micronutrients. As they are contained in fruit and vegetables and thus part of human diet, a regular low-dose intake from natural sources is normally assured. In the last decade high-dose supplementation with synthetic carotenoids has been used successfully in the treatment of diseases believed to be associated with oxidative stress. However, in a few clinical studies harmful effects have been observed as well, e.g., a higher incidence of lung cancer after BC was given in high doses to smokers. Our studies aim at shedding light on the causal mechanisms of the known side effects that we have investigated. Possibilities of preventing them are discussed. Obviously, on certain conditions of high-dose carotenoid supplementation, both the antioxidant and prooxidant reactions may arise. Carotenoid breakdown products (CBP) including very reactive aldehydes and epoxides are formed during oxidative attack in the course of antioxidative action. Carotenoid breakdown products inhibit state 3 respiration of isolated rat liver mitochondria at concentrations between 0.5 and 20 microM. In vivo stimulated neutrophils might represent an important source for the generation of CBP, and the lung might be a critical organ in CBP formation. The inhibition of mitochondrial state 3 respiration by CBP is accompanied by a reduced content of protein sulfhydryl groups, decreasing glutathione levels and redox state, and also elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favour functional deterioration of the adenine nucleotide translocator (ANT). The findings reflect a basic mechanism of the side effects of BC supplementation in circumstances of severe oxidative stress induced by CBP representing a class of lipid oxidation products. We are striving for safe conditions of carotenoid supplementation in order to protect patients in need of this kind of medical treatment from possible side effects, such as unwanted prooxidative reactions.

F2-isoprostanes as biomarkers of lipid peroxidation in patients with chronic renal failure.

Chronic renal failure patients on long-term hemolysis are found to be under increased oxidative stress, caused by antioxidant deficiency, neutrophil activation during hemodialysis (HD), platelet activation and/or chronic inflammation. Increased levels of oxidants (e.g. malondialdehyde, 4-hydroxynonenal, hydrocarbons, lipohydroperoxides, oxycholesterols, carbonyls) in HD patients are thought to play an important role in the development of endothelial dysfunction, atherogenesis and cardiovascular disease, which is a frequent condition in end-stage renal disease. F2-isoprostanes have been established as chemically stable, highly specific and reliable biomarkers of in vivo oxidative stress which can very sensitively measured by gas chromatography-mass spectrometry (Morrow et al. [17]). An up to 6-fold increase of plasma F2-isoprostanes in HD patients is accompanied by an enhanced formation of indicators of inflammation (e.g. C-reactive protein) and decreases of endogenous antioxidants (e.g. ascorbate, alpha-tocopherol). In their esterified form F2-isoprostanes may be a useful criteria to evaluate the effectiveness of clinical interventions to diminish oxidant stress and associated inflammation. Furthermore, F2-isoprostanes possess potent biological activities (e.g. 8-iso-PGF2alpha is known as a renal vasoconstrictor) suggesting that they may also act as mediators of the cellular effects of oxidative stress and inflammation.

Flavanol-rich cocoa drink lowers plasma F(2)-isoprostane concentrations in humans.

Flavan-3-ols are potent antioxidants in vitro, but convincing evidence for antioxidant action in vivo is lacking. We examined whether an oxidative stress-mediated increase in plasma F(2)-isoprostanes is counteracted by a flavanol-rich cocoa beverage. Twenty volunteers were examined in a comparative randomized double-blind crossover design with respect to ingestion of high-flavanol cocoa drink (HFCD; 187 mg flavan-3-ols/100 ml) vs. low-flavanol cocoa drink (LFCD; 14 mg/100 ml). With 10 individuals, the treatment was combined with strenuous physical exercise. Total (esterified plus nonesterified) F(2)-isoprostanes were analyzed by GC/MS. LFCD caused a slight increase in the mean (+/- SEM) plasma concentrations of F(2)-isoprostanes 2 and 4 h after intake (2.16 +/- 0.19 nM at 4 h vs. 1.76 +/- 0.11 nM at 0 h, n = 10), which may be attributable to postprandial oxidative stress. This increase did not occur with HFCD (1.57 +/- 0.06 nM at 4 h vs. 1.65 +/- 0.10 nM at 0 h, n = 10). The difference in F(2)-isoprostanes 2 and 4 h after intake of HFCD vs. LFCD became statistically significant when the intake was combined with physical exercise (P < 0.01, ANOVA). We conclude that dietary flavanols, using cocoa drink as example, can lower the plasma level of F(2)-isoprostanes, indicators of in vivo lipid peroxidation.

Hypoxia/reoxygenation-induced damage to mitochondrial activity is determined by glutathione threshold in astroglia-rich cell cultures.

It has been shown that astrocytes play an important role during ischemia/reperfusion and in neurodegenerative diseases by supporting neuronal functions, but the effect of these pathophysiological conditions on this particular cell type is still unclear. Here, we investigated the ischemia/reperfusion-induced damage to astroglia-rich cells. For that purpose, we studied the effects of substrate deprivation and hypoxia/reoxygenation on total cellular glutathione contents, and mitochondrial function. Substrate deprivation as well as increasing time of cultivation in vitro (from 2 to 4 weeks) induced a decrease in the total glutathione content. Three qualitative distinct concentration ranges of the glutathione pool with respect to the effect of hypoxia/reoxygenation on the glutathione content were found: (i) high glutathione levels above 40 nmol per mg protein remained unchanged during hypoxia/reoxygenation. (ii) Hypoxia/reoxygenation was accompanied by higher glutathione levels in comparison to controls at intermediate initial glutathione concentrations of about 20 up to 40 nmol per mg protein. (iii) Below an initial glutathione threshold concentration of about 20 nmol per mg protein, hypoxia/reoxygenation led to a stronger decrease of glutathione levels in comparison to controls. Decrease of mitochondrial respiratory chain activity during hypoxia/reoxygenation only occurred at low initial glutathione concentrations below 20 nmol per mg protein. Our data emphasize the important role of glutathione with respect to the defense of mitochondria against oxidative stress in astroglia cells during hypoxia/reoxygenation.

The role of superoxide dismutase and alpha-tocopherol in the development of seizures and kindling induced by pentylenetetrazol - influence of the radical scavenger alpha-phenyl-N-tert-butyl nitrone.

Previous experiments have shown that the generation of free hydroxyl radicals in rat brain homogenates is increased following pentylenetetrazol (PTZ) kindling. The present study was performed in order to evaluate the involvement of endogeneous radical defence systems as the superoxide dismutase (SOD) and the level of alpha-tocopherol, an important lipid-soluble and membrane-bound antioxidant in brain homogenate of rats after acute seizure and kindling induced by PTZ. The activities of the total SOD were significantly reduced after acute seizure and tend towards an enhancement in kindled animals. Western blot analysis shows an upregulation of Mn-SOD in rat brain homogenates after kindling. The level of the chain-breaking antioxidant alpha-tocopherol was reduced in acutely convulsing rats and was not modified in kindled rats. Second, we studied the influence of exogeneously supplied radical scavenger alpha-phenyl-N-tert-butyl-nitrone (PBN) on seizure and kindling following PTZ treatment. After a single injection of PTZ at a dose evoking clonic-tonic seizures, PBN did not modify either the formation of free hydroxyl radicals measured by the levels of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA or the susceptibility to PTZ. In the kindling group, subchronic treatment with PBN (over a period of 4 weeks) prevented the increase in the formation of free hydroxyl radicals, and the susceptibility to PTZ was transiently decreased during the development of kindling, but PBN did not influence the susceptibility to PTZ in fully kindled rats. Pretreatment with PBN increased the activities of total SOD and the protein content of Mn-SOD and decreased the level of alpha-tocopherol in comparison to saline controls. The results suggest that the formation of free hydroxyl radicals is not reflected by an enhanced susceptibility to PTZ classified according to the modified RACINE scale. Additionally, it may be assumed that the increased generation of hydroxyl radicals in kindled animals is not primary caused by an exhaustion of both the defence systems measured. Adaptive mechanisms, as the induction of Mn-SOD, may be taken into consideration to counteract oxidative stress-mediated free radical formation.

Analysis of monohydroxyeicosatetraenoic acids and F2-isoprostanes as markers of lipid peroxidation in rat brain mitochondria.

We have introduced two specific techniques for the quantitative measurement of monohydroxyeicosatetraenoic acids (HETEs) and F2-isoprostanes by gas chromatography-mass spectrometry/negative ion chemical ionization (GC-MS/NICI) to study lipid peroxidation in isolated rat brain mitochondria by iron/ascorbate. The analysis of HETEs involved hydrogenation, solid phase extraction on a C18-cartridge, formation of pentafluorobenzyl bromide and trimethylsilyl ether derivatives. In the case of F2-isoprostanes, the analytical procedure was similar to that of HETEs except that the hydrogenation step was omitted. We found that HETE content (sum of 5-, 8-12-, and 15-isomers) in freshly prepared rat brain mitochondria was 220 +/- 40pmol/mg protein. The corresponding content for the F2-isoprostane, 8-iso-PGF2alpha, was 0.21 +/-+/- 0.10 pmol/mg protein. HETEs and 8-iso-PGF2alpha were predominantly present in the esterified form. The content of both HETEs and 8-iso-PGF2alpha were increased in presence of iron/ascorbate as oxidation system. After 30 min incubation with Fe2+ ascorbate, the content of HETE isomers was increased about 6-fold compared with baseline levels whereas that for 8-iso-PGF2alpha was elevated 100-fold. Formation of HETEs and F2-isoprostanes corresponded to the consumption of arachidonic acid (AA) and alpha-tocopherol, respectively. There were almost no changes in the content of free (non-esterified) HETEs and 8-iso-PGF2alpha during the course of iron/ascorbate induced oxidation of the brain mitochondria. Our data provide the first direct evidence for the presence of HETEs and F2-isoprostanes in freshly isolated rat brain mitochondria and that esterified HETEs and 8-iso-PGF2alpha are predominantly generated during iron/ascorbate induced lipid peroxidation. Sensitive quantification of these products of non-enzymatic lipid peroxidation as indicators of oxidant injury opens new areas of investigation regarding the role of free radicals in the pathogenesis of human diseases. In addition, HETEs and F2-isoprostanes may be important mediators for mitochondrial functions.

Biomarkers of oxidative stress study V: ozone exposure of rats and its effect on lipids, proteins, and DNA in plasma and urine.

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.

Composition of molecular cardiolipin species correlates with proliferation of lymphocytes.

The mitochondrial phospholipid cardiolipin (CL) is required for oxidative phosphorylation. Oxidation of CL results in the disruption of CL-cytochrome c binding and the induction of apoptosis. Large variations in the acyl-chain residues of CL have been reported, but evidence as to whether these variants exert distinct biological effects has been limited. We have studied the acyl-chain composition of CL in lymphocytes, and found marked differences between highly and slowly proliferating cells. In fast growing cells, we detected a decreased number of double bonds, and a higher amount of C16 acyl-chain residues in CL, compared with slower growing cells. However, fewer C18 acyl-chain residues were found in CL from fast growing cells compared with slower proliferating cells. Our results suggest a functional link between acyl-chain composition of CL and cell proliferation.

A new preservation solution for lung transplantation: evaluation in a porcine transplantation model.

BACKGROUND: Lung preservation injury is still a major problem in lung transplantation. The aim of the current study was to evaluate the effects of a new preservation solution (Custodiol-N) for lung preservation. METHODS: Using an in vivo pig model, 7 lungs each were preserved for 24 hours after perfusion with: low-potassium dextran (LPD) solution as control (Group I); base solution of Custodiol-N without iron chelators (Group II); Custodiol-N (Group III); or Custodiol-N supplemented with dextran 40 (Group IV). Four animals received a sham operation. After left lung transplantation and contralateral lung exclusion, hemodynamics and blood gases were monitored for 6 hours; tissue samples were taken at the end of the experiments. RESULTS: All animals survived the transplantation procedure. Base solution- and Custodiol-N-preserved lungs (Groups II and III) showed graft function similar to that of LPD-preserved lungs (Group I), showing a trend toward improved values. Custodiol-N with dextran (Group IV) led to a significant reduction of mean pulmonary arterial pressure (20 ± 2 vs 28 ± 3 mm Hg, p < 0.01) and pulmonary vascular resistance (410 ± 51 vs 588 ± 83 dyne/s/cm(5), p < 0.01), and oxygenation ratio was significantly higher (536 ± 52 vs 313 ± 107 mm Hg at 6 hours, p < 0.01) and PCO(2) values were significantly lower (51 ± 9 vs 77 ± 5 mm Hg at 6 hours, p < 0.01) at 6 hours compared with LPD (Group I). Custodiol-N (Groups II to IV) showed a trend toward a lower wet/dry ratio and reduced oxidative stress; in the presence of dextran (Group IV), the difference was again statistically significant, when compared with LPD (Group I). CONCLUSIONS: Custodiol-N solution is a new alternative preservation solution for lung transplantation that offers significantly superior protection compared with LPD when dextran 40 is added.

Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype-studies in targeted gene replacement mice.

An apoE4 genotype is an important risk factor for cardiovascular and other chronic diseases. The higher cardiovascular disease risk of apoE4 carriers as compared to the apoE3 genotype has been mainly attributed to the differences in blood lipids between the two genotype subgroups. Recently, a potential protective role of the transcription factor Nrf2 in cardiovascular disease prevention has been suggested. In this study, we show that Nrf2-dependent gene expression is affected by the apoE genotype. ApoE4 vs. apoE3 mice exhibited lower hepatic Nrf2 nuclear protein levels. Furthermore, mRNA and protein levels of Nrf2 target genes including glutathione-S-transferase, heme oxygenase-1 and NAD(P)H dehydrogenase, quinone 1 were significantly lower in apoE4 as compared to apoE3 mice. Lower hepatic mRNA levels of phase II enzymes, as observed in apoE4 vs. apoE3 mice, were accompanied by higher mRNA levels of phase I enzymes including Cyp26a1 and Cyp3a16. Furthermore, miRNA-144, miRNA-125b, and miRNA-29a involved in Nrf2 signaling, inflammation, and regulation of phase I enzyme gene expression were affected by the apoE genotype. We provide first evidence that Nrf2 is differentially regulated in response to the apoE genotype.