Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Titanium dioxide food additive (E171) induces ROS formation and genotoxicity: contribution of micro and nano-sized fractions.

Since 1969, the European Union approves food-grade titanium dioxide (TiO2), also known as E171 colouring food additive. E171 is a mixture of micro-sized particles (MPs) and nano-sized particles (NPs). Previous studies have indicated adverse effects of oral exposure to E171, i.e. facilitation of colon tumour growth. This could potentially be partially mediated by the capacity to induce reactive oxygen species (ROS). The aim of the present study is to determine whether E171 exposure induces ROS formation and DNA damage in an in vitro model using human Caco-2 and HCT116 cells and to investigate the contribution of the separate MPs and NPs TiO2 fractions to these effects. After suspension of the particles in Hanks' balanced salt solution buffer and cell culture medium with either bovine serum albumin (BSA) or foetal bovine serum, characterization of the particles was performed by dynamic light scattering, ROS formation was determined by electron spin/paramagnetic resonance spectroscopy and DNA damage was determined by the comet and micronucleus assays. The results showed that E171, MPs and NPs are stable in cell culture medium with 0.05% BSA. The capacity for ROS generation in a cell-free environment was highest for E171, followed by NPs and MPs. Only MPs were capable to induce ROS formation in exposed Caco-2 cells. E171, MPs and NPs all induced single-strand DNA breaks. Chromosome damage was shown to be induced by E171, as tested with the micronucleus assay in HCT116 cells. In conclusion, E171 has the capability to induce ROS formation in a cell-free environment and E171, MPs and NPs have genotoxic potential. The capacity of E171 to induce ROS formation and DNA damage raises concerns about potential adverse effects associated with E171 (TiO2) in food.

Pattern recognition methods to relate time profiles of gene expression with phenotypic data: a comparative study.

MOTIVATION: Comparing time courses of gene expression with time courses of phenotypic data may provide new insights in cellular mechanisms. In this study, we compared the performance of five pattern recognition methods with respect to their ability to relate genes and phenotypic data: one classical method (k-means) and four methods especially developed for time series [Short Time-series Expression Miner (STEM), Linear Mixed Model mixtures, Dynamic Time Warping for -Omics and linear modeling with R/Bioconductor limma package]. The methods were evaluated using data available from toxicological studies that had the aim to relate gene expression with phenotypic endpoints (i.e. to develop biomarkers for adverse outcomes). Additionally, technical aspects (influence of noise, number of time points and number of replicates) were evaluated on simulated data. RESULTS: None of the methods outperforms the others in terms of biology. Linear modeling with limma is mostly influenced by noise. STEM is mostly influenced by the number of biological replicates in the dataset, whereas k-means and linear modeling with limma are mostly influenced by the number of time points. In most cases, the results of the methods complement each other. We therefore provide recommendations to integrate the five methods. AVAILABILITY: The Matlab code for the simulations performed in this research is available in the Supplementary Data (Word file). The microarray data analysed in this paper are available at ArrayExpress (E-TOXM-22 and E-TOXM-23) and Gene Expression Omnibus (GSE39291). The phenotypic data are available in the Supplementary Data (Excel file). Links to the pattern recognition tools compared in this paper are provided in the main text. CONTACT: d.hendrickx@maastrichtuniversity.nl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The exposome concept in a human nutrigenomics study: evaluating the impact of exposure to a complex mixture of phytochemicals using transcriptomics signatures.

The application of transcriptome analyses in molecular epidemiology studies has become a promising tool in order to evaluate the impact of environmental exposures. These analyses have a great value in establishing the exposome, the totality of human exposures, both by identifying the chemical nature of the exposures and the induced molecular responses. Transcriptomic signatures can be regarded as biomarker of exposure as well as markers of effect which reflect the interaction between individual genetic background and exposure levels. However, the biological interpretation of modulated gene expression profiles is a challenging task and translating affected molecular pathways into risk assessment, for instance in terms of cancer promoting or disease preventing responses, is a far from standardised process. Here, we describe the in-depth analyses of the gene expression responses in a human dietary intervention in which the interaction between genotype and exposure to a blueberry-apple juice containing a complex mixture of phytochemicals is investigated. We also describe how data on differences in genetic background combined with different effect markers can provide a better understanding of gene-environment interactions. Pathway analyses of differentially expressed genes in combination with gene were used to identify complex but strong changes in several biological processes like immune response, cell adhesion, lipid metabolism and apoptosis. These observed changes may lead to upgraded growth control, induced immunity, reduced platelet aggregation and activation, diminished production of reactive oxidative species by platelets, blood glucose homeostasis, regulation of blood lipid levels and increased apoptosis. Our findings demonstrate that applying transcriptomics to well-controlled human dietary intervention studies can provide insight into mechanistic pathways involved in disease prevention by dietary factors.

Elevated oxidative stress in patients with congenital heart disease and the effect of cyanosis: a meta-analysis.

Oxidative stress is an important pathophysiological mechanism in the development of numerous cardiovascular disorders. To improve therapy and preventive strategies, clinicians need a better understanding of the underlying pathophysiological mechanisms of congenital heart diseases (CHD). The objective of this meta-analysis was to determine whether oxidative stress is elevated in patients with CHD compared to healthy controls, and to evaluate whether a difference in oxidative stress parameters can be observed between patients with cyanotic (cCHD) and acyanotic CHD (aCHD). Therefore, 21 studies investigating oxidative stress in peripheral blood of both children and adults with CHD were reviewed. Different methods to assess the oxidant status were compared and divided into three categories: pro-oxidative or anti-oxidative stress markers and the ratio of pro-to-anti oxidative stress markers. This meta-analysis showed elevated oxidative stress levels in patients with CHD, and more specifically in patients with cCHD. Moreover, this indicates that there could be potential in oxidative stress measurements as a new biomarker of disease severity. Further research will be needed to clarify the exact role of oxidative stress and its contributors in CHD in order to get a better and more in-depth understanding of the underlying pathophysiology of CHD, especially the higher susceptibility of the right ventricle (RV) to progress to heart failure (HF). This could facilitate the development of antioxidant treatments and RV-specific HF therapies, which are necessary to improve survival in these patients and could be of particular importance in cCHD.

Circulating Reactive Oxygen Species in Adults with Congenital Heart Disease.

Oxidative stress is an important pathophysiological mechanism in the development of numerous cardiovascular disorders, but few studies have examined the levels of oxidative stress in adults with congenital heart disease (CHD). The objective of this study was to investigate oxidative stress levels in adults with CHD and the association with inflammation, exercise capacity and endothelial function. To this end, 36 adults with different types of CHD and 36 age- and gender-matched healthy controls were enrolled. Blood cell counts, hs-CRP, NT-proBNP, fasting glucose, cholesterol levels, iron saturation and folic acid concentrations were determined in venous blood samples. Levels of superoxide anion radical in whole blood were determined using electron paramagnetic resonance spectroscopy in combination with the spin probe CMH. Physical activity was assessed with the IPAQ-SF questionnaire. Vascular function assessment (EndoPAT) and cardiopulmonary exercise testing were performed in the patient group. Superoxide anion radical levels were not statistically significantly different between adults with CHD and the matched controls. Moreover, oxidative stress did not correlate with inflammation, or with endothelial function or cardiorespiratory fitness in CHD; however, a significant negative correlation with iron saturation was observed. Overall, whole blood superoxide anion radical levels in adults with CHD were not elevated, but iron levels seem to play a more important role in oxidative stress mechanisms in CHD than in healthy controls. More research will be needed to improve our understanding of the underlying pathophysiology of CHD.

Beta-carotene affects oxidative stress-related DNA damage in lung epithelial cells and in ferret lung.

Beta-carotene (BC) was found to enhance lung cancer risk in smokers. This adverse effect was unexpected because BC was thought to act as an anti-oxidant against cigarette smoke-derived radicals. These radicals can directly or indirectly damage DNA, leading to the formation of pro-mutagenic DNA lesions such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one deoxyguanosine (M(1)dG). Later, it was suggested that high concentrations of BC could also result in pro-oxidant effects. Therefore, we investigated whether high but physiologically feasible concentrations of BC were able to alter (i) the formation of radicals in vitro assessed by electron spin resonance spectroscopy, (ii) the levels of 8-oxo-dG and M(1)dG in vitro in lung epithelial cells after incubation with hydrogen peroxide (H(2)O(2)) and the smoke-derived carcinogen benzo[a]pyrene (B[a]P) and (iii) the levels of 8-oxo-dG and M(1)dG in vivo in ferrets' lung after chronic exposure to B[a]P. BC increased in vitro hydroxyl radical formation in the Fenton reaction but inhibited the formation of carbon-centered radicals. Similarly, BC was able to enhance 8-oxo-dG in vitro in lung epithelial cells. On the other hand, BC significantly inhibited M(1)dG formation in lung epithelial cells, especially after induction of M(1)dG by H(2)O(2) or B[a]P. Finally, BC supplementation of ferrets also resulted in a significant decrease in M(1)dG, but in contrast to the in vitro experiments, no effect was observed on 8-oxo-dG levels, probably because of increased base excision repair capacities as assessed by a modified comet assay. These data indicate that the fate of BC being a pro- or anti-oxidant strongly depends on the type of radical involved.

The R439C mutation in LMNA causes lamin oligomerization and susceptibility to oxidative stress.

Dunnigan-type familial partial lipodystrophy (FPLD) is a laminopathy characterized by an aberrant fat distribution and a metabolic syndrome for which oxidative stress has recently been suggested as one of the disease-causing mechanisms. In a family affected with FPLD, we identified a heterozygous missense mutation c.1315C>T in the LMNA gene leading to the p.R439C substitution. Cultured patient fibroblasts do not show any prelamin A accumulation and reveal honeycomb-like lamin A/C formations in a significant percentage of nuclei. The mutation affects a region in the C-terminal globular domain of lamins A and C, different from the FPLD-related hot spot. Here, the introduction of an extra cysteine allows for the formation of disulphide-mediated lamin A/C oligomers. This oligomerization affects the interaction properties of the C-terminal domain with DNA as shown by gel retardation assays and causes a DNA-interaction pattern that is distinct from the classical R482W FPLD mutant. Particularly, whereas the R482W mutation decreases the binding efficiency of the C-terminal domain to DNA, the R439C mutation increases it. Electron spin resonance spectroscopy studies show significantly higher levels of reactive oxygen species (ROS) upon induction of oxidative stress in R439C patient fibroblasts compared to healthy controls. This increased sensitivity to oxidative stress seems independent of the oligomerization and enhanced DNA binding typical for R439C, as both the R439C and R482W mutants show a similar and significant increase in ROS upon induction of oxidative stress by H2O2.

Transcriptome changes in undifferentiated Caco-2 cells exposed to food-grade titanium dioxide (E171): contribution of the nano- and micro- sized particles.

The food additive titanium dioxide (TiO2), or E171, is a white food colorant. Recent studies showed after E171 ingestion a significantly increased number of colorectal tumours in a colorectal cancer mouse model as well as inflammatory responses and dysregulation of the immune system in the intestine of rats. In the mouse colon, E171 induced gene expression changes related to oxidative stress, impairment of the immune system, activation of signalling and cancer-related processes. E171 comprises nanoparticles (NPs) and microparticles (MPs). Previous in vitro studies showed that E171, NPs and MPs induced oxidative stress responses, DNA damage and micronuclei formation. This study aimed to investigate the relative contribution of the NPs and MPs to effects of E171 at the transcriptome level in undifferentiated Caco-2 cells by genome wide microarray analysis. The results showed that E171, NPs, and MPs induce gene expression changes related to signalling, inflammation, immune system, transport and cancer. At the pathway level, metabolism of proteins with the insulin processing pathway and haemostasis were specific to E171 exposure. The gene expression changes associated with the immune system and inflammation induced by E171, MPs, and NPs suggest the creation of a favourable environment for colon cancer development.

Mitochondrial function, content and ROS production in rat skeletal muscle: effect of high-fat feeding.

A high intake of dietary fat has been suggested to diminish mitochondrial functioning in skeletal muscle, possibly attributing to muscular fat accumulation. Here we show however, that an 8-week high-fat dietary intervention did not affect intrinsic functioning of rat skeletal muscle mitochondria assessed by respirometry, neither on a carbohydrate- nor on a lipid-substrate. Interestingly, PPARGC1A protein increased by approximately 2-fold upon high-fat feeding and we observed inconsistent results on different markers of mitochondrial density. Mitochondrial ROS production, assessed by electron spin resonance spectroscopy remained unaffected. Intramyocellular lipid levels increased significantly illustrating that a reduced innate mitochondrial function is not a prerequisite for intra-muscular fat accumulation.

Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro.

Antioxidants play a vital role in the cellular protection against oxidative damage. Quercetin is a well-investigated antioxidant and known to be able to protect against cellular oxidative DNA damage. In this study, we tried to relate the protection by quercetin pre-treatment against oxidative DNA damage in human leucocytes in vitro to the interaction of quercetin in solution with hydroxyl and superoxide anion radicals as measured by electron spin resonance (ESR) spectrometry, using DMPO as a spin trap. Further, scavenging capacity of quercetin-treated leucocytes in vitro was evaluated by ESR spectrometry. Quercetin appears capable of protecting human leucocytes against oxidative DNA damage caused by hydrogen peroxide in a dose-dependent manner. The protection of leucocytes against superoxides is ambiguous. Incubation concentrations of quercetin (1, 10, and 50 microM) reduced levels of superoxide-induced oxidative DNA damage, while at 100 microM the amount of damage was increased. These results are supported by ESR-findings on quercetin in solution, also showing a prooxidant effect at 100 microM. ESR spectroscopy showed rate constant values for the reaction kinetics of quercetin in lowering iron-dependent hydroxyl radical formation and NADH-dependent superoxide anion formation of respectively 3.2 x 10(12)M(-1)s(-1) and 1.1 x 10(4)M(-1)s(-1). This shows that quercetin is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions.

Improved Preventive Effects of Combined Bioactive Compounds Present in Different Blueberry Varieties as Compared to Single Phytochemicals.

Blueberries contain many different phytochemicals which might be responsible for their disease preventive properties. In a previously conducted human dietary intervention study, we showed that a 4-week intervention with blueberry⁻apple juice protected the participants against oxidative stress and modulated expression of genes involved in different genetic pathways contributing to the antioxidant response. The present study investigates the effect of different blueberry varieties (Elliot, Draper, Bluecrop, and Aurora, and the blueberry⁻apple juice from our previous human dietary intervention study), and four different single compounds (vitamin C, peonidin, cyanidin, and quercetin) on antioxidant capacity and gene expression changes in colonic cells in vitro, and compares the outcome with the earlier in vivo findings. The results demonstrate that all blueberry varieties as well as the blueberry⁻apple juice were more effective in reducing oxidative stress as compared to the single compounds (e.g., DNA strand break reduction: EC50: Elliot 8.3 mg/mL, Aurora and Draper 11.9 mg/mL, blueberry⁻apple juice 12.3 mg/mL, and Bluecrop 12.7 mg/mL; single compounds). In addition, the gene expression profiles (consisting of 18 selected genes from the in vivo study) induced by the blueberry varieties were more similar to the profile of the human intervention study (range 44⁻78%). The blueberry variety Elliot showed the strongest and most similar effects, almost 80% of gene expression modulations were similar compared to the in vivo results. From the single compounds (range 17⁻44%), quercetin induced the most comparable gene expression changes, i.e., 44%. This approach could be useful in agriculture for identifying crop varieties containing combinations of phytochemicals which show optimal preventive capacities.

A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases.

Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.

Transcriptomics analysis reveals new insights in E171-induced molecular alterations in a mouse model of colon cancer.

Titanium dioxide as a food additive (E171) has been demonstrated to facilitate growth of chemically induced colorectal tumours in vivo and induce transcriptomic changes suggestive of an immune system impairment and cancer development. The present study aimed to investigate the molecular mechanisms behind the tumour stimulatory effects of E171 in combination with azoxymethane (AOM)/dextran sodium sulphate (DSS) and compare these results to a recent study performed under the same conditions with E171 only. BALB/c mice underwent exposure to 5 mg/kgbw/day of E171 by gavage for 2, 7, 14, and 21 days. Whole genome mRNA microarray analyses on the distal colon were performed. The results show that E171 induced a downregulation of genes involved in the innate and adaptive immune system, suggesting impairment of this system. In addition, over time, signalling genes involved in colorectal cancer and other types of cancers were modulated. In relation to cancer development, effects potentially associated with oxidative stress were observed through modulation of genes related to antioxidant production. E171 affected genes involved in biotransformation of xenobiotics which can form reactive intermediates resulting in toxicological effects. These transcriptomics data reflect the early biological responses induced by E171 which precede tumour formation in an AOM/DSS mouse model.

Time course gene expression data in colon of mice after exposure to food-grade E171.

We investigated gene expression responses in BALB/c mice exposed by gavage to 5 mg/kg bw/day of E171 for 2, 7, 14 and 21 days. Food additive E171 (titanium dioxide) has been shown to induce oxidative stress and DNA damage in vitro as well as facilitating growth of colorectal tumours in vivo. Full genome expression changes of the colon of mice were investigated by using Agilent SurePrint G3 mouse Gene exp 60kv2 microarrays slides. The data presented in this DiB include all differentially expressed for each time point with EntrezGeneID, gene symbols, gene names and Log2FC as well as genes included in pathways after over-representation analysis in ConsensusPathDataBase. The functions of these genes in relation to the colon were described in our associated article (Proquin et al., 2017 in press) [1]. Raw and normalized gene expression data are available through NCBI GEO (GEO accession: GSE92563).

Oxidative stress in healthy pregnancy and preeclampsia is linked to chronic inflammation, iron status and vascular function.

BACKGROUND: During normal pregnancy, placental oxidative stress (OS) is present during all three trimesters and is necessary to obtain normal cell function. However, if OS reaches a certain level, pregnancy complications might arise. In preeclampsia (PE), a dangerous pregnancy specific hypertensive disorder, OS induced in the ischemic placenta causes a systemic inflammatory response and activates maternal endothelial cells. In this study, we aimed to quantify superoxide concentrations (as a measure of systemic OS) using electron paramagnetic resonance (EPR) and correlate them to markers of systemic inflammation, iron status and vascular function. METHODS: Fifty-nine women with a healthy pregnancy (HP), 10 non-pregnant controls (NP) and 28 PE patients (32±3.3weeks) were included. During HP, blood samples for superoxide, neutrophil to lymphocyte ratio (NLR), mean platelet volume (MPV) and iron status were taken at 10, 25 and 39 weeks. Vascular measurements for arterial stiffness (carotid-femoral pulse wave velocity (CF-PWV), augmentation index (AIx), augmentation Pressure (AP)) and microvascular endothelial function (reactive hyperemia index (RHI)) were performed at 35 weeks. In PE, all measurements were performed at diagnosis. CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) was used as spin probe for EPR, since the formed CM radical corresponds to the amount of superoxide. RESULTS: Superoxide concentration remains stable during pregnancy (p = 0.92), but is significantly higher compared to the NP controls (p<0.0001). At 25 weeks, there is a significant positive correlation between superoxide and ferritin concentration. (p = 0.04) In PE, superoxide, systemic inflammation and iron status are much higher compared to HP (all p<0.001). During HP, superoxide concentrations correlate significantly with arterial stiffness (all p<0.04), while in PE superoxide is significantly correlated to microvascular endothelial function (p = 0.03). CONCLUSIONS: During HP there is an increased but stable oxidative environment, which is correlated to ferritin concentration. If superoxide levels increase, there is an augmentation in arterial stiffness. In PE pregnancies, systemic inflammation and superoxide concentrations are higher and result in a deterioration of endothelial function. Together, these findings support the hypothesis that vascular function is directly linked to the amount of OS and that measurement of OS in combination with vascular function tests might be used in the prediction of PE.

Gene expression profiling in colon of mice exposed to food additive titanium dioxide (E171).

Dietary factors that may influence the risks of colorectal cancer, including specific supplements, are under investigation. Previous studies showed the capacity of food additive titanium dioxide (E171) to induce DNA damage in vitro and facilitate growth of colorectal tumours in vivo. This study aimed to investigate the molecular mechanisms behind these effects after E171 exposure. BALB/c mice were exposed by gavage to 5 mg/kgbw/day of E171 for 2, 7, 14, and 21 days. Transcriptome changes were studied by whole genome mRNA microarray analysis on the mice's distal colons. In addition, histopathological changes as well as a proliferation marker were analysed. The results showed significant gene expression changes in the olfactory/GPCR receptor family, oxidative stress, the immune system and of cancer related genes. Transcriptome analysis also identified genes that thus far have not been included in known biological pathways and can induce functional changes by interacting with other genes involved in different biological pathways. Histopathological analysis showed alteration and disruption in the normal structure of crypts inducing a hyperplastic epithelium. At cell proliferation level, no consistent increase over time was observed. These results may offer a mechanistic framework for the enhanced tumour growth after ingestion of E171 in BALB/c mice.

Ophthalmic acid as a read-out for hepatic glutathione metabolism in humans.

BACKGROUND AND AIM: Animal studies indicated that systemic ophthalmic acid (OPH) is a biomarker for hepatic glutathione (GSH) homeostasis, an important determinant of liver function. We aimed to clarify whether OPH levels can be used as a read-out for hepatic GSH homeostasis after paracetamol (APAP) challenges during pylorus-preserving pancreaticoduodenectomy (PPPD) or partial hepatectomy (PH). METHODS: Nineteen patients undergoing PPPD (n=7, control group) or PH (n=12) were included. APAP (1000 mg) was administered intravenously before resection (first challenge), and six and twelve hours later, with sequential blood sampling during this period. Arterial, hepatic and portal venous blood samples and liver biopsies were taken on three occasions during the first APAP challenge. Plasma and hepatic OPH and GSH levels were quantified, and venous-arterial differences were calculated to study hepatic release. RESULTS: Systemic GSH levels decreased during the course of the APAP challenge in both surgical groups, without notable change in OPH levels. Hepatic GSH and OPH content was not affected within ˜3 hours after administration of the first APAP dose in patients undergoing PPPD or PH. In this period, net release of OPH by the liver was observed only in patients undergoing PPPD. CONCLUSION: The drop in circulating GSH levels following APAP administrations, did not result in an increase in plasma OPH in both patients with an intact liver and in those undergoing liver resection. Hepatic content of GSH and OPH was not affected during the first APAP dose. It is uncertain whether hepatic GSH homeostasis was sufficiently challenged in the present study. RELEVANCE FOR PATIENTS: In the present study, plasma OPH seemed not useful as a marker for GSH depletion because APAP administration during liver surgery did not lead to (immediate) GSH depletion or increased OPH levels. Based on stable levels of hepatic GSH, OPH and thiyl radicals during surgery, standard APAP administration seems to be safe in a postoperative care program with regards to GSH homeostasis in this specific population. However, no general statements can be made on the basis of the current experiment, since GSH homeostasis and susceptibility to xenobiotic toxicity are influenced by several metabolic and genetic factors.

Pathogenetic role of endothelial nitric oxide synthase uncoupling during lung ischaemia-reperfusion injury.

OBJECTIVES: Ischaemia-reperfusion injury is a necessary part of organ transplantation and a key determinant of both acute and chronic graft failure. We have assessed the contribution of endothelial nitric oxide synthase (eNOS) and eNOS uncoupling to oxidative and nitrosative stress formation during lung ischaemia-reperfusion injury dependent on ischaemia time. METHODS: Forty eNOS wild-type mice (eNOS +/+ ) and 40 eNOS knock-out mice (eNOS -/- ) received either a sham thoracotomy or 60 or 90 min of ischaemia, followed by 0, 1 or 24 h of reperfusion. Lung tissue was analysed with electron spin resonance for NO production and reactive oxygen species content. Protein nitrosation, eNOS and eNOS uncoupling were determined using western blotting. In peripheral blood, arterial blood gases were taken and reactive oxygen species content was determined. RESULTS: eNOS +/+ mice had lower reactive oxygen species production in their peripheral circulation but worse blood gas values after 1 h of reperfusion. Lung tissue of eNOS -/- mice showed lower reactive oxygen species and NO production and lower protein nitrosation compared with wild-type mice. Longer ischaemia times result in more elaborate oxidative and nitrosative stress dependent on eNOS genotype. Structural eNOS uncoupling was present after 60 min of ischaemia but diminished after 90 min of ischaemia. CONCLUSIONS: eNOS uncoupling may contribute to lung ischaemia-reperfusion injury and inflammation. This ultimately leads to worse clinical outcome. Stabilizing eNOS may therefore be a new approach to extend pulmonary graft survival.