Ischemia-Induced Metabolic Patterns Associate With Kidney Function During Normothermic Kidney Perfusion, a Preclinical Study.

OBJECTIVE: To investigate if ischemia alters donor kidney metabolism and whether these changes associate with organ function. SUMMARY BACKGROUND DATA: An unmet need in kidney transplantation is the ability to predict post-transplant organ function before transplantation. Key to such viability testing is a profound understanding of the organ's complex biochemistry and how ischemia, inevitable during the transplantation process, influences this. METHODS: First, metabolic changes in glucose, lactate and 20 amino acids induced by no, 1h of warm, or 22h of cold ischemia were investigated during 4h perfusion of pig kidneys with autologous whole blood (n=6/group), simulating the ischemia-reperfusion phase of transplantation. Next, we confirmed similar metabolic changes during normothermic preservation of pig (n=3/group; n=4 for cold ischemia) and discarded human kidneys (n=6) perfused with a red-blood cell based perfusate. RESULTS: At 2h of perfusion with autologous whole blood, abundances of 17/20 amino acids were significantly different between groups, reflecting the type of ischemia. Amino acid changes at 15 min and 2h of perfusion correlated with future kidney function during perfusion. Similar metabolic patterns were observed during perfusion preservation of pig and discarded human donor kidneys, suggesting an opportunity to assess kidney viability before transplantation. CONCLUSIONS: Perfusate metabolite changes during normothermic kidney perfusion represent a unique non-invasive opportunity to assess graft viability. These findings now need validation in transplant studies.

TraVis Pies: A Guide for Stable Isotope Metabolomics Interpretation Using an Intuitive Visualization.

Tracer metabolomics is a powerful technology for the biomedical community to study and understand disease-inflicted metabolic mechanisms. However, the interpretation of tracer metabolomics results is highly technical, as the metabolites' abundances, tracer incorporation and positions on the metabolic map all must be jointly interpreted. The field is currently lacking a structured approach to help less experienced researchers start the interpretation of tracer metabolomics datasets. We propose an approach using an intuitive visualization concept aided by a novel open-source tool, and provide guidelines on how researchers can apply the approach and the visualization tool to their own datasets. Using a showcase experiment, we demonstrate that the visualization approach leads to an intuitive interpretation that can ease researchers into understanding their tracer metabolomics data.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Using Large-Scale NO2 Data from Citizen Science for Air-Quality Compliance and Policy Support.

Citizen science projects that monitor air quality have recently drastically expanded in scale. Projects involving thousands of citizens generate spatially dense data sets using low-cost passive samplers for nitrogen dioxide (NO2), which complement data from the sparse reference network operated by environmental agencies. However, there is a critical bottleneck in using these citizen-derived data sets for air-quality policy. The monitoring effort typically lasts only a few weeks, while long-term air-quality guidelines are based on annual-averaged concentrations that are not affected by seasonal fluctuations in air quality. Here, we describe a statistical model approach to reliably transform passive sampler NO2 data from multiweek averages to annual-averaged values. The predictive model is trained with data from reference stations that are limited in number but provide full temporal coverage and is subsequently applied to the one-off data set recorded by the spatially extensive network of passive samplers. We verify the assumptions underlying the model procedure and demonstrate that model uncertainty complies with the EU-quality objectives for air-quality monitoring. Our approach allows a considerable cost optimization of passive sampler campaigns and removes a critical bottleneck for citizen-derived data to be used for compliance checking and air-quality policy use.

Exposure to Environmental Pollutants and Their Association with Biomarkers of Aging: A Multipollutant Approach.

Mitochondrial DNA (mtDNA) content and telomere length are putative aging biomarkers and are sensitive to environmental stressors, including pollutants. Our objective was to identify, from a set of environmental exposures, which exposure is associated with leukocyte mtDNA content and telomere length in adults. This study includes 175 adults from 50 to 65 years old from the cross-sectional Flemish Environment and Health study, of whom leukocyte telomere length and mtDNA content were determined using qPCR. The levels of exposure of seven metals, 11 organohalogens, and four perfluorinated compounds (PFHxS, PFNA, PFOA, PFOS) were measured. We performed sparse partial least-squares regression analyses followed by ordinary least-squares regression to assess the multipollutant associations. While accounting for possible confounders and coexposures, we identified that urinary cadmium (6.52%, 95% confidence interval, 1.06, 12.28), serum hexachlorobenzene (2.89%, 018, 5.68), and perfluorooctanesulfonic acid (11.38%, 5.97, 17.08) exposure were positively associated ( p < 0.05) with mtDNA content, while urinary copper (-9.88%, -14.82, -4.66) and serum perfluorohexanesulfonic acid (-4.75%, -8.79, -0.54) exposure were inversely associated with mtDNA content. Urinary antimony (2.69%, 0.45, 4.99) and mercury (1.91%, 0.42, 3.43) exposure were positively associated with leukocyte telomere length, while urinary copper (-3.52%, -6.60, -0.34) and serum perfluorooctanesulfonic acid (-3.64%, -6.60, -0.60) showed an inverse association. Our findings support the hypothesis that environmental pollutants interact with molecular hallmarks of aging.

Neonatal exposure to environmental pollutants and placental mitochondrial DNA content: A multi-pollutant approach.

BACKGROUND: Placental mitochondrial DNA (mtDNA) content can be indicative of oxidative damage to the placenta during fetal development and is responsive to external stressors. In utero exposure to environmental pollutants that may influence placental mtDNA needs further exploration. OBJECTIVES: We evaluated if placental mtDNA content is altered by environmental pollution in newborns and identified pollutants independently associated to alterations in placental mtDNA content. METHODS: mtDNA content was measured in placental tissue of 233 newborns. Four perfluoroalkyl compounds and nine organochlorine compounds were quantified in cord blood plasma samples and six toxic metals in whole cord blood. We first applied a LASSO (least absolute shrinkage and selection operator) penalized regression model to identify independent associations between environmental pollutants and placental mtDNA content, without penalization of several covariates. Then adjusted estimates were obtained using an ordinary least squares (OLS) regression model evaluating the pollutants' association with placental mtDNA content, adjusted for several covariates. RESULTS: Based on LASSO penalized regression, oxychlordane, p,p'-dichlorodiphenyldichloroethylene, ß-hexachlorocyclohexane, perfluorononanoic acid, arsenic, cadmium and thallium were identified to be independently associated with placental mtDNA content. The OLS model showed a higher placental mtDNA content of 2.71% (95% CI: 0.3 to 5.2%; p=0.03) and 1.41% (0.1 to 2.8%, p=0.04) for a 25% concentration increase of respectively cord blood ß-hexachlorocyclohexane and arsenic. For a 25% concentration increase of cord blood thallium, a 4.88% lower placental mtDNA content (95% CI: -9.1 to -0.5%, p=0.03) was observed. CONCLUSION: In a multi-pollutant approach, low fetal exposure levels of environmental organic and inorganic pollutants might compromise placental mitochondrial function as exemplified in this study by alterations in mtDNA content.

Metals, hormones and sexual maturation in Flemish adolescents in three cross-sectional studies (2002-2015).

Sex hormone levels and timing of sexual maturation are considered important markers for health status of adolescents in puberty, and previous research suggests they might be influenced by metal exposure. In three campaigns of the Flemish Environment and Health Study (FLEHS I 2002-2006; FLEHS II 2007-2011 and FLEHS III 2012-2015), data were collected on internal exposure to metals (Cd, Cu, Pb, Cr, Mn, Tl, Ni, Sb, Hg, As and As species) and sexual maturation in 2671 14-15years old adolescents. All metals were measured in blood and/or urine, except total- and methylmercury which were measured in hair samples. Sex hormone levels were measured in blood serum of adolescent males of the cohorts of FLEHS I and FLESH II. The use of a uniform methodology in successive campaigns allows to confirm associations between exposure and health in different cohorts and over time. Furthermore, mathematical and statistical density correction methods using creatinine or specific gravity were tested for urinary markers. Significant associations between sex hormones and maturity markers were observed in the FLEHS I and II campaigns, when both were assessed together. Regardless of the applied correction method, creatinine correction systematically introduced bias due to associations of creatinine with sex hormones and maturation markers, especially in adolescent males, while this is not the case for specific gravity. A series of exposure-response associations were found, but several involving Cd, Pb, As, Tl and Cu persisted in different FLEHS campaigns. The effects of Pb and Cu on luteinizing hormone, (free) testosterone, (free) oestradiol and maturation support a xenoestrogenic agonistic action on the feedback of oestradiol to the hypothalamus-pituitary-gonadal axis. Our results suggest that specific care should be taken when selecting urine density correction for investigating associations with hormonal and maturation markers in adolescent males. Furthermore, the possibility of xenoestrogenic effects of certain metals in environmentally exposed adolescents warrants further investigation.

Three cycles of human biomonitoring in Flanders - Time trends observed in the Flemish Environment and Health Study.

To follow time trends in exposure to environmental chemicals, three successive campaigns of the Flemish Environment and Health Study (FLEHS) have recruited and sampled in total 5825 participants between 2002 and 2014. Cord samples from newborns, urine and blood samples from 14 to 15 years old adolescents and from adults between 50 and 65 years old were analysed in geographical representative samples of the Flemish population. The data of the different campaigns were considered per age group and per biomarker after adjustment for predefined covariates to take into account differences in characteristics of the study populations over time. Geometric means were calculated. Multiple linear regression was used to evaluate time trends. The concentration of serum biomarkers for persistent organic pollutants (POPs), such as marker polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p'-DDE), the major metabolite of dichlorodiphenyltrichloroethane (DDT), and hexachlorobenzene (HCB) expressed per g lipid, decreased significantly with time. The levels of DDE in all age groups and those of PCBs in cord and adolescent serum samples were almost halved in a time period of ten years. HCB levels were reduced by a factor of 4 in adolescents and in adults. Mean serum concentrations of the more recently regulated perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were significantly lower in cord samples of 2013 compared to samples of 2007. The decline was more pronounced for PFOS than for PFOA. In the same period, mean metabolite levels of di-2-ethylhexyl phthalate (DEHP) and of di-n-butyl phthalate (DBP) decreased significantly in urine samples of adolescents with sharper declines for DEHP than for DBP. Cadmium and lead levels in cord and adolescent blood samples were significantly lower in the recent campaigns than 10 years before. Also the mean urinary cadmium level in adults was 35% lower compared to adult samples of 2002. Such favourable trends were not observed for arsenic and thallium measured in cord blood. Similar, the concentrations of 1-hydroxypyrene, a marker for exposure to polycyclic aromatic hydrocarbons (PAHs), was not lower in urine from adolescents sampled in 2013 compared to 2003. In contrast, concentrations of t,t'-muconic acid, a marker of benzene exposure, showed clearly reduced levels. The FLEHS program shows that concentrations of well-regulated chemicals especially traditional POPs and cadmium and lead are decreasing in the population of Flanders. Response to regulatory measures seems to happen rapid, since concentrations in humans of specific regulated perfluorinated compounds and phthalates were significantly reduced in five years time. Biomarker concentrations for arsenic, thallium, and polyaromatic hydrocarbons are not decreasing in this time span and further follow up is warranted.

Investigating unmetabolized polycyclic aromatic hydrocarbons in adolescents' urine as biomarkers of environmental exposure.

Polycyclic aromatic hydrocarbons (PAHs) are of interest to human biomonitoring studies due to their carcinogenic potential. Traditionally metabolites of these compounds, like 1-hydroxypyrene, are monitored in urine, but recent methods allow the determination of the parent compounds in urine, which give additional information regarding sources and toxicity of PAHs. In order to assess the feasibility of incorporating these methods in a human biomonitoring study, the 16 USEPA parent PAHs were determined in 20 urine samples. These samples were obtained from 10 boys and 10 girls aged 14-16 years, participating in the third Flemish Environment and Health Study (Flanders, Belgium). Of these 16 parent PAHs, nine could be determined in more than 95% of the samples and three (including benzo(a)pyrene) in more than 50%. Several correlations were found between different PAHs, but not between pyrene and its metabolite 1-hydroxypyrene. Diagnostic PAH ratios in urine and air samples pointed towards combustion sources and are in line with the ratios in environmental samples. Benzo(a)pyrene, naphthalene and fluorene have the highest carcinogenic potential in our cohort, when using toxic equivalency factors. Some associations between PAH congeners and determinants of exposure were found, while fluorene and acenaphthylene were positively associated with thyroid hormone levels and benzo(a)pyrene showed a positive correlation with DNA damage by comet assay. These results confirm that parent PAHs in urine are useful as biomarkers of exposure in biomonitoring studies.

A novel method for the determination of dissolved methylmercury concentrations using diffusive gradients in thin films technique.

A novel DGT probe and analysis protocol were developed for the determination of MeHg concentrations in aquatic system. The DGT probe consisted of an agarose (AG) gel as the diffusive hydrogel and a 3-mercaptoproply functionalised silica resin gel as the resin gel. The polyacrylamide (PA) hydrogel which is commonly used in DGT probes to assess trace metal concentrations in aquatic system appeared to be unsuitable for the determination of MeHg. The affinity of the PA hydrogel for MeHg is very high reducing its accumulation by the resin. In contrast, the AG hydrogel presents a by far lower affinity towards MeHg, which makes it suitable as diffusive layer in a DGT probe for MeHg determinations. Two extraction procedures to liberate MeHg from the resin were studied: one is involving thiourea as complexing agent, the other a simple acidic extraction. The extraction step was followed by an ethylation reaction of the liberated MeHg to determine low concentrations of MeHg species by Headspace-Gas Chromatography-Atomic Fluorescence (HS-GC-AFS). With the thiourea extraction method the recovery of the adsorbed MeHg compounds was extremely low while the recovery with the acid extraction method was 100%. The reliability of the novel DGT probe and analysis protocol was studied. A linear dependency between the amount of MeHg accumulated on the resin gel and both the deployment time and the gel thickness were demonstrated. From those experiments a diffusion coefficient of MeHg in AG gel was determined: 5.1±0.20×10(-6) cm(2) s(-1). Additional experiments showed that the new DGT method can be used in most natural waters independent of the ionic strength and within a pH range of 3-8.