Assessing Oxidative Stress in Tumors by Measuring the Rate of Hyperpolarized [1-13^{13}C] Dehydroascorbic Acid Reduction Using 13^{13}C Magnetic Resonance Spectroscopy

Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species (ROS). ROS levels increase in response to chemotherapy and cell death while an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway (PPP) is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1-$^{13}$C]dehydroascorbic acid (DHA) reduction, which can be measured $\textit{in vivo}$ using non-invasive $^{13}$C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1-$^{13}$C]DHA reduction is increased in tumors that have been oxidatively pre-stressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in PPP flux, assessed using $^{13}$C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction does not depend only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-$^{13}$C]DHA can be used therefore to assess the capacity of tumor cells to resist oxidative stress in vivo. However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic.Work in K.M. Brindle’s laboratory is supported by a Cancer Research UK Programme grant (17242) and the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465). K.N. Timm was in receipt of MRC and Cancer Research UK studentships, B.W.C. Kennedy and P. Dzien Cancer Research UK studentships and F. Bulat a CRUK -EPSRC Imaging Centre imaging center studentship. I. Marco -Rius acknowledges the European Union Seventh Framework Programme (FP7/2007-2013) for support under the M arie Curie Initial Training Network METAFLUX (project number 264780)

Analysis of heterogeneity in T2_2-weighted MR images can differentiate pseudoprogression from progression in glioblastoma

$\textbf{Purpose}$ To develop an image analysis technique that distinguishes pseudoprogression from true progression by analyzing tumour heterogeneity in $T_2$-weighted images using topological descriptors of image heterogeneity called Minkowski functionals (MFs). $\textbf{Methods}$ Using a retrospective patient cohort ($n$ = 50), and blinded to treatment response outcome, unsupervised feature estimation was performed to investigate MFs for the presence of outliers, potential confounders, and sensitivity to treatment response. The progression and pseudoprogression groups were then unblinded and supervised feature selection was performed using MFs, size and signal intensity features. A support vector machine model was obtained and evaluated using a prospective test cohort. $\textbf{Results}$ The model gave a classification accuracy, using a combination of MFs and size features, of more than 85% in both retrospective and prospective datasets. A different feature selection method (Random Forest) and classifier (Lasso) gave the same results. Although not apparent to the reporting radiologist, the $T_2$-weighted hyperintensity phenotype of those patients with progression was heterogeneous, large and frond-like when compared to those with pseudoprogression. $\textbf{Conclusion}$ Analysis of heterogeneity, in $T_2$-weighted MR images, which are acquired routinely in the clinic, has the potential to detect an earlier treatment response allowing an early change in treatment strategy. Prospective validation of this technique in larger datasets is required.Funded by Medical Research Council/ Royal College of Radiologists (UK) Clinical Research Fellowship (G1000265); Cancer Research UK Clinical Research Fellowship; Addenbrookes Charitable Trust Award to TCB. Cancer Research UK Programme grant (C197/ A3514) to KMB

Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines

Combined bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) exert unexpected antileukaemic activities against acute myeloid leukaemia (AML) and these activities are associated with the generation of reactive oxygen species (ROS) within the tumor cells. Although the generation of ROS by these drugs is supported by preceding studies including our own, the interrelationship between the cellular effects of the drugs and ROS generation is not well understood. Here we report the use of NMR metabolomic profiling to further study the effect of BEZ and MPA on three AML cell lines and to shed light on the underlying mechanism of action. For this we focused on drug effects induced during the initial 24 hours of treatment prior to the onset of overt cellular responses and examined these in the context of basal differences in metabolic profiles between the cell lines. Despite their ultimately profound cellular effects, the early changes in metabolic profiles engendered by these drugs were less pronounced than the constitutive metabolic differences between cell types. Nonetheless, drug treatments engendered common metabolic changes, most markedly in the response to the combination of BEZ and MPA. These responses included changes to TCA cycle intermediates consistent with recently identified chemical actions of ROS. Notable amongst these was the conversion of α-ketoglutarate to succinate which was recapitulated by the treatment of cell extracts with exogenous hydrogen peroxide. These findings indicate that the actions of combined BEZ and MPA against AML cells are indeed mediated downstream of the generation of ROS rather than some hitherto unsuspected mechanism. Moreover, our findings demonstrate that metabolite profiles represent highly sensitive markers for genomic differences between cells and their responses to external stimuli. This opens new perspectives to use metabolic profiling as a tool to study the rational redeployment of drugs in new disease settings

Cardiovascular Applications of Hyperpolarized MRI

Many applications of MRI are limited by an inherently low sensitivity. Previous attempts to overcome this insensitivity have focused on the use of MRI systems with stronger magnetic fields. However, the gains that can be achieved in this way are relatively small and increasing the magnetic field invariably leads to greater technical challenges. More recently, the development of a range of techniques, which can be gathered under the umbrella term of “hyperpolarization,” has offered potential solutions to the low sensitivity. Hyperpolarization techniques have been demonstrated to temporarily increase the signal available in an MRI experiment by as much as 100,000-fold. This article outlines the main hyperpolarization techniques that have been proposed and explains how they can increase MRI signals. With particular emphasis on the emerging technique of dynamic nuclear polarization, the existing preclinical cardiovascular applications are reviewed and the potential for clinical translation is discussed

Properties of local interactions and their potential value in complementing genome-wide association studies

Local interactions between neighbouring SNPs are hypothesized to be able to capture variants missing from genome-wide association studies (GWAS) via haplotype effects but have not been thoroughly explored. We have used a new high-throughput analysis tool to probe this underexplored area through full pair-wise genome scans and conventional GWAS in diastolic and systolic blood pressure and six metabolic traits in the Northern Finland Birth Cohort 1966 (NFBC1966) and the Atherosclerosis Risk in Communities study cohort (ARIC). Genome-wide significant interactions were detected in ARIC for systolic blood pressure between PLEKHA7 (a known GWAS locus for blood pressure) and GPR180 (which plays a role in vascular remodelling), and also for triglycerides as local interactions within the 11q23.3 region (replicated significantly in NFBC1966), which notably harbours several loci (BUD13, ZNF259 and APOA5) contributing to triglyceride levels. Tests of the local interactions within the 11q23.3 region conditional on the top GWAS signal suggested the presence of two independent functional variants, each with supportive evidence for their roles in gene regulation. Local interactions captured 9 additional GWAS loci identified in this study (3 significantly replicated) and 73 from previous GWAS (24 in the eight traits and 49 in related traits). We conclude that the detection of local interactions requires adequate SNP coverage of the genome and that such interactions are only likely to be detectable between SNPs in low linkage disequilibrium. Analysing local interactions is a potentially valuable complement to GWAS and can provide new insights into the biology underlying variation in complex traits

A reactivity-selectivity study of the Friedel-Crafts acetylation of 3,3′-dimethylbiphenyl and the oxidation of the acetyl derivatives

<p>Abstract</p> <p>Background</p> <p>Friedel-Crafts acetylation is an important route to aromatic ketones, in research laboratories and in industry. The acetyl derivatives of 3,3′-dimethylbiphenyl (3,3′-dmbp) have applications in the field of liquid crystals and polymers and may be oxidized to the dicarboxylic acids and derivatives that are of interest in cancer treatment.</p> <p>Findings</p> <p>The effect of solvent and temperature on the selectivity of monoacetylation of 3,3’-dmbp by the Perrier addition procedure was studied using stoichiometric amounts of reagents. 4-Ac-3,3′-dmbp was formed almost quantitatively in boiling 1,2-dichloroethane and this is almost twice the yield hitherto reported. Using instead a molar ratio of substrate:AcCl:AlCl₃ equal to 1:4:4 or 1:6:6 in boiling 1,2-dichloroethane, acetylation afforded 4,4′- and 4,6′-diacetyl-3,3′-dmbp in a total yield close to 100%. The acetyl derivatives were subsequently converted to the carboxylic acids by hypochlorite oxidation. The relative stabilities of the isomeric products and the corresponding σ-complexes were studied by DFT calculations and the data indicated that mono- and diacetylation followed different mechanisms.</p> <p>Conclusions</p> <p>Friedel-Crafts acetylation of 3,3′-dmbp using the Perrier addition procedure in boiling 1,2-dichloroethane was found to be superior to other recipes. The discrimination against the 6-acetyl derivative during monoacetylation seems to reflect a mechanism including an AcCl:AlCl₃ complex or larger agglomerates as the electrophile, whereas the less selective diacetylations of the deactivated 4-Ac-3,3′-dmbp are suggested to include the acetyl cation as the electrophile. The DFT data also showed that complexation of intermediates and products with AlCl₃ does not seem to be important in determining the mechanism.</p

Genome-wide characterization of circulating metabolic biomarkers

Genome-wide association analyses using high-throughput metabolomics platforms have led to novel insights into the biology of human metabolism. This detailed knowledge of the genetic determinants of systemic metabolism has been pivotal for uncovering how genetic pathways influence biological mechanisms and complex diseases. Here we present a genome-wide association study for 233 circulating metabolic traits quantified by nuclear magnetic resonance spectroscopy in up to 136,016 participants from 33 cohorts. We identify more than 400 independent loci and assign probable causal genes at two-thirds of these using manual curation of plausible biological candidates. We highlight the importance of sample and participant characteristics that can have significant effects on genetic associations. We use detailed metabolic profiling of lipoprotein- and lipid-associated variants to better characterize how known lipid loci and novel loci affect lipoprotein metabolism at a granular level. We demonstrate the translational utility of comprehensively phenotyped molecular data, characterizing the metabolic associations of intrahepatic cholestasis of pregnancy. Finally, we observe substantial genetic pleiotropy for multiple metabolic pathways and illustrate the importance of careful instrument selection in Mendelian randomization analysis, revealing a putative causal relationship between acetone and hypertension. Our publicly available results provide a foundational resource for the community to examine the role of metabolism across diverse diseases

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention