Clementina Mesaros

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.

Targeted quantitative analysis of eicosanoid lipids in biological samples using liquid chromatography-tandem mass spectrometry

The eicosanoids are a large family of arachidonic acid oxidation products that contain 20 carbon atoms. Cyclooxygenase (COX)-derived eicosanoids have important roles as autacoids involved in the regulation of cardiovascular function and tumor progression. Lipoxygenase (LO)-derived eicosanoids have been implicated as important mediators of inflammation, asthma, cardiovascular disease and cancer. Cytochrome P-450 (P450)-derived eicosanoids are both vasodilators and vasoconstrictors. There is intense interest in the analysis of reactive oxygen species (ROS)-derived isoprostanes (isoPs) because of their utility as biomarkers of oxidative stress. Enzymatic pathways of eicosanoid formation are regioselective and enantioselective, whereas ROS-mediated eicosanoid formation proceeds with no stereoselectivity. Many of the eicosanoids are also present in only pM concentrations in biological fluids. This presents a formidable analytical challenge because methodology is required that can separate enantiomers and diastereomers with high sensitivity and specificity. However, the discovery of atmospheric pressure ionization (API)/MS methodology of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron capture (EC) APCI has revolutionized our ability to analyze endogenous eicosanoids. LC separations of eicosanoids can now be readily coupled with API ionization, collision induced dissociation (CID) and tandem MS (MS/MS). This makes it possible to efficiently conduct targeted eicosanoid analyses using LC-multiple reaction motoring (MRM)/MS. Several examples of targeted eicosanoid lipid analysis using conventional LC-ESI/MS have been discussed and some new data on the analysis of eicosanoids using chiral LC-ECAPCI/MS has been presented.

Evidence for Intramyocardial Disruption of Lipid Metabolism and Increased Myocardial Ketone Utilization in Advanced Human Heart Failure.

Background— The failing human heart is characterized by metabolic abnormalities, but these defects remains incompletely understood. In animal models of heart failure there is a switch from a predominance of fatty acid utilization to the more oxygen-sparing carbohydrate metabolism. Recent studies have reported decreases in myocardial lipid content, but the inclusion of diabetic and nondiabetic patients obscures the distinction of adaptations to metabolic derangements from adaptations to heart failure per se. Methods and Results— We performed both unbiased and targeted myocardial lipid surveys using liquid chromatography-mass spectroscopy in nondiabetic, lean, predominantly nonischemic, advanced heart failure patients at the time of heart transplantation or left ventricular assist device implantation. We identified significantly decreased concentrations of the majority of myocardial lipid intermediates, including long-chain acylcarnitines, the primary subset of energetic lipid substrate for mitochondrial fatty acid oxidation. We report for the first time significantly reduced levels of intermediate and anaplerotic acyl-coenzyme A (CoA) species incorporated into the Krebs cycle, whereas the myocardial concentration of acetyl-CoA was significantly increased in end-stage heart failure. In contrast, we observed an increased abundance of ketogenic &bgr;-hydroxybutyryl-CoA, in association with increased myocardial utilization of &bgr;-hydroxybutyrate. We observed a significant increase in the expression of the gene encoding succinyl-CoA:3-oxoacid-CoA transferase, the rate-limiting enzyme for myocardial oxidation of &bgr;-hydroxybutyrate and acetoacetate. Conclusions— These findings indicate increased ketone utilization in the severely failing human heart independent of diabetes mellitus, and they support the role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart.

CYP3A4 Mediates Growth of Estrogen Receptor-positive Breast Cancer Cells in Part by Inducing Nuclear Translocation of Phospho-Stat3 through Biosynthesis of (±)-14,15-Epoxyeicosatrienoic Acid (EET)

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G2/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ∼2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.

Antioxidant protection by PECAM-targeted delivery of a novel NADPH-oxidase inhibitor to the endothelium in vitro and in vivo

Oxidant stress caused by pathological elevation of reactive oxygen species (ROS) production in the endothelial cells lining the vascular lumen is an important component of many vascular and pulmonary disease conditions. NADPH oxidase (NOX) activated by pathological mediators including angiotensin and cytokines is a major source of endothelial ROS. In order to intercept this pathological pathway, we have encapsulated an indirect NOX inhibitor, MJ33, into immunoliposomes (Ab-MJ33/IL) targeted to endothelial marker platelet endothelial cell adhesion molecule (PECAM-1). Ab-MJ33/IL, but not control IgG-MJ33/IL are specifically bound to endothelium and attenuated angiotensin-induced ROS production in vitro and in vivo. Additionally, Ab-MJ33/IL inhibited endothelial expression of the inflammatory marker vascular cell adhesion molecule (VCAM) in cells and animals challenged with the cytokine TNF. Furthermore, Ab-MJ33/IL alleviated pathological disruption of endothelial permeability barrier function in cells exposed to vascular endothelial growth factor (VEGF) and in the lungs of mice challenged with lipopolysaccharide (LPS). Of note, the latter beneficial effect has been achieved both by prophylactic and therapeutic injection of Ab-MJ33/IL in animals. Therefore, specific suppression of ROS production by NOX in endothelium, attainable by Ab-MJ33/IL targeting, may help deciphering mechanisms of vascular oxidative stress and inflammation, and potentially improve treatment of these conditions.

Stable Isotope Labeling by Essential Nutrients in Cell Culture for Preparation of Labeled Coenzyme A and Its Thioesters

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [13C315N1]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [13C315N1]-pantothenate after three passages of the murine cells in culture. Charcoal−dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse Lung

1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02–0.5 µmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67–87% of the injected dose for i.t. and 23–42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24–72 hours. Mice treated with MJ33 at 12.5–25 µmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 µmol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 µmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation.

Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography-tandem mass spectrometry

An ultrasensitive stable isotope dilution liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1fg on column, and was 10fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200pg/mL (5-2000pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7pg/mL) were very similar to those obtained by highly sensitive gas chromatography-mass spectrometry (GC-MS) methodology. However, the level obtained in serum from older men (mean 9.5pg/mL) was lower than has been reported previously by both GC-MS and LC-MS procedures. The total (unconjugated+conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases.

Prediction of Postoperative Recurrence-Free Survival in Non-small Cell Lung Cancer by Using an Internationally Validated Gene Expression Model

Purpose: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non–small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations. Experimental Design: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts. Results: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010). Conclusion: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients. Clin Cancer Res; 17(9); 2934–46. ©2011 AACR.

Secondhand smoke inhibits both Cl - and K + conductances in normal human bronchial epithelial cells

Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure.