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1.  Stable Isotope- and Mass Spectrometry-based Metabolomics as Tools in Drug Metabolism: A Study Expanding Tempol Pharmacology 
Journal of proteome research  2013;12(3):1369-1376.
The application of mass spectrometry-based metabolomics in the field of drug metabolism has yielded important insights not only into the metabolic routes of drugs but has provided unbiased, global perspectives of the endogenous metabolome that can be useful for identifying biomarkers associated with mechanism of action, efficacy, and toxicity. In this report, a stable isotope- and mass spectrometry-based metabolomics approach that captures both drug metabolism and changes in the endogenous metabolome in a single experiment is described. Here the antioxidant drug tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was chosen because its mechanism of action is not completely understood and its metabolic fate has not been studied extensively. Furthermore, its small size (MW = 172.2) and chemical composition (C9H18NO2) makes it challenging to distinguish from endogenous metabolites. In this study, mice were dosed with tempol or deuterated tempol (C9D17HNO2) and their urine profiled using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Principal component analysis of the urinary metabolomics data generated a Y-shaped scatter plot containing drug metabolites (protonated and deuterated) that were clearly distinct from the endogenous metabolites. Ten tempol drug metabolites, including eight novel metabolites, were identified. Phase II metabolism was the major metabolic pathway of tempol in vivo, including glucuronidation and glucosidation. Urinary endogenous metabolites significantly elevated by tempol treatment included 2,8-dihydroxyquinoline (8.0-fold, P<0.05) and 2,8-dihydroxyquinoline-β-D-glucuronide (6.8-fold, P<0.05). Urinary endogenous metabolites significantly attenuated by tempol treatment including pantothenic acid (1.3-fold, P<0.05) and isobutrylcarnitine (5.3-fold, P<0.01). This study underscores the power of a stable isotope- and mass spectrometry-based metabolomics in expanding the view of drug pharmacology.
doi:10.1021/pr301023x
PMCID: PMC3594779  PMID: 23301521
Tempol; Stable Isotope; Metabolomics; Mass spectrometry; Drug Metabolism
2.  Metabolomics: An Essential Tool to Understand the Function of Peroxisome Proliferator–Activated Receptor Alpha 
Toxicologic pathology  2012;41(2):410-418.
The peroxisome proliferator–activated receptor (PPAR) family of nuclear hormone transcription factors (PPARα, PPARβ/δ, and PPARγ) is regulated by a wide array of ligands including natural and synthetic chemicals. PPARs have important roles in control of energy metabolism and are known to influence inflammation, differentiation, carcinogenesis, and chemical toxicity. As such, PPARs have been targeted as therapy for common disorders such as cancer, metabolic syndrome, obesity, and diabetes. The recent application of metabolomics, or the global, unbiased measurement of small molecules found in biofluids, or extracts from cells, tissues, or organisms, has advanced our understanding of the varied and important roles that the PPARs have in normal physiology as well as in pathophysiological processes. Continued development and refinement of analytical platforms, and the application of new bioinformatics strategies, have accelerated the widespread use of metabolomics and have allowed further integration of small molecules into systems biology. Recent studies using metabolomics to understand PPARα function, as well as to identify PPARα biomarkers associated with drug efficacy/toxicity and drug-induced liver injury, will be discussed.
doi:10.1177/0192623312466960
PMCID: PMC3690496  PMID: 23197196
metabolomics; liver; PPARα; chromatography; mass spectrometry
3.  Metabolomics identifies an inflammatory cascade involved in dioxin- and diet-induced steatohepatitis 
Cell metabolism  2012;16(5):634-644.
Summary
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent environmentally toxic compounds. Serum metabolomics identified azelaic acid-mono esters as significantly increased metabolites after TCDD treatment, due to down-regulation of hepatic carboxylesterase 3 (CES3, also known as triglyceride hydrolase) expression in an arylhydrocarbon receptor (AhR)-dependent manner in mice. The decreased CES3 expression was accomplished by TCDD-stimulated TGFβ-SMAD3 and IL6-STAT3 signaling, but not by direct AhR signaling. Methionine- and choline-deficient (MCD) diet-treated mice also showed enhanced serum azelaic acid-mono ester levels following attenuation of hepatic CES3 expression, while db/db mice did not, thus suggesting an association with steatohepatitis. Forced expression of CES3 reversed serum azelaic acid-mono ester/azelaic acid ratios and hepatic TGFβ mRNA levels in TCDD- and MCD diet-treated mice and ameliorated steatohepatitis induced by MCD diet. These results support the view that azelaic acid-mono esters are possible indicators of TCDD exposure and steatohepatitis, and suggest a link between CES3, TGFβ, and steatohepatitis.
doi:10.1016/j.cmet.2012.10.006
PMCID: PMC3496181  PMID: 23140643
4.  Biochemistry and Physiology of the β Class Carbonic Anhydrase (Cpb) from Clostridium perfringens Strain 13 
Journal of Bacteriology  2013;195(10):2262-2269.
The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the β class, the first β class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO2 and 4.8 × 106 s−1 M−1, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO2. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO2 in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.
doi:10.1128/JB.02288-12
PMCID: PMC3650550  PMID: 23475974
5.  An Objective Method to Optimize the MR Sequence Set for Plaque Classification in Carotid Vessel Wall Images Using Automated Image Segmentation 
PLoS ONE  2013;8(10):e78492.
A typical MR imaging protocol to study the status of atherosclerosis in the carotid artery consists of the application of multiple MR sequences. Since scanner time is limited, a balance has to be reached between the duration of the applied MR protocol and the quantity and quality of the resulting images which are needed to assess the disease. In this study an objective method to optimize the MR sequence set for classification of soft plaque in vessel wall images of the carotid artery using automated image segmentation was developed. The automated method employs statistical pattern recognition techniques and was developed based on an extensive set of MR contrast weightings and corresponding manual segmentations of the vessel wall and soft plaque components, which were validated by histological sections. Evaluation of the results from nine contrast weightings showed the tradeoff between scan duration and automated image segmentation performance. For our dataset the best segmentation performance was achieved by selecting five contrast weightings. Similar performance was achieved with a set of three contrast weightings, which resulted in a reduction of scan time by more than 60%. The presented approach can help others to optimize MR imaging protocols by investigating the tradeoff between scan duration and automated image segmentation performance possibly leading to shorter scanning times and better image interpretation. This approach can potentially also be applied to other research fields focusing on different diseases and anatomical regions.
doi:10.1371/journal.pone.0078492
PMCID: PMC3806831  PMID: 24194941
6.  Identification of serum insulin-like growth factor binding protein 1 as diagnostic biomarker for early-stage alcohol-induced liver disease 
Background
Alcohol consumption is a major cause of liver disease in humans. The use and monitoring of biomarkers associated with early, pre-clinical stages of alcohol-induced liver disease (pre-ALD) could facilitate diagnosis and treatment, leading to improved outcomes.
Methods
We investigated the pathological, transcriptomic and protein changes in early stages of pre-ALD in mice fed the Lieber-Decarli liquid diet with or without alcohol for four months to identify biomarkers for the early stage of alcohol induced liver injury. Mice were sampled after 1, 2 and 4 months treatment.
Results
Pathological examination revealed a modest increase in fatty liver changes in alcohol-treated mice. Transcriptomics revealed gene alterations at all time points. Most notably, the Igfbp1 (Insulin-Like Growth Factor Binding Protein 1) was selected as the best candidate gene for early detection of liver damage since it showed early and continuously enhanced induction during the treatment course. Consistent with the microarray data, both Igfbp1mRNA expression in the liver tissue and the IGFBP1 serum protein levels showed progressive and significant increases over the course of pre-ALD development.
Conclusions
The results suggest that in conjunction with other tests, serum IGFBPI protein could provide an easily measured biomarker for early detection of alcohol-induced liver injury in humans.
doi:10.1186/1479-5876-11-266
PMCID: PMC4016206  PMID: 24152801
Alcohol-induced liver disease; Transcriptomics; Biomarker; Igfbp1; IGFBP1 protein
7.  Role of the Ah Receptor in Homeostatic Control of Fatty Acid Synthesis in the Liver 
Toxicological Sciences  2012;129(2):372-379.
We have previously demonstrated a role for the aryl hydrocarbon receptor (AHR) in the attenuation of the cholesterol biosynthesis pathway. This regulation did not require that the AHR binds to its cognate response element. Based on these observations and other reports depicting a role for AHR in lipid metabolism, we chose to investigate the involvement of the receptor in the regulation of the fatty acid synthesis pathway in mice and humans. For this purpose, C57BL/6J, liver-specific transgenic DRE-binding mutant AhR (A78D-Ahr Ttr Cre Alb Ahr fx/fx) and Cre Alb Ahr fx/fx mice were treated with an AHR ligand, and hepatic mRNA expression levels of key fatty acid genes (e.g., Acaca, Fasn, Scd1) were measured. The basal levels of those genes were also compared between C57BL6/J and hepatic AHR-deficient mice, as well as between Ah b and Ah d congenic mice. To extend these results to humans, fatty acid gene expression in human cells were compared with AHR-silenced cells. In addition, primary human hepatocytes were treated with an AHR ligand to assess alterations in gene expression and fatty acid synthesis. These studies indicated that the AHR constitutively attenuates the expression of key fatty acid synthesis genes in the absence of binding to its cognate response element. In addition, activation of AHR led to further repression of the expression of these genes and a decrease in overall fatty acid synthesis and secretion in human hepatocytes. Based on our results, we can conclude that increased AHR activity represses fatty acid synthesis, suggesting it may be a future therapeutic target.
doi:10.1093/toxsci/kfs204
PMCID: PMC3491957  PMID: 22696238
AHR; Ah receptor; fatty acids; DRE
8.  Radiation Metabolomics. 5. Identification of Urinary Biomarkers of Ionizing Radiation Exposure in Nonhuman Primates by Mass Spectrometry-Based Metabolomics 
Radiation research  2012;178(4):328-340.
Mass spectrometry-based metabolomics has previously demonstrated utility for identifying biomarkers of ionizing radiation exposure in cellular, mouse and rat in vivo radiation models. To provide a valuable link from small laboratory rodents to humans, γ-radiation-induced urinary biomarkers were investigated using a nonhuman primate total-body-irradiation model. Mass spectrometry-based metabolomics approaches were applied to determine whether biomarkers could be identified, as well as the previously discovered rodent biomarkers of γ radiation. Ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry analysis was carried out on a time course of clean-catch urine samples collected from nonhuman primates (n = 6 per cohort) exposed to sham, 1.0, 3.5, 6.5 or 8.5 Gy doses of 60Co γ ray (~0.55 Gy/min) ionizing radiation. By multivariate data analysis, 13 biomarkers of radiation were discovered: N-acetyltaurine, isethionic acid, taurine, xanthine, hypoxanthine, uric acid, creatine, creatinine, tyrosol sulfate, 3-hydroxytyrosol sulfate, tyramine sulfate, N-acetylserotonin sulfate, and adipic acid. N-Acetyltaurine, isethionic acid, and taurine had previously been identified in rats, and taurine and xanthine in mice after ionizing radiation exposure. Mass spectrometry-based metabolomics has thus successfully revealed and verified urinary biomarkers of ionizing radiation exposure in the nonhuman primate for the first time, which indicates possible mechanisms for ionizing radiation injury.
PMCID: PMC3498937  PMID: 22954391
10.  Metabolomics Reveals the Heterogeneous Secretome of Two Entomopathogenic Fungi to Ex Vivo Cultured Insect Tissues 
PLoS ONE  2013;8(8):e70609.
Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.
doi:10.1371/journal.pone.0070609
PMCID: PMC3734240  PMID: 23940603
11.  PPARα-dependent Induction of Uncoupling Protein 2 Protects Against Acetaminophen-Induced Liver Toxicity 
Hepatology (Baltimore, Md.)  2012;56(1):281-290.
Acetaminophen (APAP) overdose causes acute liver failure in humans and rodents due in part to the destruction of mitochondria as a result of increased oxidative stress followed by hepatocellular necrosis. Activation of the peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor superfamily that controls the expression of genes encoding peroxisomal and mitochondrial fatty acid β-oxidation enzymes, with the experimental ligand Wy-14,643 or the clinically-used fibrate drug fenofibrate, fully protects mice from APAP-induced hepatotoxicity. PPARα-humanized mice were also protected while Ppara-null mice were not, thus indicating that the protection extends to human PPARα and is PPARα-dependent. This protection is due in part to induction of the PPARα target gene encoding mitochondrial uncoupling protein 2 (UCP2). Forced overexpression of UCP2 protected wild-type mice against APAP-induced hepatotoxicity in the absence of PPARα activation. Ucp2-null mice, however, were sensitive to APAP induced hepatotoxicity despite activation of PPARα with Wy-14,643. Protection against hepatotoxicity by UCP2-induction through activation of PPARα, is associated with decreased APAP-induced c-jun and c-fos expression, decreased phosphorylation of JNK and c-jun, lower mitochondrial H2O2 levels, increased mitochondrial glutathione in liver, and decreased levels of circulating fatty acyl-carnitines. These studies indicate that the PPARα target gene UCP2 protects against elevated reactive oxygen species generated during drug-induced hepatotoxicity and suggest that induction of UCP2 may also be a general mechanism for protection of mitochondria during fatty acid β-oxidation.
doi:10.1002/hep.25645
PMCID: PMC3378765  PMID: 22318764
12.  The Role of Mass Spectrometry-Based Metabolomics in Medical Countermeasures Against Radiation 
Mass spectrometry reviews  2010;29(3):503-521.
Radiation metabolomics can be defined as the global profiling of biological fluids to uncover latent, endogenous small molecules whose concentrations change in a dose-response manner following exposure to ionizing radiation. In response to the potential threat of nuclear or radiological terrorism, the Center for High-Throughput Minimally Invasive Radiation Biodosimetry (CMCR) was established to develop field-deployable biodosimeters based, in principle, on rapid analysis by mass spectrometry of readily and easily obtainable biofluids. In this review, we briefly summarize radiation biology and key events related to actual and potential nuclear disasters, discuss the important contributions the field of mass spectrometry has made to the field of radiation metabolomics, and summarize current discovery efforts to use mass spectrometry-based metabolomics to identify dose-responsive urinary constituents, and ultimately to build and deploy a noninvasive high-throughput biodosimeter.
doi:10.1002/mas.20272
PMCID: PMC3690279  PMID: 19890938
Biodosimetry; ionizing radiation; metabolomics
13.  Metabolomics reveals the metabolic map of procainamide in humans and mice 
Biochemical pharmacology  2012;83(10):1435-1444.
Procainamide, a type I antiarrhythmic agent, is used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25–30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice as well as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation.
doi:10.1016/j.bcp.2012.02.013
PMCID: PMC3665348  PMID: 22387617
Procainamide; Systemic lupus erythematosus; Metabolomics; N-Oxidation; Ultra-performance liquid chromatography; Time-of-flight mass spectrometry
14.  Postischemic Brain Injury Is Attenuated in Mice Lacking the β2-Adrenergic Receptor 
Anesthesia and analgesia  2009;108(1):280-287.
Background
Several β-adrenergic receptor (βAR) antagonists have been shown to have neuroprotective effects against cerebral ischemia. However, clenbuterol, a β2AR agonist, was shown to have neuroprotective activity by increasing nerve growth factor expression. We used β2AR knockout mice and a β2 selective antagonist to test the effect of loss of β2ARs on outcome from transient focal cerebral ischemia.
Methods
Ischemia was induced by the intraluminal suture method, for 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Neurological score was determined at 24 h reperfusion and infarct size was determined by cresyl violet or 2,3,5-triphenyltetrazolium chloride staining. β2AR knockout mice and wild-type congenic FVB/N controls were studied, as well as 2 groups of wild type mice given either ICI 118,551 (0.2 mg/kg) or 0.9% saline intraperitoneally 30 min before MCAO (n = 10 per group). Changes in expression of heat shock protein (Hsp)72 after ischemia were examined by immunohistochemistry and western blots.
Results
Compared with wild type littermates, infarct volume was decreased by 22.3% in β2AR knockout mice (39.7 ± 10.7 mm3 vs 51.0 ± 11.4 mm3, n = 10/group, P = 0.034) after 60 min of MCAO followed by 24 h reperfusion. Pretreatment with a β2AR selective antagonist, ICI 118,551, also decreased infarct size significantly, by 25.1%, compared with the saline control (32.8 ± 11.9 mm3 vs 43.8 ± 10.3 mm3, n = 10/group, P = 0.041). Neurological scores were also significantly improved in mice lacking the β2AR or pretreated with ICI 118,551. After cerebral ischemia, total levels of Hsp72 and the number of Hsp72 immunopositive cells were greater in mice lacking β2 AR.
Conclusion
Brain injury is reduced and neurological outcome improved after MCAO in mice lacking the β2AR, or in wild type mice pretreated with a selective β2AR antagonist. This is consistent with a shift away from prosurvival signaling to prodeath signaling in the presence of β2AR activation in cerebral ischemia. Protection is associated with higher levels of Hsp72, a known antideath protein. The effect of β2AR signaling in the setting of cerebral ischemia is complex and warrants further study.
Implications
Brain injury is reduced and neurological outcome improved after middle cerebral artery occlusion in mice lacking the β2AR or in wild type mice pretreated with a selective β2AR antagonist. This is consistent with a shift away from prosurvival signaling to prodeath signaling in the presence of β2AR activation in cerebral ischemia.
doi:10.1213/ane.0b013e318187ba6b
PMCID: PMC3661414  PMID: 19095863
15.  FXR Agonist INT-747 Upregulates DDAH Expression and Enhances Insulin Sensitivity in High-Salt Fed Dahl Rats 
PLoS ONE  2013;8(4):e60653.
Aims
Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.
Methods and Results
In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.
Conclusion
Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.
doi:10.1371/journal.pone.0060653
PMCID: PMC3617194  PMID: 23593273
16.  Hypoxia-derived oxidative stress mediates epigenetic repression of PKCɛ gene in foetal rat hearts 
Cardiovascular Research  2011;93(2):302-310.
Aims
Hypoxia causes protein kinase C epsilon (PKCɛ) gene repression in foetal hearts, resulting in heightened cardiac susceptibility to ischaemic injury in offspring. We tested the hypothesis that hypoxia inducible factor 1 (HIF-1) and/or reactive oxygen species (ROS) mediate hypoxia-induced PKCɛ gene repression.
Methods and results
Hypoxia induced in vivo to pregnant rats, ex vivo to isolated foetal rat hearts, and in vitro in the rat embryonic ventricular myocyte cell line H9c2 resulted in a comparable decrease in PKCɛ protein and mRNA abundance in foetal hearts and H9c2 cells, which was associated with a significant increase in CpG methylation of the SP1-binding sites at the PKCɛ promoter. In H9c2 cells and foetal hearts, hypoxia caused nuclear accumulation of HIF-1α, which was inhibited by 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole and 2-methoxy estradiol. The HIF-1α inhibitors had no significant effect on hypoxia-induced PKCɛ mRNA repression. Hypoxia produced a time-dependent increase in ROS production in H9c2 cells and foetal hearts that was blocked by ROS scavengers N-acetyl-cysteine or tempol. In accordance, N-acetyl-cysteine and tempol, but not apocynin, inhibited the hypoxic effect and restored PKCɛ protein and mRNA expression to the control values in foetal hearts and H9c2 cells. The ROS scavengers blocked hypoxia-induced CpG methylation of the SP1-binding sites, restored SP1 binding to the PKCɛ promoter, and abrogated the hypoxia-induced increase in the susceptibility of the heart to ischaemic injury in offspring.
Conclusions
The results demonstrate that hypoxia induces epigenetic repression of the PKCɛ gene through a NADPH oxidase-independent ROS-mediated pathway in the foetal heart, leading to heightened heart vulnerability to ischaemic injury in offspring.
doi:10.1093/cvr/cvr322
PMCID: PMC3258654  PMID: 22139554
Hypoxia; Heart; Protein kinase C; Epigenetic; Oxidative stress
17.  Network Analysis of a Pkd1-Mouse Model of Autosomal Dominant Polycystic Kidney Disease Identifies HNF4α as a Disease Modifier 
PLoS Genetics  2012;8(11):e1003053.
Autosomal Dominant Polycystic Kidney Disease (ADPKD; MIM ID's 173900, 601313, 613095) leads to end-stage kidney disease, caused by mutations in PKD1 or PKD2. Inactivation of Pkd1 before or after P13 in mice results in distinct early- or late-onset disease. Using a mouse model of ADPKD carrying floxed Pkd1 alleles and an inducible Cre recombinase, we intensively analyzed the relationship between renal maturation and cyst formation by applying transcriptomics and metabolomics to follow disease progression in a large number of animals induced before P10. Weighted gene co-expression network analysis suggests that Pkd1-cystogenesis does not cause developmental arrest and occurs in the context of gene networks similar to those that regulate/maintain normal kidney morphology/function. Knowledge-based Ingenuity Pathway Analysis (IPA) software identifies HNF4α as a likely network node. These results are further supported by a meta-analysis of 1,114 published gene expression arrays in Pkd1 wild-type tissues. These analyses also predict that metabolic pathways are key elements in postnatal kidney maturation and early steps of cyst formation. Consistent with these findings, urinary metabolomic studies show that Pkd1 cystic mutants have a distinct profile of excreted metabolites, with pathway analysis suggesting altered activity in several metabolic pathways. To evaluate their role in disease, metabolic networks were perturbed by inactivating Hnf4α and Pkd1. The Pkd1/Hnf4α double mutants have significantly more cystic kidneys, thus indicating that metabolic pathways could play a role in Pkd1-cystogenesis.
Author Summary
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common genetic cause of polycystic kidney disease and is responsible for 4.6% of the end-stage renal disease (ESRD) cases in the United States. It is most often caused by mutation in the PKD1 gene. To understand this disease, we made a mouse model in which we could delete the Pkd1 gene and study the animal as its kidney becomes cystic. Using this model, we had previously found that the maturation status of the animal determines whether cysts form within days or within months, and we had narrowed down this switch to a two-day interval. In the current study, we used the rapid cyst-forming model to analyze the expression pattern of thousands of genes in mutant and control kidneys, and metabolites excreted in the urine. Our results identify a number of genes that may be involved in cyst formation and suggest that metabolic changes may play a role in ADPKD and could alter disease progression. These analyses also predict that metabolic pathways are key elements in normal postnatal kidney maturation.
doi:10.1371/journal.pgen.1003053
PMCID: PMC3510057  PMID: 23209428
18.  Aberrant Lipid Metabolism in Hepatocellular Carcinoma Revealed by Plasma Metabolomics and Lipid Profiling 
Cancer research  2011;71(21):6590-6600.
There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here we report the findings of a plasma metabolomic investigation of HCC patients using ultraperformance liquid chromatography-electrospray ionization-quadrupole mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also performed using ultra-performance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). Using this method we found that that HCC was associated also with reduced levels of lysophosphocholines (LPC) and in 4/20 patients with increased levels of lysophosphatidic acid (LPA(16:0)), where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GCMS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GCMS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.
doi:10.1158/0008-5472.CAN-11-0885
PMCID: PMC3206149  PMID: 21900402
metabolomics; hepatocellular carcinoma; LPC; LPA; VLCFA
19.  Chemotherapeutic Evaluation of a Novel Synthetic Tubulysin Analogue-Dendrimer Conjugate in C26 Tumor Bearing Mice 
ChemMedChem  2011;6(1):49-53.
doi:10.1002/cmdc.201000377
PMCID: PMC3470805  PMID: 20973119
Tubulysin; dendrimer; drug delivery; antitumor; chemotherapy; colon carcinoma
20.  Regulatory T cells limit vascular endothelial injury and prevent pulmonary hypertension. 
Circulation research  2011;109(8):867-879.
Rationale
Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood.
Objective
To determine whether immune dysregulation due to absent T cell populations directly contributes to the development of PAH.
Methods and Results
Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell deficient rats but not in immune-reconstituted (IR) rats. T cell-lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4+CD25hi or CD4+CD25- T cell subsets prior to vascular injury attenuated the development of PAH. Immune reconstitution limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3+-, IL-10- and TGF-β– expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)-expressing cells, a receptor that activates endothelial cell survival pathways.
Conclusions
PAH may arise when regulatory T cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.
doi:10.1161/CIRCRESAHA.110.236927
PMCID: PMC3204361  PMID: 21868697
pulmonary arterial hypertension; inflammation; regulatory T cell; bone morphogenetic protein receptor type 2
21.  OME Remote Objects (OMERO): a flexible, model-driven data management system for experimental biology 
Nature methods  2012;9(3):245-253.
Data-intensive research depends on tools that manage multi-dimensional, heterogeneous data sets. We have built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices, and tables. OMERO’s design and flexibility have enabled its use for light microscopy, high content screening, electron microscopy, and even non-image genotype data. OMERO is open source software and available at http://openmicroscopy.org.
doi:10.1038/nmeth.1896
PMCID: PMC3437820  PMID: 22373911
22.  Mice lacking the β2 adrenergic receptor have a unique genetic profile before and after focal brain ischaemia 
ASN NEURO  2012;4(5):e00096.
The role of the β2AR (β2 adrenergic receptor) after stroke is unclear as pharmacological manipulations of the β2AR have produced contradictory results. We previously showed that mice deficient in the β2AR (β2KO) had smaller infarcts compared with WT (wild-type) mice (FVB) after MCAO (middle cerebral artery occlusion), a model of stroke. To elucidate mechanisms of this neuroprotection, we evaluated changes in gene expression using microarrays comparing differences before and after MCAO, and differences between genotypes. Genes associated with inflammation and cell deaths were enriched after MCAO in both genotypes, and we identified several genes not previously shown to increase following ischaemia (Ccl9, Gem and Prg4). In addition to networks that were similar between genotypes, one network with a central core of GPCR (G-protein-coupled receptor) and including biological functions such as carbohydrate metabolism, small molecule biochemistry and inflammation was identified in FVB mice but not in β2KO mice. Analysis of differences between genotypes revealed 11 genes differentially expressed by genotype both before and after ischaemia. We demonstrate greater Glo1 protein levels and lower Pmaip/Noxa mRNA levels in β2KO mice in both sham and MCAO conditions. As both genes are implicated in NF-κB (nuclear factor κB) signalling, we measured p65 activity and TNFα (tumour necrosis factor α) levels 24 h after MCAO. MCAO-induced p65 activation and post-ischaemic TNFα production were both greater in FVB compared with β2KO mice. These results suggest that loss of β2AR signalling results in a neuroprotective phenotype in part due to decreased NF-κB signalling, decreased inflammation and decreased apoptotic signalling in the brain.
doi:10.1042/AN20110020
PMCID: PMC3436074  PMID: 22867428
β2 adrenergic receptor; Glo1; microarray; Noxa; nuclear factor κB; stroke; tumour necrosis factor α; AGE, advanced glycation end-product; β2AR, β2 adrenergic receptor; CNS, central nervous system; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPCR, G-protein-coupled receptor; IL, interleukin; IPA, ingenuity pathway analysis; Iκbα, inhibitory κB α; LPS, lipopolysaccharide; MCAO, middle cerebral artery occlusion; MMP9, matrix metalloproteinase 9; NF-κB, nuclear factor κB; RT–qPCR, reverse transcription quantitative real-time-PCR; SAGAT, singular value decomposition augmented gene expression analysis tool; SAM, significance analysis of microarrays; TNFα, tumour necrosis factor α; WT, wild-type
23.  UPLC-MS-based Urine Metabolomics Reveals Indole-3-lactic acid and Phenyllactic acid as Conserved Biomarkers for Alcohol-induced Liver Disease in the Ppara-null Mouse Model 
Journal of proteome research  2011;10(9):4120-4133.
Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as, ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine were background-dependent. Elevation of ethyl-β-D-glucuronide and N-acetylglycine was found to be common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.
doi:10.1021/pr200310s
PMCID: PMC3170755  PMID: 21749142
Metabolomics; alcohol; Ppara-null mouse; genetic background; liver disease; UPLC-ESI-QTOF mass spectrometry; biomarker
24.  A Cyp2a polymorphism predicts susceptibility to NNK-induced lung tumorigenesis in mice 
Carcinogenesis  2011;32(8):1279-1284.
Lung tumors from smokers as well as lung tumors from mice exposed to tobacco carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), often carry mutations in K-ras, which activates downstream-signaling pathways such as PI3K/AKT/mTOR pathway. Mice with genetic deletion of one of three isoforms of AKT were used to investigate the role of AKT in mutant K-ras-induced lung tumorigenesis in mice. Although deletion of Akt1 or Akt2 decreased NNK-induced lung tumor formation by 90%, deletion of Akt2 failed to decrease lung tumorigenesis in two other mouse models driven by mutant K-ras. Genetic mapping showed that Akt2 was tightly linked to the cytochrome P450 Cyp2a locus on chromosome 7. Consequently, targeted deletion of Akt2 created linkage to a strain-specific Cyp2a5 polymorphism that decreased activation of NNK in vitro. Mice with this Cyp2a5 polymorphism had decreased NNK-induced DNA adduct formation in vivo and decreased NNK-induced lung tumorigenesis. These studies support human epidemiological studies linking CYP2A polymorphisms with lung cancer risk in humans and highlight the need to confirm phenotypes of genetically engineered mice in multiple mouse strains.
doi:10.1093/carcin/bgr097
PMCID: PMC3149208  PMID: 21625009
25.  Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis 
Hepatology (Baltimore, Md.)  2011;53(4):1282-1293.
Lithocholic acid (LCA) is an endogenous compound associated with hepatic toxicity during cholestasis. LCA exposure in mice resulted in decreased serum lysophosphatidylcholine (LPC) and sphingomyelin levels due to elevated lysophosphatidylcholine acyltransferase (LPCAT) and sphingomyelin phosphodiesterase (SMPD) expression. Global metabolome analysis indicated significant decreases in serum palmitoyl-, stearoyl-, oleoyl- and linoleoyl-LPC levels after LCA exposure. LCA treatment also resulted in decreased serum sphingomyelin levels and increased hepatic ceramide levels, and induction of LPCAT and SMPD mRNAs. Transforming growth factor-β TGF-β) induced Lpcat2/4 and Smpd3 gene expression in primary hepatocytes and the induction was diminished by pretreatment with the SMAD3 inhibitor SIS3. Furthermore, alteration of the LPC metabolites and Lpcat1/2/4 and Smpd3 expression was attenuated in LCA-treated farnesoid X receptor-null mice that are resistant to LCA-induced intrahepatic cholestasis. This study revealed that LCA induced disruption of phospholipid/sphingolipid homeostasis through TGF-β signaling and that serum LPC is a biomarker for biliary injury.
doi:10.1002/hep.24193
PMCID: PMC3077083  PMID: 21480330

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