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1.  Hypoxia-Inducible Factor/MAZ-Dependent Induction of Caveolin-1 Regulates Colon Permeability through Suppression of Occludin, Leading to Hypoxia-Induced Inflammation 
Molecular and Cellular Biology  2014;34(16):3013-3023.
Caveolae are specialized microdomains on membranes that are critical for signal transduction, cholesterol transport, and endocytosis. Caveolin-1 (CAV1) is a multifunctional protein and a major component of caveolae. Cav1 is directly activated by hypoxia-inducible factor (HIF). HIFs are heterodimers of an oxygen-sensitive α subunit, HIF1α or HIF2α, and a constitutively expressed β subunit, aryl hydrocarbon receptor nuclear translocator (ARNT). Whole-genome expression analysis demonstrated that Cav1 is highly induced in mouse models of constitutively activated HIF signaling in the intestine. Interestingly, Cav1 was increased only in the colon and not in the small intestine. Currently, the mechanism and role of HIF induction of CAV1 in the colon are unclear. In mouse models, mice that overexpressed HIF1α or HIF2α specifically in intestinal epithelial cells demonstrated an increase in Cav1 gene expression in the colon but not in the duodenum, jejunum, or ileum. HIF2α activated the Cav1 promoter in a HIF response element-independent manner. myc-associated zinc finger (MAZ) protein was essential for HIF2α activation of the Cav1 promoter. Hypoxic induction of CAV1 in the colon was essential for intestinal barrier integrity by regulating occludin expression. This may provide an additional mechanism by which chronic hypoxia can activate intestinal inflammation.
doi:10.1128/MCB.00324-14
PMCID: PMC4135598  PMID: 24891620
2.  Quercetin Inhibits Radiation-Induced Skin Fibrosis 
Radiation research  2013;180(2):205-215.
Radiation induced fibrosis of the skin is a late toxicity that may result in loss of function due to reduced range of motion and pain. The current study sought to determine if oral delivery of quercetin mitigates radiation-induced cutaneous injury. Female C3H/HeN mice were fed control chow or quercetin-formulated chow (1% by weight). The right hind leg was exposed to 35 Gy of X rays and the mice were followed serially to assess acute toxicity and hind leg extension. Tissue samples were collected for assessment of soluble collagen and tissue cytokines. Human and murine fibroblasts were subjected to clonogenic assays to determine the effects of quercetin on radiation response. Contractility of fibroblasts was assessed with a collagen contraction assay in the presence or absence of quercetin and transforming growth factor-β (TGF-β). Western blotting of proteins involved in fibroblast contractility and TGF-β signaling were performed. Quercetin treatment significantly reduced hind limb contracture, collagen accumulation and expression of TGF-β in irradiated skin. Quercetin had no effect on the radioresponse of fibroblasts or murine tumors, but was capable of reducing the contractility of fibroblasts in response to TGF-β, an effect that correlated with partial stabilization of phosphorylated cofilin. Quercetin is capable of mitigating radiation induced skin fibrosis and should be further explored as a therapy for radiation fibrosis.
doi:10.1667/RR3237.1
PMCID: PMC4281888  PMID: 23819596
4.  CYP2E1 potentiates binge alcohol-induced gut leakiness, steatohepatitis and apoptosis 
Free radical biology & medicine  2013;65:10.1016/j.freeradbiomed.2013.09.009.
Ethanol-inducible cytochrome P450 2E1 (CYP2E1) contributes to increased oxidative stress and steatosis in chronic alcohol-exposure models. However, its role in binge ethanol-induced gut leakiness and hepatic injury is unclear. This study was aimed to investigate the role of CYP2E1 in binge alcohol-induced gut leakiness and the mechanisms of steatohepatitis. Female wild-type (WT) and Cyp2e1-null mice were treated with three doses of binge ethanol (WT-EtOH or Cyp2e1-null-EtOH) (6 g/kg oral gavage at 12-h intervals) or dextrose (negative control). Intestinal histology of only WT-EtOH exhibited epithelial alteration and blebbing of lamina propria while liver histology obtained at 6 h after the last ethanol dose showed elevated steatosis with scattered inflammatory foci. These were accompanied by increased levels of serum endotoxin, hepatic enterobacteria and triglycerides. All these changes including the intestinal histology and hepatic apoptosis, determined by TUNEL assay, were significantly reversed when WT-EtOH mice were treated with the specific inhibitor of CYP2E1 chlormethiazole and the antioxidant N-acetyl-cysteine, both of which suppressed the oxidative markers including intestinal CYP2E1. WT-EtOH also exhibited elevated amounts of serum TNF-α, hepatic cytokines, CYP2E1 and lipid peroxidation with decreased levels of mitochondrial superoxide dismutase and suppressed aldehyde dehydrogenase 2 activity. Increased hepatocyte apoptosis with elevated levels of pro-apoptotic proteins and decreased levels of active (phosphorylated) p-AKT, p-AMPK and peroxisome proliferator-activated receptor-alpha (PPAR-α), all of which are involved in fat metabolism and inflammation, were observed in WT-EtOH. These changes were significantly attenuated in the corresponding Cyp2e1-null-EtOH mice. These data indicate that both intestinal and hepatic CYP2E1 induced by binge alcohol seem critical in the binge alcohol-mediated increased nitroxidative stress, gut leakage, endotoxemia, and altered fat metabolism, and inflammation, contributing to hepatic apoptosis and steatohepatitis.
doi:10.1016/j.freeradbiomed.2013.09.009
PMCID: PMC3859835  PMID: 24064383
Liver; binge ethanol; CYP2E1; oxidative stress; gut leakiness; steatohepatitis; apoptosis
5.  MiR-193b and miR-365-1 are not required for the development and function of brown fat in the mouse 
RNA Biology  2013;10(12):1807-1814.
Generating heat and maintaining body temperature is the primary function of brown adipose tissue (BAT). Previous studies have implicated microRNAs, including miR-193b and miR-365-1, in BAT differentiation. We used mouse genetics to further understand the specific contributions of these two miRs. BAT function in mice with an inactivated miR-193b-365-1 locus, as determined by their response to the selective β3 adrenergic receptor agonist CL316.243 and their tolerance to cold exposure, was normal and expression of genes associated with functional BAT, including Prdm16 and Ucp1, was unaffected. In addition, genome-wide expression profiles of miRNAs and mRNAs in BAT in the presence and absence of miR-193b-365-1 were determined. In summary, these data demonstrate, in contrast to earlier work, that the development, differentiation, and function of BAT do not require the presence of miR-193b and miR-365-1.
doi:10.4161/rna.27239
PMCID: PMC3917983  PMID: 24356587
RNA-seq; brown fat; gene knock-out; miR-193b; mouse
6.  Potential role of CYP2D6 in the central nervous system 
Cytochrome P450 2D6 (CYP2D6) is a pivotal enzyme responsible for a major human drug oxidation polymorphism in human populations. Distribution of CYP2D6 in brain and its role in serotonin metabolism suggest this CYP2D6 may have a function in central nervous system.To establish an efficient and accurate platform for the study of CYP2D6 in vivo, a transgenic human CYP2D6 (Tg-2D6) model was generated by transgenesis in wild-type C57BL/6 (WT) mice using a P1 phage artificial chromosome clone containing the complete human CYP2D locus, including CYP2D6 gene and 5’- and 3’- flanking sequences.Human CYP2D6 was expressed not only in the liver, but also in brain. The abundance of serotonin and 5-hydroxyindoleacetic acid in brain of Tg-2D6 is higher than in WT mice either basal levels or after harmaline induction. Metabolomics of brain homogenate and cerebrospinal fluid revealed a significant up-regulation of l-carnitine, acetyl-l-carnitine, pantothenic acid, dCDP, anandamide, N-acetylglucosaminylamine, and a down-regulation of stearoyl-l-carnitine in Tg-2D6 mice compared with WT mice. Anxiety tests indicate Tg-2D6 mice have a higher capability to adapt to anxiety.Overall, these findings indicate that the Tg-2D6 mouse model may serve as a valuable in vivo tool to determine CYP2D6-involved neurophysiological metabolism and function.
doi:10.3109/00498254.2013.791410
PMCID: PMC3750078  PMID: 23614566
CYP2D6; brain; serotonin; anxiety
7.  Loss of von Hippel-Lindau Protein (VHL) Increases Systemic Cholesterol Levels through Targeting Hypoxia-Inducible Factor 2α and Regulation of Bile Acid Homeostasis 
Molecular and Cellular Biology  2014;34(7):1208-1220.
Cholesterol synthesis is a highly oxygen-dependent process. Paradoxically, hypoxia is correlated with an increase in cellular and systemic cholesterol levels and risk of cardiovascular diseases. The mechanism for the increase in cholesterol during hypoxia is unclear. Hypoxia signaling is mediated through hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. The present study demonstrates that activation of HIF signaling in the liver increases hepatic and systemic cholesterol levels due to a decrease in the expression of cholesterol hydroxylase CYP7A1 and other enzymes involved in bile acid synthesis. Specifically, activation of hepatic HIF-2α (but not HIF-1α) led to hypercholesterolemia. HIF-2α repressed the circadian expression of Rev-erbα, resulting in increased expression of E4BP4, a negative regulator of Cyp7a1. To understand if HIF-mediated decrease in bile acid synthesis is a physiologically relevant pathway by which hypoxia maintains or increases systemic cholesterol levels, two hypoxic mouse models were assessed, an acute lung injury model and mice exposed to 10% O2 for 3 weeks. In both models, cholesterol levels increased with a concomitant decrease in expression of genes involved in bile acid synthesis. The present study demonstrates that hypoxic activation of hepatic HIF-2α leads to an adaptive increase in cholesterol levels through inhibition of bile acid synthesis.
doi:10.1128/MCB.01441-13
PMCID: PMC3993569  PMID: 24421394
8.  Identification of 2-Piperidone as a Biomarker of CYP2E1 Activity Through Metabolomic Phenotyping 
Toxicological Sciences  2013;135(1):37-47.
Cytochrome P450 2E1 (CYP2E1) is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, a mass spectrometry–based metabolomic approach was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type (WT), Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from WT and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of WT and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the WT mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity.
doi:10.1093/toxsci/kft143
PMCID: PMC3748767  PMID: 23811823
CYP2E1; biomarker; metabolomics; 2-piperidone; cadaverine.
9.  Heterogeneities in regional volumes of distribution and flows in rabbit heart 
The American journal of physiology  1990;258(4 0 2):H1012-H1024.
The heterogeneity of volumes of distribution in the heart influences the rates of uptake and washout of substrates and metabolites; thus it is important to evaluate their variability in the normal heart. Several tracers were injected intravenously into anesthetized adult closed-chest rabbits, and time was allowed for equilibration in the heart. Tracer microspheres were injected into the left ventricular cavity at the apex for the measurement of regional flows, the chest was opened, another set of microspheres was injected, and the heart was frozen rapidly in situ with liquid nitrogen-cooled Freon-22. Each heart was divided into 72 pieces of <0.1 g weight, and the tracer content of each was determined by multichannel γ-counting and the water content by desiccation. The regional myocardial flows were (closed chest) 0.62 ± 0.16 ml·g−1·min−1 and (open chest) 0.63 ± 0.37 ml·g−1·min−1. The volumes of distribution (ml/g) for the 432 pieces for six rabbits, given as mean ± SD (% coefficient of variation), were as follows: for plasma, VP = 0.11 ± 0.03 (26%); erythrocytes, VRBC = 0.041 ± 0.015 (37%); vascular space, VV = 0.15 ± 0.04 (26%); extracellular space, VECF = 0.33 ± 0.05 (15%); interstitial space, VISF = 0.21 ± 0.03 (15%); and water space, VW − 0.79 ± 0.022 (2.8%). Regional hematocrits were 77% ± 9% of the large-vessel hematocrits.
PMCID: PMC4138226  PMID: 2109937
extracellular space; interstitial fluid; plasma volume; erythrocyte space; myocardial blood volume; heart water content; capillary hematocrit; tissue variability; sucrose; radiocobaltic ethylenediaminetetraacetic acid
10.  PPARβ/δ activation of CD300a controls intestinal immunity 
Scientific Reports  2014;4:5412.
Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARβ/δ (peroxisome proliferator-activated receptor β/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a−/− mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a−/− macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.
doi:10.1038/srep05412
PMCID: PMC4067692  PMID: 24958459
11.  Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins 
Four dioxin-inducible enzymes—NAD(P)H:quinone oxidoreductase-1 (NQO1) and three cytochromes P450 (CYP1A1, CYP1A2 & CYP1B1)—are implicated in both detoxication and metabolic activation of various endobiotics and xenobiotics. NQO1 is generally regarded as a cytosolic enzyme; whereas CYP1 proteins are located primarily in endoplasmic reticulum (ER), CYP1A1 and CYP1A2 proteins are also targeted to mitochondria. This lab has generated Cyp1a1(mc/mc) and Cyp1a1(mtt/mtt) knock-in mouse lines in which CYP1A1 protein is targeted exclusively to ER (microsomes) and mitochondria, respectively. Comparing dioxin-treated Cyp1(+/+) wild-type, Cyp1a1(mc/mc), Cyp1a1(mtt/mtt), and Cyp1a1(−/−), Cyp1b1(−/−) and Nqo1(−/−) knockout mice, in the present study we show that [a] NQO1 protein locates to cytosol, ER and mitochondria, [b] CYP1B1 protein (similar to CYP1A1 and CYP1A2 proteins) traffics to mitochondria as well as ER, and [c] NQO1 and CYP1B1 targeting to mitochondrial or ER membranes is independent of CYP1A1 presence in that membrane.
doi:10.1016/j.bbrc.2013.05.051
PMCID: PMC3735136  PMID: 23692925
NAD(P)H:quinone oxidoreductase-1 (NQO1); Cytochrome P450 1B1 (CYP1B1); CYP1A1; subcellular localization; mitochondrial targeting; endoplasmic reticulum targeting; dioxin-inducible enzymes; mouse tissues (lung, spleen, uterus, small intestine, liver, kidney)
12.  Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells 
Analytical and bioanalytical chemistry  2013;405(15):5279-5289.
In this study, a liquid chromatography mass spectrometry (LC/MS)-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the human pancreatic cancer cell line Panc-1. Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in water were compared for sample harvesting. Four different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Global metabolomics profiles were compared; the number of total detected features and the recovery and relative extraction efficiencies of target metabolites were assessed. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method therefore provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further LC/MS-based global metabolomics study on Panc-1 cell lines and possibly other cancer cell lines with similar chemical and physical properties.
doi:10.1007/s00216-013-6927-9
PMCID: PMC3678261  PMID: 23604415
Metabolomics; Sample preparation; Metabolite extraction; Panc-1 cell line; HILIC
13.  Metabolomics reveals aging-associated attenuation of noninvasive radiation biomarkers in mice: potential role of polyamine catabolism and incoherent DNA damage-repair 
Journal of proteome research  2013;12(5):2269-2281.
Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sub-lethal irradiation was examined in mice using UPLC-ESI-TOF mass spectrometry. Aging attenuated post-exposure elevation in excretions of DNA damage biomarkers as well as N1-acetylspermidine. Although N1-acetylspermidine and 2’-deoxyuridine elevation was highly correlated in all age groups, xanthine and N1-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N1-acetylspermidine, 2’-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2’-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2’-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sub-lethal radiation exposure.
doi:10.1021/pr400161k
PMCID: PMC3678303  PMID: 23586774
Ionizing radiation; age; exposure history; biomarker; metabolomics; UPLC-ESIQTOF-MS; DNA damage-repair; polyamine metabolism
14.  FXR signaling in the enterohepatic system 
Enterohepatic circulation serves to capture bile acids and other steroid metabolites produced in the liver and secreted to the intestine, for reabsorption back into the circulation and reuptake to the liver. This process is under tight regulation by nuclear receptor signaling. Bile acids, produced from cholesterol, can alter gene expression in the liver and small intestine via activating the nuclear receptors farnesoid X receptor (FXR; NR1H4), pregnane X receptor (PXR; NR1I2), vitamin D receptor (VDR; NR1I1), G protein coupled receptor TGR5, and other cell signaling pathways (JNK1/2, AKT and ERK1/2). Among these controls, FXR is known to be a major bile acid-responsive ligand-activated transcription factor and a crucial control element for maintaining bile acid homeostasis. FXR has a high affinity for several major endogenous bile acids, notably cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid. By responding to excess bile acids, FXR is a bridge between the liver and small intestine to control bile acid levels and regulate bile acid synthesis and enterohepatic flow. FXR is highly expressed in the liver and gut, relative to other tissues, and contributes to the maintenance of cholesterol/bile acid homeostasis by regulating a variety of metabolic enzymes and transporters. FXR activation also affects lipid and glucose metabolism, and can influence drug metabolism.
doi:10.1016/j.mce.2012.05.004
PMCID: PMC3491147  PMID: 22609541
15.  Bisphenol A Increases Atherosclerosis in Pregnane X Receptor‐Humanized ApoE Deficient Mice 
Background
Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA has recently been associated with increased risk of cardiovascular disease (CVD) in multiple large‐scale human population studies, but the underlying mechanisms remain elusive. We previously reported that BPA activates the pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism and has pro‐atherogenic effects in animal models upon activation. Interestingly, BPA is a potent agonist of human PXR but does not activate mouse or rat PXR signaling, which confounds the use of rodent models to evaluate mechanisms of BPA‐mediated CVD risk. This study aimed to investigate the atherogenic mechanism of BPA using a PXR‐humanized mouse model.
Methods and Results
A PXR‐humanized ApoE deficient (huPXR•ApoE−/−) mouse line was generated that respond to human PXR ligands and feeding studies were performed to determine the effects of BPA exposure on atherosclerosis development. Exposure to BPA significantly increased atherosclerotic lesion area in the aortic root and brachiocephalic artery of huPXR•ApoE−/− mice by 104% (P<0.001) and 120% (P<0.05), respectively. By contrast, BPA did not affect atherosclerosis development in the control littermates without human PXR. BPA exposure did not affect plasma lipid levels but increased CD36 expression and lipid accumulation in macrophages of huPXR•ApoE−/− mice.
Conclusion
These findings identify a molecular mechanism that could link BPA exposure to increased risk of CVD in exposed individuals. PXR is therefore a relevant target for future risk assessment of BPA and related environmental chemicals in humans.
doi:10.1161/JAHA.113.000492
PMCID: PMC4187496  PMID: 24755147
atherosclerosis; cells; receptors; risk factors
16.  Cancer-produced metabolites of 5-lipoxygenase induce tumor-evoked Bregs via peroxisome proliferator-activated receptor alpha 
Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report here that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase (5-LO) pathway such as leukotriene B4 (LTB4) to activate the proliferator-activated receptor alpha (PPARα) in B cells. Inactivation of LTB4 signaling or genetic deficiency of PPARα in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARα initiates programs required for differentiation of tBregs. We propose that PPARα in B cells or/and tumor 5-LO pathways represents new targets for pharmacological control of tBreg –mediated cancer escape.
doi:10.4049/jimmunol.1201920
PMCID: PMC3594535  PMID: 23408836
tBreg induction; metastasis; PPARα; leukotriene
17.  PXR/CYP3A4-humanized mice for studying drug-drug interactions involving intestinal P-glycoprotein 
Molecular pharmaceutics  2013;10(3):1056-1062.
Rodent models are less suitable for predicting drug-drug interactions at the level of the human intestinal mucosa, especially when nuclear receptors like pregnane X receptor (PXR) are involved. Recently, a transgenic mouse model, expressing both human PXR and CYP3A4, was developed and shown to be a better predictor of CYP3A4 induction by xenobiotics in humans as compared to wild-type mice. In the present study, we tested the hypothesis that this mouse model can also predict PXR-mediated induction of intestinal P-gp in humans. By use of the in situ intestinal perfusion technique with mesenteric blood sampling, the effect of oral rifampicin treatment on intestinal permeability for the HIV protease inhibitor darunavir, a dual CYP3A4/P-gp substrate, was investigated. Rifampicin treatment lowered the intestinal permeability for darunavir by 50 % compared to non-treated mice. The P-gp inhibitor GF120918 increased the permeability for darunavir by 400 % in rifampicin-treated mice, while this was only 56 % in mice that were not treated, thus indicating P-gp induction by rifampicin. The non-specific P450 inhibitor aminobenzotriazole (100 μM) did not affect the permeability for darunavir. Quantitative Western blot analysis of the intestinal tissue showed that rifampicin treatment induced intestinal P-gp levels four-fold, while CYP3A4 levels remained unchanged. Oral co-administration of rifampicin with the phytochemical sulforaphane for three days increased the permeability for darunavir by 50 % compared to rifampicin treatment alone. These data show that PXR/CYP3A4-humanized mice can be used to study the inducing effects of xenobiotics on intestinal P-gp.
doi:10.1021/mp300512r
PMCID: PMC3594649  PMID: 23360470
In situ intestinal perfusion; humanized mouse; PXR; P-glycoprotein; rifampicin; darunavir
18.  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
19.  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
20.  CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid 
Toxicology and applied pharmacology  2012;266(2):245-253.
Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation.
doi:10.1016/j.taap.2012.10.024
PMCID: PMC3661416  PMID: 23142471
Isoniazid; CYP2E1; Metabolomics; Drug-induced hepatotoxicity
21.  Metabolomics Reveals a Role for the Chromatin-Binding Protein HMGN5 in Glutathione Metabolism 
PLoS ONE  2014;9(1):e84583.
High mobility group nucleosome-binding protein 5 (HMGN5) is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5 in vivo, mice lacking the nucleosome-binding domain of HMGN5 were generated and characterized. Serological analysis revealed that compared to wild-type littermates (Hmgn5+/Y), mice with a targeted mutation in the HMGN5 gene (Hmgn5tm1/Y), had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between Hmgn5tm1/Y and their Hmgn5+/Y littermates. Hmgn5tm1/Y mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), was significantly decreased in Hmgn5tm1/Y mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of Hmgn5tm1/Y mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.
doi:10.1371/journal.pone.0084583
PMCID: PMC3879345  PMID: 24392144
22.  Implication of intestinal VDR deficiency in inflammatory bowel disease 
Biochimica et biophysica acta  2012;1830(1):2118-2128.
BACKGROUND
To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice.
METHODS
VdrΔIEpC mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics.
RESULTS
VdrΔIEpC mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, VdrΔIEpC mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, VdrΔIEpC mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in VdrΔIEpC mice.
CONCLUSIONS
Disruption of the intestinal Vdr gene showed phenotypical changes that may exacerbate IBD. These results suggest that VDR may play an important role in IBD.
GENERAL SIGNIFICANCE
VDR function has been implicated in IBD. This is of value for understanding the etiology of IBD and for development of diagnostic biomarkers for IBD.
doi:10.1016/j.bbagen.2012.09.020
PMCID: PMC3508150  PMID: 23041070
Vitamin D receptor (VDR); inflammatory bowel disease (IBD); bile acids
23.  Emerging Concepts About Prenatal Genesis, Aberrant Metabolism and Treatment Paradigms in Polycystic Ovary Syndrome 
Endocrine  2012;42(3):526-534.
The interactive nature of the 8th Annual Meeting of the Androgen Excess & PCOS Society Annual Meeting in Munich, Germany (AEPCOS 2010) and subsequent exchanges between speakers led to emerging concepts in PCOS regarding its genesis, metabolic dysfunction, and clinical treatment of inflammation, metabolic dysfunction, anovulation and hirsutism. Transition of care in congenital adrenal hyperplasia from pediatric to adult providers emerged as a potential model for care transition involving PCOS adolescents.
doi:10.1007/s12020-012-9701-4
PMCID: PMC3485440  PMID: 22661293
Developmental programming; clomiphene citrate; aromatase inhibitors; metformin; lifestyle intervention; advanced glycated end products; inflammation; statins; congenital adrenal hyperplasia; hirsutism
24.  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
25.  Beta-Cell ARNT Is Required for Normal Glucose Tolerance in Murine Pregnancy 
PLoS ONE  2013;8(10):e77419.
Aims
Insulin secretion increases in normal pregnancy to meet increasing demands. Inability to increase beta-cell function results in gestational diabetes mellitus (GDM). We have previously shown that the expression of the transcription factor ARNT (Aryl-hydrocarbon Receptor Nuclear Translocator) is reduced in the islets of humans with type 2 diabetes. Mice with a beta-cell specific deletion of ARNT (β-ARNT mice) have impaired glucose tolerance secondary to defective insulin secretion. We hypothesised that ARNT is required to increase beta-cell function during pregnancy, and that β-ARNT mice would be unable to compensate for the beta-cell stress of pregnancy. The aims of this study were to investigate the mechanisms of ARNT regulation of beta-cell function and glucose tolerance in pregnancy.
Methods
β-ARNT females were mated with floxed control (FC) males and FC females with β-ARNT males.
Results
During pregnancy, β-ARNT mice had a marked deterioration in glucose tolerance secondary to defective insulin secretion. There was impaired beta-cell proliferation in late pregnancy, associated with decreased protein and mRNA levels of the islet cell-cycle regulator cyclinD2. There was also reduced expression of Irs2 and G6PI. In contrast, in control mice, pregnancy was associated with a 2.1-fold increase in ARNT protein and a 1.6-fold increase in cyclinD2 protein, and with increased beta-cell proliferation.
Conclusions
Islet ARNT increases in normal murine pregnancy and beta-cell ARNT is required for cyclinD2 induction and increased beta-cell proliferation in pregnancy.
doi:10.1371/journal.pone.0077419
PMCID: PMC3812008  PMID: 24204824

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