bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution and Determinants of Obesity in New York City, 2003–2007
Authors: Black Jennifer L. , Macinko James
Published: June 2021

OBJECTIVES

This study characterized the individual and neighborhood‐level determinants and distribution of obesity (BMI≥30 kg/m2) in New York City (NYC) from 2003 to 2007.

METHODS

Individual‐level data from the Community Health Survey (n=48,506 adults, 34 neighborhoods) were combined with neighborhood measures. Multilevel regression assessed changes in obesity over time and associations with neighborhood‐level income and food and physical activity amenities, controlling for age, racial/ethnic identity, education, employment, US nativity and marital status, and stratified by gender.

RESULTS

Obesity risk increased by 1.6% (P<0.05) each year, but changes over time differed significantly between neighborhoods and by gender. Obesity risk increased for women, even after controlling for individual and neighborhood‐level factors (Prevalence Ratio (PR)=1.021, P<0.05), whereas no significant changes were reported for men. Neighborhood factors including increased area income (PR=0.932) and availability of local food and fitness amenities (PR=0.890) were significantly associated with reduced obesity (P<0.001).

CONCLUSIONS

Findings suggest that policies intended to decrease obesity in urban environments should be informed by up to date surveillance data, and may require a variety of initiatives that respond to both the individual and contextual determinants of obesity.
Dietary food sources of potassium and the sodium‐potassium ratio of foods consumed in the U.S.: NHANES, 2003–2006
Authors: Keast Debra R. , Fulgoni Victor L.
Published: June 2021

Diets containing foods that are good sources of potassium and low in sodium may reduce the risk of high blood pressure and stroke. The aim of this study was to examine dietary food sources of potassium (K), and the sodium‐potassium (Na/K) ratio of foods consumed in the U.S. NHANES, 2003–2006, 24‐hr recall data from subjects 2+ years (n=16,822) were analyzed using SUDAAN. Foods as consumed, including mixtures, were classified by their main ingredient to USDA Dietary Sources of Nutrients food groups. Mean K and Na intakes were 2619±25 and 3422±28 mg/d, and the Na/K ratio for the diet was 1.3. Percent contributions to total dietary intakes contributed from grain products, meat/poultry/fish, dairy products, vegetables, fruit, eggs, legumes, nuts/seeds, fats/oils, desserts/sweets, beverages, and other foods were 20.3, 16.2, 13.9, 13.7, 11.1, 4.7, 0.8, 5.2, 10.4, and 3.7%, for K; 39.7, 22.9, 7.3, 7.1, 0.1, 4.2, 4.2, 5.1, 2.6, and 6.8%, for Na; and the Na/K ratios were 2.6, 1.8, 0.7, 0.7, 0.01, 1.2, 6.5, 1.3, 0.3, and 2.4, respectively. Examples of foods with low Na/K ratios were fruit (0.01), fruit juice (0.02), lettuce (0.09), coffee/tea (0.16), alcoholic beverages (0.23), and milk (0.28), and those with high Na/K ratios were pizza/turnovers (3.17), condiments/sauces (3.26), quick breads (3.32), processed meats (3.50), breads/rolls (4.16), and cheese (5.23). In conclusion, because food sources of potassium and sodium are equally ubiquitous in the food supply, foods with varying Na/K ratios can be combined in mixtures or as meal accompaniments to balance the sodium‐potassium ratio of the diet. (Support: International Life Sciences Institute – North America)
Modeling whole grain intake with whole grains replacing refined grains in foods consumed by children aged 9 to 18 years: NHANES, 2003–2004
Authors: Keast Debra R. , Rosen Renee A. , Arndt Elizabeth A. , Marquart Leonard F.
Published: June 2021

The impact of substituting whole grains for refined grains in the food supply was examined using 24‐hour dietary recall data from children 9–18 years of age in NHANES, 2003–2004 (n=2,349). Varying proportions of refined wheat flour were replaced depending on the acceptability of whole grain foods tested among children in schools. A proportion of white rice was replaced by brown rice as well. The whole grain composition of survey foods was modeled using the MyPyramid Equivalents Database 2.0 and the SR‐Link of FNDDS 2.0. Sample‐weighted means and standard errors were determined using SUDAAN. Pre‐modeled whole grain intake differed by poverty level, but post‐modeled whole grain intake did not. Overall, whole grain intake increased by 1.7 ounce equivalents per day (from 0.5 to 2.2 oz equiv/d). Major food sources of the increase in whole grain intakes were white bread and rolls, including sandwiches (30.2%), pizza (18.3%), rice and pasta, including mixtures (13.1%), tortilla products, muffins, waffles (12.6%), other baked goods (10.2%), and savory snacks (7.7%). Pre‐ and post‐modeled whole grain intakes were 6% and 28% of total grain intake, respectively, thus, by gradually increasing the replacement level, whole grain intakes approach recommended amounts (50% of total grains). A variety of grain products enriched with whole grain contribute a large proportion to an increase in whole grain intake, and the increase is equally distributed across all income groups, because the modified foods are widely consumed by the target population. (Supported by Grains for Health Foundation and ConAgra Foods, Inc.)
Contributions of milk, dairy products, and other foods to vitamin D intakes in the U.S.: NHANES, 2003–2006
Authors: Keast Debra R. , Fulgoni Victor L. , Quann Erin E. , Auestad Nancy
Published: June 2021

Many Americans have vitamin D intakes below the Adequate Intake level. This study examined food sources of vitamin D using NHANES, 2003–2006, data and the recently released Vitamin D Addendum to USDA Food and Nutrient Database for Dietary Studies 3.0. Vitamin D intakes were determined for all subjects 2 yrs and older with complete, reliable 24‐hr recall interviews on Day 1 (n=16,822). With food mixtures/mixed dishes classified by their main ingredient, the percent contributions to vitamin D intake from dairy products; grain products; meat, poultry, and fish; eggs, legumes, nuts and seeds; fruits and fruit juices; vegetables; and other foods was, 68.1,12.7, 9.2, 3.0, 3.0, 0.6, and 3.5%, in children 2–18 yrs (n=7,332), and 46.0, 10.7, 23.4, 5.7, 3.6, 1.4, and 9.1% in adults 19+ yrs (n=9,490), respectively. Using current food composition data, fluid milk/milk drinks provided 49.7, 65.8, and 43.2% of vitamin D intake in all subjects 2+ yrs, children 2–18 yrs and adults 19+ yrs, respectively. While still the primary source, a lower percent of daily vitamin D was provided by milk/milk drinks in Non‐Hispanic Black as compared to Non‐Hispanic White and Mexican American subjects (2–18 yrs: 57.0 vs. 67.9 and 65.1 %, respectively; and 19+ yrs: 28.7 vs. 45.3 and 48.5%, respectively). In conclusion, milk and milk products are the primary sources of vitamin D from food in the U.S. diet. (Supported by the National Dairy Council)
USDA Nutrition Evidence Library: Support to the 2010 Dietary Guidelines Advisory Committee
Authors: MacNeil Patricia Carrera , Lyon Joan , Altman Jean M. , Blum‐Kemelor Donna , Essery Eve V. , Fungwe Thomas V. , McGrane Mary , Obbagy Julie E. , Spahn Joanne , Wong Yat Ping
Published: June 2021

The USDA Nutrition Evidence Library (NEL) aims to be a trusted national source of evidence‐based research to inform nutrition policies and programs. NEL is also an on‐line resource that empowers scientists and decision makers by illuminating the strength of the evidence supporting nutrition policy and identifying research opportunities to facilitate improvement of Americans’ health and well‐being. The Dietary Guidelines Advisory Committee (DGAC) is charged with reviewing current science related to nutrition and health, and providing recommendations for the Dietary Guidelines for Americans. An integral part of NEL's mission is to provide ongoing support for the DGAC's scientific review process. This presentation describes the evidence‐based methodology used to support the 2010 DGAC effort. The 2010 DGAC divided the broad Dietary Guidelines into seven major topic areas, which paralleled seven subcommittees. NEL staff managed the systematic review process for each. This included assisting with question formulation; developing and implementing literature search plans; evaluating methodological rigor of studies; preparing portfolios of evidence worksheets, overview tables, and evidence summaries; and ensuring compliance with the NEL methodology each step of the way. NEL ensured that the 2010 DGAC scientific review process was consistent, rigorous, transparent, unbiased, and reproducible.
Prevention of Fas‐Induced Liver Injury by cytosolic Phospholipase A2α
Authors: Li Guiying , Han Chang , Wu Tong
Published: June 2021

The goal of this study was to explore the role of cytosolic phospholipase A2α (cPLA2α) in Fas‐mediated apoptosis, in vivo. We generated transgenic (Tg) mice with targeted expression of cPLA2α in the liver by using the albumin promoter‐enhancer driven vector. The cPLA2α Tg mice developed normally under normal housing conditions. Within 10 h of Jo2 injection, 100% wild type mice died of acute hemorrhagic liver failure; in contrast only 20% cPLA2α Tg mice died during the same time period (p<0.01). 4 hours after Jo2 challenge, the wild type mice showed significantly higher serum ALT and AST levels, caspase‐8, 9, 3 activity, more TUNEL‐positive hepatocytes and more prominent liver tissue damage when compared to the cPLA2α Tg mice (p<0.01). Higher levels of EGFR, gp130, phosphorylated PTEN, phosphorylated Akt, Akt, phosphorylated STAT3, STAT3, Mcl‐1 were observed in the liver tissues from the cPLA2α Tg mice than in the wild type mice. Pretreatment of wild type mice with the cPLA2 inhibitor, AACOCF3 (3 mg/kg body weight), aggravated Jo2‐mediated liver injury and hepatocyte apoptosis. In parallel, pretreatment of cPLA2α Tg mice with the STAT3 inhibitor, Stattic (25 mg/kg body weight), exacerbates Jo2‐mediated liver injury and hepatocyte apoptosis. These results demonstrate that hepatocyte cPLA2α protected against Fas‐induced liver injury at least in part through activation of STAT3 pathway.
A role for IFITM proteins in angiogenesis
Authors: Popson Stephanie Ann , Hughes Christopher C.W.
Published: June 2021

The Interferon Inducible Transmembrane (IFITM) proteins were originally identified as part of a multi‐protein complex on the surface of lymphocytes that mediates homotypic adhesion and the transmission of antiproliferative signals. Recently, we have identified the IFITMs as regulators of endothelial cell (EC) sprouting in vitro, and angiogenesis in vivo. Angiogenesis is a multistep process whereby new blood vessels are formed from the preexisting vasculature in response to various angiogenic stimuli. During angiogenesis, EC shift from a quiescent phenotype, to migrating and proliferating cells as they sprout from parent vessels, and back to quiescent cells as the new vessels mature and stabilize. Both IFITM1 and IFITM2 are rapidly induced as EC sprout in vitro and form lumenized vessels, and knockdown of either disrupts both sprouting and lumen formation. Using a vascular bed xeno‐transplant model, we have also identified a role for the human IFITMs during in vivo angiogenesis. Immunohistochemical staining of multiple human tissues identified widespread IFITM expression on vessels. Our data suggest that IFITMs may regulate the transition of EC from a quiescent to an angiogenic state.
Cleaved high molecular weight kininogen accelerates endothelial progenitor cell senescence via induction of reactive oxygen species
Authors: Dai Jihong , Wu Yi
Published: June 2021

High‐molecular‐weight kininogen (HK) is a component of the plasma kallikrein‐kinin system (KKS) and plays a central role in the activation of this system. Upon cleavage by plasma kellikrein, HK is converted into cleaved form of HK (HKa), which gains a function of antiadhesion and antiangiogenesis. In this study, we further investigated whether HKa regulates the functions of endothelial progenitor cells (EPCs). Our observations demonstrate that HKa at 50 mM markedly inhibited colony formation as well as proliferation of EPCs as assessed by BrdU incorporation assay. This effect did not result from its antiadhesive activity and induction of apoptosis, because HKa failed to inhibit EPC adhesion to collagen surfaces and did not increase the percentage of apoptotic cells. In the absence of HKa, EPCs became slightly senescent as determined by acidic ¦Â‐galactosidase staining. HKa, however, significantly accelerated the onset of EPC senescence. Since reduced form of glutathione, an ROS scavenger, prevented the increase in acidic ¦Â‐galactosidase‐positive cells, HKa acceleration of EPC senescence is through ROS production. Taken together, the present study demonstrates a novel role for HKa in inhibition of EPC function, and suggests the likelihood for HKa serving as endogenous inducer of EPC dysfunction by accelerating the onset of EPC senescence.
Gene expression analysis of Mas‐related gene receptors in the non‐inflamed and inflamed murine ileum
Authors: Avula Leela Rani , Knapen Dries , Van Op den bosch Joeri , Vergauwen Lucia , Blust Ronny , Van Nassauw Luc , Timmermans Jean‐Pierre
Published: June 2021

Due to the lack of data on the expression of Mas‐related gene receptors (Mrgs), a family of G protein‐coupled orphan receptors, we investigated their expression in the non‐inflamed and inflamed murine ileum. We performed a gene expression analysis using the Agilent Whole‐Mouse Genome Oligo Microarray. Relative expression levels of 20 Mrgs sequenced in mouse, were analysed using Real‐Time PCR, and immunohistochemical analyses with antibodies against MrgE and F were performed. Microarray analyses resulted in ~3000 and ~5000 differentially expressed genes in the acute phases of intestinal schistosomiasis and TNBS‐induced ileitis, respectively. No altered gene expression of any of the Mrgs was detected, whereas Real‐Time PCR revealed significant increased expression of MrgB1 and D during intestinal schistosomiasis and of MrgA2 during ileitis. The expression of MrgE, F, G, H and X1 was significantly reduced during ileitis. MrgX2 was found to be only expressed during inflammation. MrgE and F were both detected in a subpopulation of enteric neurons. In conclusion, the results indicate that, in accordance to what has been proposed earlier, some Mrgs appear to be involved in intestinal inflammation.
Myxopyronin induced bacterial RNA polymerase dynamics and computer aided drug discovery
Authors: Swift Robert V , Amaro Rommie E
Published: June 2021

Bacterial DNA‐dependent RNA polymerase (RNAP) is composed of a core of five subunits with the composition, 2α, β, β′, and ω. Each subunit has a highly conserved sequence among bacteria that is not shared among eukaryotes, characteristics that allow development of broad‐spectrum antibiotics. Here, we use two 175ns molecular dynamics simulations of MYX‐bound and unbound RNAP to show that MYX binding likely leads to an overall decrease in RNAP mobility and shortens the average distance and root mean square fluctuations between the β and β′ domains by one angstrom. These results are consistent with the view that MYX binding prevents opening of the DNA binding gate formed by the β and β′ domains, disallowing DNA binding and leading to inhibition. Additionally, we report a set of computationally predicted inhibitors found by integrating the molecular dynamics generated RNAP conformational distributions with a physics‐based virtual screen of the NCI diversity set.
The Role of MUC1 Splice Variants in Cellular Inflammation
Authors: Imbert Yoannis , Klinge Carolyn M
Published: June 2021

MUC 1 is a transmembrane mucin that contains a region of 30–100 variable number of tandem repeats (VNTR) where the majority of the O‐glycosylation sites are found. Two full length variants, MUC1/B and MUC1/A, differ by the inclusion of 27 bp from intron 1 in MUC1/A and a SNP, but have identical intracellular cytoplasmic domain (CTD) sequences. The frequency of the MUC1/A splice variant expression was lower in patients with non‐Sjögren's aqueous deficient dry eye compared to controls, suggesting a role for MUC1/A in the symptoms associated with dry eye disease. The objective of the present study was to clone the MUC1/A splice variant and determine whether the MUC1/A and MUC1/B splice variants differentially modulate inflammatory responses in transfected cells.

Methods

The additional 27bp present in the MUC1/A variant were cloned into the MUC1/B construct. MUC1/A and MUC1/B constructs were transiently transfected into COS‐7 cells. After 48 hrs the cells were treated with 10ng/ml TNFα for 4 hrs. The levels of IL1‐beta and IL8 were measured using real‐time RT‐PCR.

Results

Here we report that MUC1/B and MUC1/A splice variants show equal plasma membrane localization but differ in their ability to modulate the TNFα‐induced inflammatory response. The MUC1/B splice variant, and not MUC1/A, inhibited the induction of IL1‐beta and IL8 expression by TNFα. These data suggest that MUC1/B has a stronger anti‐inflammatory activity.
Hypoxia and adrenergic function: HIF1a, a potential regulatory switch
Authors: Wong Dona Lee , Tai T.C. , Wong‐Faull David C. , Claycomb Robert
Published: June 2021

Stress hormones epinephrine (epi) and cortisol, initiate behavioral and physiological changes for coping with and overcoming stress. Knowledge of molecular events whereby stress controls epi may permit early intervention in stress‐related disorders. Immobilization stress (IMMO) in rats controls epi released from the adrenal medulla, the source of circulating epi, in part through biosynthesis by phenylethanolamine N‐methyltransferase (PNMT). IMMO stress regulates PNMT through transcriptional activators, Egr‐1 and Sp1. Hypoxia (5% O2) also activates PNMT via Egr‐1 and Sp1 and HIF1a, a factor controlling genes under hypoxic stress. A hypoxia response element exists in the proximal PNMT promoter but HIF1a seems to mediate PNMT promoter transcriptional activity indirectly by stimulating Egr‐1 and Sp1. If Egr‐1 and Sp1 elements in the promoter are mutated or Egr‐1or Sp1 siRNA is present in PC12 cells harboring a PNMT promoter‐luciferase construct, promoter stimulation by hypoxia or HIF1a is prevented. Selective inhibitors of cAMP and PLC as well as PKA, PKC, PI3K, ERK1/2 and p38 block hypoxia stimulated promoter activity and the rise in Egr‐1 and Sp1. Currently HIF1a siRNA effects on hypoxic induction of the PNMT promoter and Egr‐1 and Sp1 are being investigated. Under stress HIF1a may act as a transcriptional switch elevating Egr‐1 and Sp1, and thereby PNMT expression.

Emerald Foundation, Sobel and Keller Fund, McLean Hospital
Fluorine Magnetic Resonance Assessment of siRNA Delivery to Endothelial Cells via Perfluorocarbon Nanoparticles
Authors: Myerson Jacob Wheatley , Kaneda Megan M , Lanza Gregory M , Wickline Samuel A
Published: June 2021

RNA interference mediated by the introduction of exogenous siRNAs has been a powerful tool for the modulation of gene expression. The ability to track siRNA delivery would be advantageous for future in vivo studies.

To design a synthetic vehicle to serve as both an siRNA delivery agent and an imaging agent, perfluorocarbon nanoparticles (PFC NP) were loaded with the cationic lipid DOTAP in the lipid monolayer to form transfection complexes with siRNA to the VCAM‐1 gene. siRNA loading onto nanoparticles was measured via PAGE gel. Mouse 2F2B endothelial cells were incubated with PFC NP/siRNA for 4h. 19F MR spectroscopy was performed at 11.7 T to determine the number of NP bound to each cell. mRNA levels were measured 48h after transfection to assess knockdown.

The selected loading conditions resulted in 645 siRNA molecules per nanoparticle. Incubation with 2F2B cells led to a roughly linear increase in bound nanoparticles over the range of concentrations tested. In one condition which resulted in a 72% reduction in VCAM‐1 mRNA levels, 19F spectroscopy data reported 5563 particles per cell.

For future in vivo studies, a trackable delivery agent would aid in the determination of localization of siRNA delivery to specific tissues. To this end, we have developed a nanoparticle which is able to deliver siRNA to mouse endothelial cells and whose signal can be detected by 19F MR spectroscopy and imaging.
Rat hepatic cytochrome P450 oxidoreductase is up‐regulated by low dose dexamethasone and down‐regulated by adrenalectomy
Authors: Grey Anne K Mullen , Riddick David S
Published: June 2021

Microsomal cytochrome P450 (CYP) enzyme activity requires cytochrome P450 oxidoreductase (POR). We studied rat hepatic POR after adrenalectomy (ADX) since some CYP activities are decreased by ADX. The effects of ADX or glucocorticoids on POR are not clear. Male ADX or SHAM‐ADX rats were treated with dexamethasone (DEX) once 6 h before euthanasia, 4 days post‐ADX (acute), or once daily in the third week post‐ADX (subacute). A DEX time‐course study was also performed in intact rats. Hepatic POR was measured by quantitative RT‐PCR and by immunoblot. Hepatic POR protein but not mRNA was decreased at 4 days and 3 weeks post‐ADX. Subacute DEX dosing (1 mg/kg) returned hepatic POR protein in ADX rats to SHAM levels; a single dose (1.5 mg/kg) showed a similar trend. DEX induced hepatic POR mRNA in SHAM and ADX rats in both dosing regimens. DEX is a selective glucocorticoid receptor (GR) agonist at low doses but activates the pregnane X receptor (PXR) at higher doses. CYP3A23, a PXR target gene, was measured to determine whether DEX activated the PXR. Hepatic CYP3A23 mRNA was not induced up to 27 h post‐DEX in the time‐course but showed a modest 30% increase with subacute DEX dosing in SHAM rats. In this study, DEX induced POR mRNA and protein but not CYP3A23 mRNA, suggesting a role for GR in POR regulation. Further studies are needed to determine how DEX at low doses acts to induce the obligate redox partner for all microsomal CYPs. [Support: CIHR]
MicroRNAs negatively regulate vitamin D receptor and cytochrome P450 3A4 enzyme pathway
Authors: Pan Yuzhuo , Gao WenQing , Yu Aiming
Published: June 2021

CYP3A4 is an important hemoprotein that metabolizes many drugs. To understand posttranscriptional regulation of CYP3A4 and role of microRNAs (miRNAs) in drug metabolism, we found that two miRNAs might control posttranscriptional and transcriptional regulation of CYP3A4 through their actions on 3′UTR of CYP3A4 and vitamin D receptor (VDR/NR1I1). Luciferase assays showed that CYP3A4 3′UTR‐luciferase activity was decreased significantly after transfected with plasmid expressing miR‐27b or mmu‐miR‐298, whereas the activity was unchanged in cells transfected with miR‐122a or miR‐328. Disruption of corresponding miRNA response element (MRE) within CYP3A4 3′UTR led to 2‐ to 3‐fold increase in luciferase activity. Immunoblot analyses indicated that CYP3A4 protein expression was down‐regulated over 40% by miR‐27b and mmu‐miR‐298 in LS‐180 and PANC1 cells. Down‐regulation of VDR by the two miRNAs was supported by luciferase assay, immunoblot and qPCR analyses. Furthermore, Overexpression of miR‐27b or mmu‐miR‐298 resulted in lower cell sensitivity to cyclophosphamide. Together, these findings suggest that noncoding miRNAs may regulate CYP3A4 and VDR protein expression at the posttranscriptional level, and consequently affect drug‐metabolizing capacity. This project was supported by UB Interdisciplinary Research Development Fund, and in part by NIH R01DA021172.
Human CYP3A Expression under Hypertonic Stimuli In Vivo
Authors: Chuang Andrew I , Yang Mingdong , Dalvi Pooja , Gonzalez Frank J , Harper Patricia A , Ito Shinya
Published: June 2021

We previously showed that human CYP3A expression in vitro is under the transcriptional control mediated by NFAT5, which is a tonicity responsive transcriptional factor. Interestingly, hypertonicity increases CYP3A expression, while hypotonicity decreases it, suggesting constitutive regulation roles of the NFAT5‐CYP3A pathway. In contrast, mouse Cyp3a was unresponsive to ambient hypertonicity in vitro. In the present study, our objective was to show if human CYP3A expression can be induced by hypertonic stimuli in vivo. The humanized CYP3A4–3A7 transgenic mice were subjected to 4 cycles of 24h water deprivation every other day, or to a 7‐day high‐salt diet (8% salt content, HSD), that mimics the dehydration and hypertonic luminal content in the intestine, respectively. In the kidney of mice undergoing the cyclic dehydration, NFAT5‐regulated BGT1 mRNA was significantly increased compared to control (5.9±1.1 fold: M±SEM; p=0.003). Fold‐changes of the CYP3A4 and 3A7 were 2.5±0.5 (p=0.02) and 0.7±0.2 (p=0.13), respectively. In mice after HSD, fold‐induction of the duodenal BGT1, CYP3A4 and 3A7 expressions were 25.2±8.2 (p=0.02), 4.7±0.9 (p=0.01) and 2.0±0.3 (p=0.08), respectively. Our findings suggest that human CYP3A transcription is under the influence of ambient tonicity. Direct involvement of NFAT5 in vivo awaits further studies.

CIHR supported
Differential role of ginkgolide A and bilobalide in CYP3A and CYP2B6 induction by Ginkgo biloba extract in primary cultures of human hepatocytes
Authors: Lau Aik‐Jiang , Rajaraman Ganesh , Baucom Christie , Yang Guixiang , Chang Thomas K. H.
Published: June 2021

Ginkgo biloba extract and ginkgolide A induce CYP3A and CYP2B6 in human hepatocytes, but conflicting data exist for the effect of bilobalide on CYP3A expression. Also, it is unknown which chemicals contribute to these effects by the extract. We assessed in human hepatocytes the role of ginkgolide A and bilobalide in the modulation of CYP3A and CYP2B6 expression by G. biloba extract, and compared the effect of the extract and chemicals on these enzymes. As analyzed by HPLC, LC‐MS, and real‐time PCR, control analysis indicated that rifampicin increased CYP3A‐mediated testosterone 6β‐hydroxylation and CYP3A4 mRNA levels, and CITCO increased CYP2B6‐mediated bupropion hydroxylation and CYP2B6 mRNA levels. G. biloba extract (25–200 μg/ml) induced CYP3A to a greater extent than CYP2B6. At a level present in 100 μg/ml of extract, ginkgolide A (1.1 μg/ml) increased CYP3A but not CYP2B6 expression, whereas bilobalide (2.8 μg/ml) had no effect. Ginkgolide A (1.1–10 μg/ml) induced CYP3A to a greater extent than CYP2B6, but bilobalide (5–20 μg/ml) induced both enzymes to a similar extent. CYP3A induction by bilobalide was less than that by ginkgolide A. In summary, G. biloba extract, ginkgolide A, and bilobalide differentially induced CYP3A and CYP2B6. Among the two chemicals, only ginkgolide A contributed to CYP3A induction by the extract, whereas neither of them accounted for CYP2B6 induction. [Supported by CIHR and MSFHR]
Gender dimorphic formation of Mallory‐Denk bodies and the role of xenobiotic metabolism and oxidative stress
Authors: Snider Natasha T , Hanada Shinichiro , Brunt Elizabeth M , Hollenberg Paul F , Omary M. Bishr
Published: June 2021

Mallory‐Denk bodies (MDBs) are hepatocyte cytoplasmic inclusions associated with alcoholic steatohepatitis. Understanding the significance of MDBs and devising ways of manipulating their formation has clinical relevance to protein aggregation diseases beyond liver. Current appreciation of MDBs comes from established models whereby feeding mice the porphyrinogenic compound 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) induces inclusions resembling human MDBs. Given the significant gender differences in predisposition to liver injury, we hypothesized that gender is an important factor in MDB formation. Male DDC‐fed mice formed significantly more MDBs, which correlated with hepatocyte apoptosis, fat globules and lipid peroxidation. Female mice had significantly less MDBs and oxidative stress, but elevated ductular reaction and protoporphyrin‐IX accumulation. There were significant gender differences in the expression and activity of cytochrome‐P450 (CYP) enzymes before and after DDC treatment, resulting in differences in formation of a CYP3A‐derived DDC metabolite, which may contribute to oxidative stress. The data provide a mechanistic explanation for the gender dimorphic formation of MDBs and aid in understanding how inter‐individual differences in microsomal metabolism may alter susceptibility to diseases underlying protein aggregation. Support: DK52951 and UL1RR024986.
Species dependent substrate specificity of the human and mouse OATP2B1/Oatp2b1
Authors: Pacyniak Erik K. , Roth Megan , Guo Grace L. , Hagenbuch Bruno
Published: June 2021

Knockout mouse models are valuable tools to investigate the role of transporters in drug disposition and hepatotoxicity. However, due to limited understanding of species differences in substrate specificity, extrapolation of transporter data between species can be problematic. In vitro expression systems are a critical tool to screen for substrates and to delineate differences between human and mouse transporters. Therefore, the objective of the current study was to compare the substrate specificities of human OATP2B1 to mouse Oatp2b1. We used a CHO cell line stably expressing OATP2B1 and HEK293 cells transiently expressing Oatp2b1 and measured uptake of radiolabeled substrates. Our results confirm the model compounds estrone‐3‐sulfate and bromosulfophthalein as substrates for both the human and the mouse transporter. Additionally, we identified the anticancer drug paclitaxel and the anti‐HIV drug saquinavir as substrates for human OATP2B1. Mouse Oatp2b1 demonstrated a broader substrate specificity and transported numerous substrates including docetaxel, oleic acid, methotrexate, paclitaxel, ritonavir and saquinavir. Based on these data we conclude that the OATP2B sub‐family contains multispecific xenobiotic transporters with the mouse Oatp2b1 having a broader substrate specificity than its human ortholog.
Global gene expression profile in the liver and blood of rats administered a toxic dose of acetaminophen
Authors: Joseph Pius , Sellamuthu Rajendran , Li Shengqiao , Kashon Michael , Luster Michael , Umbright Christina
Published: June 2021

The objective of the study was to compare global gene expression profile in the target organ (liver) and a surrogate tissue (blood) in rats administered a toxic dose of the hepatotoxic drug, acetaminophen (AP). Blood and liver were obtained from rats 24‐hours following administration of an acute toxic dose of AP. AP‐induced hepatotoxicity was evidenced from a significant elevation in the serum level of transaminases and the presence of centrilobular necrosis in the liver of the rats. Microarray analysis of global gene expression profile demonstrated significant differential expression of a large number of genes in the liver and blood of the AP administered rats compared with the controls. Genes belonging to several biological processes that are known to play definite role in the hepatotoxicity induced by AP were found differentially expressed both in the liver and blood of the rats. Cellular processes such as inflammatory response, oxidative stress, apoptosis, and energy metabolism were affected in the liver and blood of the AP‐administered rats. The significant similarity noticed in global gene expression profile between liver and blood of the AP administered rats demonstrated that blood gene expression profiling is an appropriate surrogate approach not only to detect target organ toxicity but also to understand the mechanisms underlying the toxicity induced by AP ‐ a model hepatotoxic chemical.
Methamphetamine oxidatively modifies the E3 ligase parkin and attenuates the activity of 26S proteasome in vivo
Authors: Moszczynska Anna , Yamamoto Bryan K
Published: June 2021

One of the mechanisms involved in METH neurotoxicity is oxidative stress. Oxidatively damaged proteins are degraded by the ubiquitin proteasome system (UPS) which consists of the 26S proteasome and enzymes such as the E3 ligases. Disruption of the UPS promotes toxic accumulation of proteins. The objective of the present study was to examine the effect of METH on the E3 ligase parkin and proteasome in vivo. To address the hypothesis that METH decreases parkin function through oxidative stress and impairs function of the 26S proteasome, the levels of parkin, 4‐hydroxynonenal‐ and nitrosocysteine‐parkin conjugates as well as activity of 20S and 26S proteasome were examined in synaptosomes from male Sprague Dawley rats killed 1 h after the last injection of METH (4 × 10 mg/kg, 2 h apart, i.p.) or saline. METH caused a 41% decrease in parkin levels, a 28% increase in 4‐HNE‐modified parkin and a 51% decrease in 26S activity in striatal synaptosomes as compared to saline controls. Pretreatment of rats with vitamin E significantly diminished the METH‐induced changes. These results suggest that (1) there is lipid peroxidation‐mediated oxidative damage to parkin that leads to its subsequent degradation within 1 h after last dose of toxic METH and (2) decreases in parkin and 26S proteasome function might contribute to METH neurotoxicity via accumulation of oxidatively damaged proteins. Supported by NIH grants DA023085 and DA07606
H2O2 modulates the transcriptional regulation of the 3′IgH regulatory region via the NFκB Pathway
Authors: Romer Eric J , Sulentic Courtney E.W.
Published: June 2021

Transcription of the immunoglobulin heavy chain (IgH) in B lymphocytes is governed by a 3′IgH Regulatory Region (3′IgHRR) and is an essential step in antibody production. Lymphocytes are known to be functionally affected by exposure to reactive oxygen species (ROS) such as H2O2. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in IgH transcriptional activity. This study demonstrates that μM concentrations of H2O2 induces a biphasic effect on 3′IgHRR activity and that expression of a degradation resistant Inhibitory κB (IκBαAA) can suppress H2O2‐mediated activation of the 3′IgHRR. Furthermore, H2O2 concentrations that suppressed 3′IgHRR activity appeared to induce the formation of a proteolytic cleavage product of IκBα which has been shown to exhibit a stronger inhibitory function than the non‐cleaved native IκBα. These observations suggest that exposure of B lymphocytes to H2O2 can modulate IgH transcriptional activity in a concentration‐dependent manner via the 3′IgHRR and modulation of the NFκB pathway (Supported by NIEHS R01ES014676).
Modulation of the human polymorphic hs1,2 enhancer within the 3′IgH regulatory region by TCDD
Authors: Sulentic Courtney E.W. , Fernando Tharu M. , Ochs Sharon
Published: June 2021

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a potent environmental toxin known to inhibit immunoglobulin (Ig) gene expression. Transcriptional regulation of the Ig heavy chain (IgH) involves the 3′IgH regulatory region (3′IgHRR) and its enhancers (hs3, hs1,2, and hs4) which contain DNA binding sites for several transcription factors including NF‐κB and dioxin response elements (DRE). TCDD induces binding of the aryl hydrocarbon receptor complex to DREs within the 3′IgHRR and inhibits murine 3′IgHRR activation in a well characterized mouse B‐cell line (CH12.LX). In humans, a polymorphism of the hs1,2 enhancer resulting in varying numbers of a 53 bp sequence has been correlated with autoimmune diseases like IgA nephropathy and Celiac disease. The repeated sequence contains κB and DRE binding sites. The objective of this study was to comparatively evaluate the effect of TCDD and LPS on human and mouse hs1,2 enhancers in the CH12.LX model. In transient luciferase studies, an increased number of repeats in the human hs1,2 enhancer increased the sensitivity to LPS. Interestingly, TCDD also markedly enhanced human hs1,2 activity and even augmented LPS‐induced activation. This starkly contrasted with TCDD‐induced inhibition of the mouse hs1,2 and 3′IgHRR in LPS‐stimulated CH12.LX cells suggesting a species difference in IgH transcriptional regulation.

(Supported by NIEHS R01ES014676)
Acetaminophen‐induced injury in HepaRG cells: a novel human cell line for studies of drug hepatotoxicity
Authors: McGill Mitchell R , Yan Huimin , Jaeschke Hartmut
Published: June 2021

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the U.S. The mechanism of APAP hepatotoxicity in rodents involves formation of a reactive metabolite, glutathione (GSH) depletion, mitochondrial dysfunction, and oncotic necrosis. To investigate these mechanisms in a human system, a metabolically competent cell line is needed. In this study, we tested the value of a human hepatoma‐derived cell line (HepaRG) for APAP toxicity studies. Cells were treated with 20 mM APAP and the time course of cell dysfunction and injury was evaluated. APAP caused a decline in cellular GSH levels to 31% of control at 6 h and 24 % at 24 h. The mitochondrial membrane potential (JC‐1) was unaffected at 6 h but was reduced to 45% of control at 12 h and 20% at 24 h. Lactate dehydrogenase (LDH) release was not observed until 24 h (38%) and 48 h (69%) indicating cell necrosis. In addition, a clear dose‐response (5–20 mM) was observed with each of these parameters. Inhibition of drug metabolism with 2% DMSO abrogated all effects. Experiments with HepG2 cells, which are not metabolically competent, showed no GSH depletion, mitochondrial dysfunction or cell injury. Conclusion: APAP toxicity in HepaRG cells mimics closely the sequence of events observed in rodents. Thus, HepaRG cells may be a useful human model for mechanistic studies of drug hepatotoxicity. (Supported by NIH grant DK070195 and Biopredic Int.)
Confirmation of the mode of action of anesthetic‐induced developmental neurotoxicity with gene expression studies
Authors: Slikker William , Shi Qiang , Guo Lei , Patterson Tucker A , Dial Stacey , Li Quan , Sadovova Natalya , Zhang Xuan , Hanig Joseph P , Paule Merle G , Wang Cheng
Published: June 2021

Ketamine, a non‐competitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist, is associated with accelerated neuronal apoptosis in the developing rodent brain. In this study, postnatal day (PND) 7 rats were treated with 20 mg/kg ketamine or saline in six successive doses (sc) at 2‐h intervals. Brain frontal cortical areas were collected 6 h after the last dose and RNA isolated and hybridized to Illumina Rat Ref‐12 Expression BeadChips containing 22,226 probes. Many of the differentially expressed genes were associated with cell death or differentiation and receptor activity. Ingenuity Pathway Analysis software identified perturbations in NMDA‐type glutamate, GABA and dopamine receptor signaling. Q‐PCR confirmed that NMDA receptor subunits were significantly up‐regulated. Up‐regulation of NMDA receptor mRNA signaling was further confirmed by in situ hybridization. These observations support our working hypothesis that prolonged ketamine exposure produces up‐regulation of NMDA receptors and subsequent over‐stimulation of the glutamatergic system by endogenous glutamate, triggering enhanced apoptosis in developing neurons. Supported in part by an IAG (224‐07‐007) between NCTR/FDA, NIEHS/NTP, CDER/FDA and NICHD.
The effects of early life exposure to citalopram on glomerular morphology and aquaporin 2 distribution in rat kidneys
Authors: Tucci Michelle A. , Swilley Sharonda , Zifodya Jerry , Lin Rick , Simpson Kim , Benghuzzi Ham
Published: June 2021

Thirty percent of infants exposed in utero to SSRIs display behavioral and physiological abnormalities in the first few weeks after birth including increased incidence of pulmonary hypertension. Rats exposed from postnatal day (PN) 1–21 to SSRIs display persistent neurobehavioral abnormalities which include severe deficits in social interactions and expression of serotonergic neurons. Disturbances in serotonin levels result in alterations in mood, sleep, sexuality, and depression. SSRIs have been developed to restore serotonin homeostasis. Adult treatment with SSRIs cause increased water resorption by increasing ADH secretion or increased aquaporin‐2 (AQ‐2) channels in the collecting ducts. The goal of this study was to investigate the lasting effects of early life SSRI exposure on AQ‐2 distribution in the kidneys. Long Evans rats ( 30 male, 30 female) were treated from PN 1–21 with 0.1 mL s.c. of either saline or citalopram (5mg/Kg, 10 mg/Kg, or 20mg/Kg). Animals were sacrificed at PN‐180. Distribution of AQ‐2 and glomeruli size was evaluated. The results show a dose dependent increase in AQ‐2 distribution within the kidney tubules without alterations in glomerular size (p<0.05). No gender differences were observed, which is in contrast to the neurobehavioral effects of early life citalopram exposure. Additional research is needed to determine if ADH secretion or an increase in corticosteroids leads to increased aquaporin‐2 distribution. Supported by RR‐017701 to IAP and KLS and MH‐084194 to RCSL.
Approaching a New Animal Model of Amyotrophic Lateral Sclerosis
Authors: Moran Jacqueline Teresa , Dayton Robert , Henning Phillip , Wang David , Klein Ronald
Published: June 2021

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease in which the premature death of motor neurons ultimately leads to fatal paralysis. The most pronounced pathological feature of ALS is the cytoplasmic deposition and aggregation of proteolytically cleaved fragments of the TAR DNA‐binding protein 43 (TDP‐43). This recent and pivotal identification of TDP‐43 in intracytoplasmic inclusions of neuronal and glial cells has revolutionized the current field of research to focus on the role of TDP‐43 in ALS pathogenesis. Previous data has demonstrated overexpression of TDP‐43 via an adeno‐associated virus vector (AAV9) in the substantia nigra of a rat yielded cytoplasmic localization of ubiquitinated TDP‐43, indicative of ALS‐like preinclusions (Klein et al., 2008). This study aims to further the understanding of ALS pathology by overexpressing TDP‐43 in either the red nucleus or forepaw motor cortex of Sprague‐Dawley rats via stereotaxic injection of an AAV9 expressing full‐length human TDP‐43. Utilizing photobeam tracking sensors and a cylinder test to monitor fluctuations of vertical and horizontal motor movements, data for TDP‐43 and GFP rats were compared to elucidate the influence of TDP‐43 overexpression in the rubrospinal tract and forepaw motor cortex. Preliminary results have not shown aberrant changes in locomotor activity thus far (4 weeks), although future analysis of the brain tissue may discern the neuropathological consequences of TDP‐43 overexpression in the red nucleus or forepaw motor cortex.
Protective Effects of Midazolam and MMB‐4 plus Atropine Sulfate in Nerve Agent Intoxication
Authors: Shih Tsung‐Ming , Koenig Jeffrey A , Tarzia Kristin A. , Skovira Jacob W. , McDonough John H.
Published: June 2021

This study utilized the guinea pig nerve agent‐seizure model to measure the protective effectiveness of midazolam, a more rapidly acting benzodiazepine, and of MMB‐4, a more efficacious oxime ChE reactivator, in comparison with current antidotes 2‐PAM and diazepam. One week after implantation of cortical electroencephalographic (EEG) electrodes, animals were pretreated with pyridostigmine bromide (0.026 mg/kg, im) 30 min prior to exposure to a 2.0 × LD50 subcutaneous dose of a nerve agent (GB, GD, GF, or VX). One min after agent exposure, animals were treated im with atropine sulfate (0.1 mg/kg) and either 2‐PAM (25 mg/kg) or MMB‐4 (26 mg/kg). At the onset of EEG seizures, a dose of diazepam (range 1.0–10.0 mg/kg, im) was administered to those animals treated with 2‐PAM or a dose of midazolam (range 0.5–5.0 mg/kg, im) to animals treated with MMB‐4. The anticonvulsant ED50 dose of midazolam (in the presence of MMB‐4) or diazepam (in the presence of 2‐PAM) was determined. The MMB‐4/midazolam combination therapy produced similar (against GD) or significantly better (against GB, GF and VX) anticonvulsant effects than did the 2‐PAM/diazepam therapy. The MMB‐4/midazolam combination also reduced the latency to seizure termination, especially in the GF‐exposed group. These results demonstrate the effectiveness of the MMB‐4/midazolam combination over current therapy for improved protection against nerve agents.
NAD+ administration can profoundly decrease the brain injury induced by hypoxia‐ischemia
Authors: He Xin , Liu Na , Xia Weiliang , Ying Weihai
Published: June 2021

Our lab has found that NAD+ treatment can markedly decrease oxidative stress‐induced cell death, and NAD+ administration can profoundly reduce the brain injury in a rat MCAO model of transient focal ischemia. The goal of this study is to test our hypothesis that NAD+ administration can also decrease the brain injury in a neonatal mouse model of hypoxia‐ischemia (H/I). We found that permanent ischemia plus 90‐min hypoxia led to significant infarct formation of the mice as assessed by TTC staining. Administration of NAD+ led to profound decreases in infarct size, assessed at either 24 or 72 hrs after H/I. Moreover, NAD+ administration can also significantly attenuate the H/I‐induced neurological deficits of the animals. In summary, our study has provided first evidence indicating that NAD+ can profoundly decrease the H/I ‐induced brain injury, suggesting great promise of NAD+ for treating both permanent and transient cerebral ischemia (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee 08JC1415400, Pujiang Scholar Program Grant, Shanghai Med‐X Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases, and by a 973 Grant 2010CB834306).
Ability of bacterial cocaine esterase to hydrolyze active cocaine metabolites, and function in the presence of commonly co‐abused drugs
Authors: Brim Remy Leigh , Sunahara Roger K , Woods James H
Published: June 2021

Cocaine toxicity is a widespread problem in the United States, responsible for over half a million emergency department visits in 2006 (DAWN, Pub No. (SMA) 08‐4339, 2006). There is currently no FDA‐approved direct pharmacotherapy for cocaine toxicity. To this end, we have developed a mutant bacterial cocaine esterase (CocE), which hydrolyzes cocaine into inert metabolites and has low immunogenic potential. CocE prevents cocaine‐induced cardiovascular and neurological toxicities in rodent models (Jutkiewicz, Ann Emerg Med 2009; Wood, Drug and Alcohol Dependence 2009). Here we describe the ability of CocE to hydrolyze the active cocaine metabolites norcocaine, cocaethylene and benzoylecgonine, and the resilience of CocE, in that it hydrolyzes cocaine in the presence of drugs commonly co‐abused with cocaine. CocE hydrolyzes norcocaine and cocaethylene with 58% and 45% of its catalytic efficiency for cocaine in vitro (respectively), as measured by a spectrophotometric assay. The catalytic efficiency of CocE is not affected by many commonly co‐abused drugs in vitro, including alcohol, phencyclidine, morphine and nicotine. Overall, these results suggest that CocE is a good candidate pharmacotherapy for cocaine toxicity in that it hydrolyzes cocaine along with toxic metabolites, and is not inhibited by high concentrations of commonly co‐abused drugs. Research supported by USPHS grants DA021416 and GM007767.
Amelioration of the acute cardiovascular effects of cocaine by a longer acting mutant cocaine esterase in freely moving rhesus monkeys
Authors: Collins Gregory Thomas , Carey Kathy Ann , Ko Mei‐Chuan
Published: June 2021

Cocaine is the most common illicit drug related to emergency room admissions, with the majority of visits due to cocaine‐associated chest pain resulting from increases in blood pressure (BP) and heart rate (HR). These studies were aimed at characterizing the capacity of a longer acting mutant form of cocaine esterase (T172R/G173Q; DM CocE) to ameliorate the cardiovascular effects of cocaine in four freely moving rhesus monkeys implanted with radio‐telemetric probes (DSI; D70‐PCT) capable of collecting continuous measures of BP and HR. DM CocE (0, 0.032, 0.1, 0.32, 1.0, or 3.2 mg/kg; IV) was administered 10 min after a dose of 3.2 mg/kg; IV cocaine (M2 was tested at 1.0 mg/kg; IV), and BP and HR were recorded for the 120 min following cocaine administration. DM CocE dose‐dependently, and rapidly decreased cocaine‐induced elevations in BP in each of the four monkeys, with saline‐like levels observed within the first 5 min. Similarly, DM CocE dose‐dependently decreased cocaine‐induced elevations in HR in three of the four monkeys, although over a slightly longer timecourse. These results suggest that DM CocE may provide a novel, and effective therapeutic for the treatment of acute cocaine toxicity.

Supported by NIDA grant DA 023213