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Chhabra R.S.,National Health Research Institute | Streicker M.,ILS Inc. | Witt K.L.,National Health Research Institute
Environmental and Molecular Mutagenesis | Year: 2012

Styrene-acrylonitrile Trimer (SAN Trimer), a by-product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2-year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN-RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose-related increases (P < 0.0001) in MN-RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical-related bone marrow toxicity. Results of the Comet assay showed significant, dose-related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical-related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer. © 2012 Wiley Periodicals, Inc.


Huang R.,U.S. National Institutes of Health | Sakamuru S.,U.S. National Institutes of Health | Martin M.T.,U.S. Environmental Protection Agency | Reif D.M.,U.S. Environmental Protection Agency | And 18 more authors.
Scientific Reports | Year: 2014

The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα β-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ERα agonists and antagonists was evaluated using a set of 39 reference compounds with known ERα activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ERα binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ERα active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ERα signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.


PubMed | Environmental Health Science and Research Bureau, Carleton University and ILS Inc.
Type: | Journal: Toxicology | Year: 2014

Bromodeoxyuridine (BrdU) is a synthetic nucleoside used to detect cellular proliferation. BrdU incorporates in the place of thymine but pairs with guanine, thereby increasing the risk of transition mutations in dividing cells. Given its mutagenicity, standard practice is to use a second cohort of animals for parallel toxicogenomics studies; however, the impact of BrdU on global gene expression is unknown. To test this, we performed a case study to determine whether the molecular mode of action of furan, a liver carcinogen, could be detected in BrdU-treated samples. We measure global hepatic gene expression using Agilent DNA microarrays in female B6C3F1 mice that were sub-chronically exposed to 0, 1, 4, or 8mg/kg bodyweight (bw) per day furan either in the presence (+BrdU) or absence (-BrdU) of BrdU. Exposure to 0.02% BrdU in drinking water for five days resulted in minimal gene expression changes. A comparison of +BrdU versus -BrdU control mice revealed only 11 probes with fold change1.5 and false discovery rate (FDR) corrected p0.05. The same comparison in the high dose group yielded only 3 differentially expressed probes. Differentially expressed gene lists generated for furan-treated versus control mice and were compared for the -BrdU and +BrdU groups. The high dose of furan had 452 shared probes and 27 and 90 unique probes for -BrdU and +BrdU groups, respectively. These differences did not impact hierarchical clustering. Further, they did not impair detection of the previously reported furan mode of action, which was well represented in the BrdU-treated samples. Taken together, we demonstrate that BrdU treatment does not mask important furan-induced transcriptional changes. We suggest that BrdU-treated mice could be used for toxicogenomic analysis, which would generally halve the number of rodents required for toxicogenomics studies. However, we also recommend that this type of case study be repeated for other chemicals before the use of BrdU-treated animals in omics studies becomes common practice.


Onoue S.,University of Shizuoka | Sato H.,University of Shizuoka | Ogawa K.,University of Shizuoka | Kojo Y.,University of Shizuoka | And 7 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2012

The main purpose of the present study was to develop a novel respirable powder (RP) formulation of cyclosporine A (CsA) using a spray-dried O/W-emulsion (DE) system. DE formulation of CsA (DE/CsA) was prepared by spray-drying a mixture of erythritol and liquid O/W emulsion containing CsA, polyvinylpyrrolidone, and glyceryl monooleate as emulsifying agent. The DE/CsA powders were mixed with lactose carriers to obtain an RP formulation of DE/CsA (DE/CsA-RP), and its physicochemical, pharmacological, and pharmacokinetic properties were evaluated. Spray-dried DE/CsA exhibited significant improvement in dissolution behavior with ca. 4500-fold increase of dissolution rate, and then, nanoemulsified particles were reconstituted with a mean diameter of 317 nm. Laser diffraction analysis on the DE/CsA-RP suggested high dispersion of DE/CsA on the surface of the lactose carrier. Anti-inflammatory properties of the inhaled DE/CsA-RP were characterized in antigen-sensitized asthma/COPD-model rats, in which the DE/CsA-RP was more potent than the RP formulation of physical mixture containing CsA and erythritol in inhibiting inflammatory responses, possibly due to the improved dissolution behavior. Pharmacokinetic studies demonstrated that systemic exposure of CsA after intratracheal administration of the DE/CsA-RP at a pharmacologically effective dose (100 μg-CsA/rat) was 50-fold less than that of the oral CsA dosage form at a toxic dose (10 mg/kg). From these findings, use of inhalable DE formulation of CsA might be a promising approach for the treatment of airway inflammatory diseases with improved pharmacodynamics and lower systemic exposure. © 2011 Elsevier B.V. All rights reserved.


Frawley R.,National Health Research Institute | DeVito M.,National Health Research Institute | Walker J.N.,National Health Research Institute | Birnbaum L.,National Health Research Institute | And 6 more authors.
Toxicological Sciences | Year: 2014

The use of brominated flame retardants and incineration of bromine-containing materials has lead to an increase in polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in the environment. Measurable amounts of PBDD/Fs have been detected in soil, seafood, and human breast milk and serum. Studies indicate that the relative potencies of some PBDD/Fs based on enzyme induction are equivalent to those of some polychlorinated dibenzo-p-dioxins and dibenzofurans. To assess the humoral immunity relative potencies of PBDD/Fs and compare them to their chlorinated analogs, female B6C3F1/N mice received a single oral exposure to 2,3,7,8-tetrachlorodibenzop- dioxin (TCDD), 2,3,7,8-tetrabromodibenzofuran (TBDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentabromodibenzofuran (1PeBDF), 1,2,3,7,8- pentachlorodibenzofuran (1PeCDF), 2,3,4,7,8-pentabromodibenzofuran (4PeBDF), 2,3,4,7,8- pentachlorodibenzofuran (4PeCDF), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (DBDCDD), or 2,3,7-tribromodibenzop-dioxin (TriBDD). Inhibition of the immunoglobulin M (IgM) antibody forming cell response was measured 4 days following immunization with sheep red blood cells. The data were fit to a Hill model to estimate the ED50 for inhibition. Expression of xenobiotic metabolizing enzyme (XME) and thyroxine transport protein (Ttr) genes in liver was measured by PCR to assess aryl hydrocarbonmediated responses. TCDD, TBDF, TCDF, 1PeBDF, 4PeBDF, 4PeCDF, andDBDCDDsuppressed the IgM antibody response and Ttr gene expression, and upregulated phase I XME genes. 1PeCDF suppressed the IgM antibody response but only upregulated phase I XME genes; TriBDD had no effect on antibody response. The rank order of potency (ED50) for these chemicals was TCDD>TBDF>4PeBDF>TCDF/4PeCDF/1PeBDF>1PeCDF. Whereas TCDD was the most potent compound tested, the brominated analogs were more potent than their chlorinated analogs, suggesting that these compounds should be considered in toxic equivalency factor evaluation and risk assessment.


Hobbs C.A.,ILS Inc. | Swartz C.,ILS Inc. | Maronpot R.,Maronpot Consulting LLC | Davis J.,ILS Inc. | And 2 more authors.
Food and Chemical Toxicology | Year: 2012

Gum ghatti is a food additive in some parts of the world, serving as an emulsifier, a stabilizer, and a thickening agent. To evaluate its genotoxic potential, we conducted Good Laboratory Practice compliant in vitro and in vivo studies in accordance with the Organisation for Economic Co-operation and Development (OECD) guidelines. No evidence of toxicity or mutagenicity was detected in a bacterial reverse mutation assay using five tester strains evaluating gum ghatti at up to 6. mg/plate, with or without metabolic activation. Gum ghatti also did not induce chromosome structural damage in a chromosome aberration assay using Chinese hamster ovary cells. To assess the ability to induce DNA damage in rodents, a combined micronucleus/Comet assay was conducted in male B6C3F1 mice. Gum ghatti was administered at 1000, 1500, and 2000. mg/kg/day by gavage once daily for 4. days and samples were collected 4. h after the final dosing. No effect of gum ghatti was measured on micronucleated reticulocyte (MN-RET) frequency in peripheral blood, or DNA damage in blood leukocytes or liver as assessed by the Comet assay. Our results show no evidence of genotoxic potential of gum ghatti administered up to the maximum concentrations recommended by OECD guidelines. © 2011 Elsevier Ltd.


Onoue S.,University of Shizuoka | Suzuki H.,University of Shizuoka | Kojo Y.,University of Shizuoka | Matsunaga S.,University of Shizuoka | And 6 more authors.
European Journal of Pharmaceutical Sciences | Year: 2014

The present study aimed to develop a self-micellizing solid dispersion (SMSD) of cyclosporine A (CsA) using an amphiphilic block copolymer, poly[MPC-co-BMA], to improve the biopharmaceutical properties of CsA. The cytotoxicity of poly[MPC-co-BMA] was assessed in rat intestinal IEC-6 cells, and the pMB was less cytotoxic than polysorbate 80, a non-ionic surfactant with a wide safety margin. SMSD/CsA was prepared using a wet-milling system, and its physicochemical properties were characterized in terms of morphology, crystallinity, dissolution, particle size distribution, and stability. The SMSD/CsA exhibited immediate formation of fine micelles with a mean diameter of ca. 180 nm when introduced into aqueous media. There was marked improvement in the dissolution behavior of the SMSD/CsA compared with amorphous CsA. Even after storage at 40 °C/75% relative humidity, the dissolution behavior of aged SMSD/CsA seemed to be almost identical to that of its freshly prepared equivalent, and CsA in aged SMSD/CsA was still in amorphous form. After oral administration of SMSD/CsA (10 mg CsA/kg) in rats, enhanced CsA exposure was observed with increases of Cmax and BA by ca. 11- and 42-fold, respectively, compared with those of amorphous CsA. The poly[MPC-co-BMA]-based SMSD formulation system might be an efficacious dosage option for CsA to achieve improvements in oral bioavailability.


Misaka S.,University of Shizuoka | Aoki Y.,University of Shizuoka | Karaki S.-i.,Japan Institute for Environmental Sciences | Kuwahara A.,Japan Institute for Environmental Sciences | And 3 more authors.
Peptides | Year: 2010

Vasoactive intestinal peptide (VIP) exerts immunomodulating and anti-inflammatory activities through its specific receptors, such as VPAC1 and 2 receptors. Previously, a stabilized VIP derivative, [R15,20,21, L17]-VIP-GRR (IK312532), was proposed as a candidate of anti-asthma drug, and a dry powder inhaler system of IK312532 was also developed for inhalation therapy with minimal systemic side-effects. In the present study, the anti-inflammatory properties of IK312532 respirable powder (RP) were characterized in an asthma/COPD-like animal model, with the use of newly developed ovalbumin (OVA)-RP for lung inflammation. Marked inflammatory events in the lung were observed after OVA-RP challenge in rats as evidenced by significant increase of inflammatory biomarkers such as eosinophil peroxidase (EPO), myeloperoxidase (MPO) and lactate dehydrogenase (LDH). However, intratracheal administration of IK312532-RP led to significant attenuation of plasma EPO, MPO and LDH activities, as well as significant reduction of recruited inflammatory cells in BALF, especially macrophages and eosinophils. In the rats pretreated with IK312532-RP, histochemical examinations revealed that the inflammatory cells infiltrating to the lung and the epithelial wall thickness decreased significantly by 85% and 58%, respectively. Thus, inhalable powder formulation of IK312532 exerts its anti-inflammatory activity by suppressing granulocyte recruitment to the lung and epithelial hyperplasia, followed by the reduction of cytotoxic peroxidases. © 2009 Elsevier Inc. All rights reserved.


Witt K.L.,U.S. National Institutes of Health | Malarkey D.E.,U.S. National Institutes of Health | Hobbs C.A.,ILS Inc. | Davis J.P.,ILS Inc. | And 4 more authors.
Environmental and Molecular Mutagenesis | Year: 2010

Following a 2005 report of chromosomal damage in children with attention deficit/hyperactivity disorder (ADHD) who were treated with the commonly prescribed medication methylphenidate (MPH), numerous studies have been conducted to clarify the risk for MPH-induced genetic damage. Although most of these studies reported no changes in genetic damage endpoints associated with exposure to MPH, one recent study (Andreazza et al. [2007]: Prog Neuropsychopharmacol Biol Psychiatry 31:1282-1288) reported an increase in DNA damage detected by the Comet assay in blood and brain cells of Wistar rats treated by intraperitoneal injection with 1, 2, or 10 mg/kg MPH; no increases in micronucleated lymphocyte frequencies were observed in these rats. To clarify these findings, we treated adult male Wistar Han rats with 0, 2, 10, or 25 mg/kg MPH by gavage once daily for 28 consecutive days and measured micronucleated reticulocyte (MN-RET) frequencies in blood, and DNA damage in blood, brain, and liver cells 4 hr after final dosing. Flow cytometric evaluation of blood revealed no significant increases in MN-RET. Comet assay evaluations of blood leukocytes and cells of the liver, as well as of the striatum, hippocampus, and frontal cortex of the brain showed no increases in DNA damage in MPH-treated rats in any of the three treatment groups. Thus, the previously reported observations of DNA damage in blood and brain tissue of rats exposed to MPH for 28 days were not confirmed in this study. Additionally, no histopathological changes in brain or heart, or elevated serum biomarkers of cardiac injury were observed in these MPH-exposed rats.


PubMed | ILS Inc. and University of Shizuoka
Type: | Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences | Year: 2016

The present study aimed to develop an inhalable self-micellizing solid dispersion of cyclosporine A (SMSD/CsA) for the direct delivery to the respiratory system with improved therapeutic efficacy and minimized systemic exposure. SMSD/CsA was obtained by wet-milling, and then jet-milled SMSD/CsA was blended with lactose carrier, producing a respirable powder of SMSD/CsA (SMSD/CsA-RP). The physicochemical, pharmacological, and pharmacokinetic properties of SMSD/CsA-RP were characterized, and the hepatotoxic and nephrotoxic potentials were investigated by biomarker analysis. Cascade impactor analysis demonstrated that SMSD/CsA-RP had high in vitro inhalation performance, with a fine particle fraction of 36%. In simulated lung fluid, the SMSD/CsA exhibited better dissolution behavior than amorphous CsA. Pretreatment with SMSD/CsA-RP resulted in significant suppression of antigen-evoked inflammatory events in rats. After intratracheal administration of SMSD/CsA-RP at a pharmacologically effective dose (100g-CsA/rat), the AUC

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