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  Subjects -> ENVIRONMENTAL STUDIES (Total: 779 journals)
    - ENVIRONMENTAL STUDIES (715 journals)
    - POLLUTION (21 journals)
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ENVIRONMENTAL STUDIES (715 journals)            First | 1 2 3 4 5 6 7 8     

International Aquatic Research     Open Access   (Followers: 3)
International Archives of Occupational and Environmental Health     Hybrid Journal   (Followers: 5)
International Environmental Agreements: Politics, Law and Economics     Hybrid Journal   (Followers: 10)
International Gambling Studies     Hybrid Journal   (Followers: 5)
International Innovation - climate     Open Access   (Followers: 1)
International innovation. Environment     Open Access  
International Journal of Acarology     Hybrid Journal   (Followers: 1)
International Journal of Advancement in Earth and Enviromental Sciences     Open Access   (Followers: 2)
International Journal of African Renaissance Studies - Multi-, Inter- and Transdisciplinarity     Hybrid Journal   (Followers: 2)
International Journal of Agricultural and Environmental Information Systems     Full-text available via subscription   (Followers: 2)
International Journal of Alternative Propulsion     Hybrid Journal   (Followers: 1)
International Journal of Applied Psychoanalytic Studies     Hybrid Journal   (Followers: 2)
International Journal of Chinese Culture and Management     Hybrid Journal   (Followers: 1)
International Journal of Corrosion     Open Access   (Followers: 10)
International Journal of Critical Infrastructures     Hybrid Journal   (Followers: 3)
International Journal of Disaster Risk Reduction     Hybrid Journal   (Followers: 6)
International Journal of Disaster Risk Science     Open Access   (Followers: 8)
International Journal of Ecological Economics and Statistics     Full-text available via subscription   (Followers: 1)
International Journal of Ecology     Open Access   (Followers: 8)
International Journal of Ecology & Development     Full-text available via subscription   (Followers: 1)
International Journal of Energy and Environmental Engineering     Open Access   (Followers: 2)
International Journal of Environment     Open Access   (Followers: 3)
International Journal of Environment and Health     Hybrid Journal   (Followers: 7)
International Journal of Environment and Pollution     Hybrid Journal   (Followers: 6)
International Journal of Environment and Sustainable Development     Hybrid Journal   (Followers: 15)
International Journal of Environment and Waste Management     Hybrid Journal   (Followers: 6)
International Journal of Environment, Workplace and Employment     Hybrid Journal   (Followers: 3)
International Journal of Environmental Engineering     Hybrid Journal   (Followers: 5)
International Journal of Environmental Health Engineering     Open Access  
International Journal of Environmental Health Research     Hybrid Journal   (Followers: 3)
International Journal of Environmental Policy and Decision Making     Hybrid Journal   (Followers: 10)
International Journal of Environmental Protection     Open Access   (Followers: 12)
International Journal of Environmental Research and Public Health     Open Access   (Followers: 14)
International Journal of Environmental Science and Technology     Hybrid Journal   (Followers: 5)
International Journal of Environmental Studies     Hybrid Journal   (Followers: 11)
International Journal of Exergy     Hybrid Journal   (Followers: 4)
International Journal of Forest, Soil and Erosion     Open Access   (Followers: 3)
International Journal of Global Environmental Issues     Hybrid Journal   (Followers: 4)
International Journal of Global Warming     Hybrid Journal   (Followers: 5)
International Journal of Greenhouse Gas Control     Partially Free   (Followers: 6)
International Journal of Health Planning and Management     Hybrid Journal   (Followers: 6)
International Journal of Hygiene and Environmental Health     Hybrid Journal   (Followers: 6)
International Journal of Logistics Research and Applications : A Leading Journal of Supply Chain Management     Hybrid Journal   (Followers: 9)
International Journal of Philosophical Studies     Hybrid Journal   (Followers: 2)
International Journal of Phytoremediation     Hybrid Journal   (Followers: 2)
International Journal of Process Systems Engineering     Hybrid Journal   (Followers: 1)
International Journal of Recycling of Organic Waste in Agriculture     Open Access   (Followers: 1)
International Journal of Regulation and Governance     Hybrid Journal   (Followers: 2)
International Journal of Reliability and Safety     Hybrid Journal   (Followers: 6)
International Journal of Renewable Energy Development     Open Access   (Followers: 6)
International Journal of Social Sciences and Management     Open Access  
International Journal of Soil, Sediment and Water     Open Access   (Followers: 8)
International Journal of Stress Management     Full-text available via subscription   (Followers: 9)
International Journal of Sustainable Construction Engineering and Technology     Open Access   (Followers: 7)
International Journal of Sustainable Engineering     Hybrid Journal   (Followers: 7)
International Journal of Sustainable Materials and Structural Systems     Hybrid Journal   (Followers: 5)
International Journal of Sustainable Society     Hybrid Journal   (Followers: 7)
International Journal of Testing     Hybrid Journal   (Followers: 1)
International Journal of the Commons     Open Access   (Followers: 3)
International Journal of Toxicology     Hybrid Journal   (Followers: 7)
International Journal of Water Resources and Environmental Engineering     Open Access   (Followers: 1)
International Review of Environmental and Resource Economics     Full-text available via subscription  
International Studies in the Philosophy of Science     Hybrid Journal   (Followers: 9)
Interventions : International Journal of Postcolonial Studies     Hybrid Journal   (Followers: 7)
IOP Conference Series: Earth and Environmental Science     Open Access   (Followers: 7)
Iranian Studies     Hybrid Journal   (Followers: 8)
Irish Educational Studies     Hybrid Journal   (Followers: 2)
Irish Journal of Earth Sciences     Full-text available via subscription  
Irish Political Studies     Hybrid Journal   (Followers: 9)
ISLE: Interdisciplinary Studies in Literature and Environment     Hybrid Journal   (Followers: 2)
Isotopes in Environmental and Health Studies     Hybrid Journal   (Followers: 2)
Israel Studies     Full-text available via subscription   (Followers: 5)
ISRN Ecology     Open Access  
ISRN Environmental Chemistry     Open Access  
Jahangirnagar University Environmental Bulletin     Open Access  
Journal of Bioremediation & Biodegradation     Open Access   (Followers: 2)
Journal of Earth Science & Climatic Change     Open Access   (Followers: 8)
Journal of Petroleum & Environmental Biotechnology     Open Access   (Followers: 2)
Journal of Advances in Environmental Health Research     Open Access  
Journal of Agricultural and Environmental Ethics     Hybrid Journal   (Followers: 9)
Journal of Agricultural Biotechnology and Sustainable Development     Open Access  
Journal of Agricultural Chemistry and Environment     Open Access  
Journal of Agriculture and Environment     Open Access  
Journal of Agriculture and Environment for International Development     Open Access   (Followers: 5)
Journal of Agrobiology     Open Access   (Followers: 2)
Journal of Applied Ecology     Hybrid Journal   (Followers: 221)
Journal of Applied Meteorology and Climatology     Full-text available via subscription   (Followers: 7)
Journal of Applied Psychoanalytic Studies     Hybrid Journal   (Followers: 1)
Journal of Applied Sciences and Environmental Management     Open Access   (Followers: 1)
Journal of Applied Sciences in Environmental Sanitation     Open Access   (Followers: 6)
Journal of Applied Toxicology     Hybrid Journal   (Followers: 10)
Journal of Applied Volcanology     Open Access   (Followers: 8)
Journal of Arid Environments     Hybrid Journal   (Followers: 8)
Journal of Asian Studies     Full-text available via subscription   (Followers: 24)
Journal of Biochemical and Molecular Toxicology     Hybrid Journal   (Followers: 5)
Journal of Black Studies     Hybrid Journal   (Followers: 2)
Journal of Chemical Ecology     Hybrid Journal   (Followers: 2)
Journal of Chemical Health and Safety     Hybrid Journal   (Followers: 3)
Journal of Climate     Full-text available via subscription   (Followers: 24)
Journal of Coastal Research     Full-text available via subscription   (Followers: 10)

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Journal Cover Journal of Applied Toxicology
   [12 followers]  Follow    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
     ISSN (Print) 0260-437X - ISSN (Online) 1099-1263
     Published by John Wiley and Sons Homepage  [1604 journals]   [SJR: 0.689]   [H-I: 47]
  • Induction of the estrogen‐responsive genes encoding choriogenin H
           and L in the liver of male medaka (Oryzias latipes) upon exposure to
           estrogen receptor subtype‐selective ligands
    • Authors: Akemi Yamaguchi; Keisuke Kato, Koji Arizono, Nobuaki Tominaga
      Pages: n/a - n/a
      Abstract: Choriogenin (Chg) H and L are estrogen‐induced chorion precursors. We measured the induction of ChgH and ChgL mRNA in the livers of male medaka fish treated with Orthoester‐2k, a selective ligand for estrogen receptor (ER) α, and 2‐(4‐hydroxyphenyl)‐5‐hydroxy‐1,3‐benzoxazole (HPHB), a selective ligand of ERβ. Although both ChgH and ChgL mRNA were induced by treatment with Orthoester‐2k or HPHB separately, their combination induced much greater expression of each Chg. ChgH expression correlated more closely with Orthoester‐2k dosage when combined with a small fixed dose of HPHB (1 μm), whereas ChgL mRNA expression was more responsive to HPHB dose when combined with a fixed dose of Orthoester‐2k (2.8 nm). Moreover, upon long‐term treatment with Orthoester‐2k, ChgH mRNA and ERα mRNA expression showed similar patterns with peak expression between days 6 and 10. These results imply that ERβ primarily regulates ChgL mRNA expression and ERα action primarily regulates ChgH mRNA expression. Thus, it is necessary to develop screening methods for fish ER subtype‐specific ligands. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-12-10T08:14:40.91655-05:0
      DOI: 10.1002/jat.3063
  • Maternal exposure to 3,3’‐iminodipropionitrile targets
           late‐stage differentiation of hippocampal granule cell lineages to
           affect brain‐derived neurotrophic factor signaling and interneuron
           subpopulations in rat offspring
    • Authors: Megu Itahashi; Hajime Abe, Takeshi Tanaka, Sayaka Mizukami, Yoh Kikuchihara, Toshinori Yoshida, Makoto Shibutani
      Abstract: 3,3’‐Iminodipropionitrile (IDPN) causes neurofilament (NF)‐filled swellings in the proximal segments of many large‐caliber myelinated axons. This study investigated the effect of maternal exposure to IDPN on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 67 or 200 ppm IDPN in drinking water from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, female offspring subjected to analysis had decreased parvalbumin+, reelin+ and phospho‐TrkB+ interneurons in the dentate hilus at 200 ppm and increased granule cell populations expressing immediate‐early gene products, Arc or c‐Fos, at ≥  67 ppm. mRNA expression in the dentate gyrus examined at 200 ppm decreased with brain‐derived neurotrophic factor (Bdnf) and very low density lipoprotein receptor. Immunoreactivity for phosphorylated NF heavy polypeptide decreased in the molecular layer of the dentate gyrus and the stratum radiatum of the cornu ammonis (CA) 3, portions showing axonal projections from mossy cells and pyramidal neurons, at 200 ppm on PND 21, whereas immunoreactivity for synaptophysin was unchanged in the dentate gyrus. Observed changes all disappeared on PND 77. There were no fluctuations in the numbers of apoptotic cells, proliferating cells and subpopulations of granule cell lineage in the subgranular zone on PND 21 and PND 77. Thus, maternal IDPN exposure may reversibly affect late‐stage differentiation of granule cell lineages involving neuronal plasticity as evident by immediate‐early gene responses to cause BDNF downregulation resulting in a reduction in parvalbumin+ or reelin+ interneurons and suppression of axonal plasticity in the mossy cells and CA3 pyramidal neurons. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-25T07:53:45.351994-05:
      DOI: 10.1002/jat.3086
  • Successful validation of genomic biomarkers for human immunotoxicity in
           Jurkat T cells in vitro
    • Authors: Peter C. J. Schmeits; Jia Shao, Danique A. Krieken, Oscar L. Volger, Henk Loveren, Ad. A. C. M. Peijnenburg, Peter J. M. Hendriksen
      Abstract: Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non‐immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)–PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non‐immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo‐e‐pyrene). Of the four potential (non‐)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non‐immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3‐dichloro‐propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo‐e‐pyrene and anti‐CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT–PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non‐immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-25T07:38:41.511308-05:
      DOI: 10.1002/jat.3079
  • Experimentally induced, synergistic late effects of a single dose of
           radiation and aging: significance in LKS fraction as compared with mature
           blood cells
    • Authors: Yoko Hirabayashi; Isao Tsuboi, Kei Nakachi, Yoichiro Kusunoki, Tohru Inoue
      Abstract: The number of murine mature blood cells recovered within 6 weeks after 2‐Gy whole‐body irradiation at 6 weeks of age, whereas in the case of the undifferentiated hematopoietic stem/progenitor cell (HSC/HPC) compartment [cells in the lineage‐negative, c‐kit‐positive and stem‐cell‐antigen‐1‐positive (LKS) fraction], the numerical differences between mice with and without irradiation remained more than a year, but conclusively the cells showed numerical recovery. When mice were exposed to radiation at 6 months of age, acute damages of mature blood cells were rather milder probably because of their maturation with age; but again, cells in the LKS fraction were specifically damaged, and their numerical recovery was significantly delayed probably as a result of LKS‐specific cellular damages. Interestingly, in contrast to the recovery of the number of cells in the LKS fraction, their quality was not recovered, which was quantitatively assessed on the basis of oxidative‐stress‐related fluorescence intensity. To investigate why the recovery in the number of cells in the LKS fraction was delayed, expression levels of genes related to cellular proliferation and apoptosis of cells in the bone marrow and LKS fraction were analyzed by real‐time polymerase chain reaction (RT‐PCR). In the case of 21‐month‐old mice after radiation exposure, Ccnd1, PiK3r1 and Fyn were overexpressed solely in cells in the LKS fraction. Because Ccnd1and PiK3r1 upregulated by aging were further upregulated by radiation, single‐dose radiation seemed to induce the acceleration of aging, which is related to the essential biological responses during aging based on a lifetime‐dependent relationship between a living creature and xenobiotic materials. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-21T04:07:24.66173-05:0
      DOI: 10.1002/jat.3088
  • Surface‐expressed insulin receptors as well as IGF‐I receptors
           both contribute to the mitogenic effects of human insulin and its
    • Authors: Anders Lundby; Pernille Bolvig, Anne Charlotte Hegelund, Bo F. Hansen, Jesper Worm, Anne Lützen, Nils Billestrup, Christine Bonnesen, Martin B. Oleksiewicz
      Abstract: There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF‐I receptors (IGF‐IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF‐I caused phosphorylation of the IR as well as IGF‐IR. Insulin exhibited mitogenicity EC50 values in the single‐digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF‐IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA‐mediated knockdown of IR and IGF‐IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF‐IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF‐IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF‐I in cells expressing more IR than IGF‐IR, the hyper‐mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper‐mitogenic effect of X10 involves the IR as well as the IGF‐IR. These results are relevant for preclinical safety assessment of developmental insulin analogues. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-21T03:01:09.888264-05:
      DOI: 10.1002/jat.3082
  • Tributyltin alters secretion of interleukin 1 beta from human immune cells
    • Authors: Shyretha Brown; Margaret Whalen
      Abstract: Tributyltin (TBT) has been used as a biocide in industrial applications such as wood preservation, antifouling paint and antifungal agents. Owing to its many uses, it contaminates the environment and has been found in human blood samples. Interleukin‐1 beta (IL‐1β) is a pro‐inflammatory cytokine that promotes cell growth, tissue repair and immune response regulation. Produced predominately by both monocytes and macrophages, IL‐1β appears to increase the invasiveness of certain tumors. This study shows that TBT modifies the secretion of IL‐1β from increasingly reconstituted preparations of human immune cells. IL‐1β secretion was examined after 24‐, 48‐h or 6‐day exposures to TBT in highly enriched human natural killer (NK) cells, monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs), PBMCs, granulocytes and a preparation combining both PBMCs and granulocytes (PBMCs+granulocytes). TBT altered IL‐1β secretion from all of the cell preparations. The 200 nM concentration of TBT normally blocked the secretion of IL‐1β, whereas lower concentrations (usually 5–50 nM) elevated secretion of IL‐1β. Examination of the signaling pathway(s) responsible for the elevated secretion of IL‐1β was carried out in MD‐PBMCs. Pathways examined were IL‐1β processing (Caspase‐1), mitogen‐activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB). Results indicated that MAPK pathways (p44/42 and p38) appear to be the targets of TBT that lead to increased IL‐1β secretion from immune cells. These results from human immune cells show IL‐1β dysregulation by TBT is occurring ex vivo. Thus, the potential for in vivo effects on pro‐inflammatory cytokine levels may possibly be a consequence of TBT exposures. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-07T22:41:26.682232-05:
      DOI: 10.1002/jat.3087
  • 2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin induces premature
           senescence of astrocytes via WNT/β‐catenin signaling and ROS
    • Authors: Xiaoke Nie; Lingwei Liang, Hanqing Xi, Shengyang Jiang, Junkang Jiang, Cuiying Tang, Xipeng Liu, Suyi Liu, Chunhua Wan, Jianya Zhao, Jianbin Yang
      Abstract: 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental contaminant that could exert significant neurotoxicity in the human nervous system. Nevertheless, the molecular mechanism underlying TCDD‐mediated neurotoxicity has not been clarified clearly. Herein, we investigated the potential role of TCDD in facilitating premature senescence in astrocytes and the underlying molecular mechanisms. Using the senescence‐associated β‐galactosidase (SA‐β‐Gal) assay, we demonstrated that TCDD exposure triggered significant premature senescence of astrocyte cells, which was accompanied by a marked activation of the Wingless and int (WNT)/β‐catenin signaling pathway. In addition, TCDD altered the expression of senescence marker proteins, such as p16, p21 and GFAP, which together have been reported to be upregulated in aging astrocytes, in both dose‐ and time‐dependent manners. Further, TCDD led to cell‐cycle arrest, F‐actin reorganization and the accumulation of cellular reactive oxygen species (ROS). Moreover, the ROS scavenger N‐acetylcysteine (NAC) markedly attenuated TCDD‐induced ROS production, cellular oxidative damage and astrocyte senescence. Notably, the application of XAV939, an inhibitor of WNT/β‐catenin signaling pathway, ameliorated the effect of TCDD on cellular β‐catenin level, ROS production, cellular oxidative damage and premature senescence in astrocytes. In summary, our findings indicated that TCDD might induce astrocyte senescence via WNT/β‐catenin and ROS‐dependent mechanisms. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-07T22:08:59.991905-05:
      DOI: 10.1002/jat.3084
  • Acute toxicity of 50 metals to Daphnia magna
    • Authors: Akira Okamoto; Masumi Yamamuro, Norihisa Tatarazako
      Abstract: Metals are essential for human life and physiological functions but may sometimes cause disorders. Therefore, we conducted acute toxicity testing of 50 metals in Daphnia magna: EC50s of seven elements (Be, Cu, Ag, Cd, Os, Au and Hg) were  100,000 µg l−1; and. 7 elements (Ti, Zr, Bi, Nb, Hf, Re and Ta) did not show EC50 at the upper limit of respective aqueous solubility, and EC50s were not obtained. Ga, Ru and Pd adhered to the body of D. magna and physically retarded the movement of D. magna. These metals formed hydroxides after adjusting the pH. Therefore, here, we distinguished this physical effect from the physiological toxic effect. The acute toxicity results of 40 elements obtained in this study were not correlated with electronegativity. Similarly, the acute toxicity results of metals including the rare metals were also not correlated with first ionization energy, atomic weight, atomic number, covalent radius, atomic radius or ionic radius. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-11-07T21:25:55.880885-05:
      DOI: 10.1002/jat.3078
  • RNA‐Seq‐based toxicogenomic assessment of fresh frozen and
           formalin‐fixed tissues yields similar mechanistic insights
    • Authors: Scott S. Auerbach; Dhiral P. Phadke, Deepak Mav, Stephanie Holmgren, Yuan Gao, Bin Xie, Joo Heon Shin, Ruchir R. Shah, B. Alex Merrick, Raymond R. Tice
      Abstract: Formalin‐fixed, paraffin‐embedded (FFPE) pathology specimens represent a potentially vast resource for transcriptomic‐based biomarker discovery. We present here a comparison of results from a whole transcriptome RNA‐Seq analysis of RNA extracted from fresh frozen and FFPE livers. The samples were derived from rats exposed to aflatoxin B1 (AFB1) and a corresponding set of control animals. Principal components analysis indicated that samples were separated in the two groups representing presence or absence of chemical exposure, both in fresh frozen and FFPE sample types. Sixty‐five percent of the differentially expressed transcripts (AFB1 vs. controls) in fresh frozen samples were also differentially expressed in FFPE samples (overlap significance: P 
      PubDate: 2014-11-06T04:16:58.370692-05:
      DOI: 10.1002/jat.3068
  • Impact of di‐ethylhexylphthalate exposure on metabolic programming
           in P19 ECC‐derived cardiomyocytes
    • Authors: Kristina Schaedlich; Juliane‐Susanne Schmidt, Wing Yee Kwong, Kevin D. Sinclair, Randy Kurz, Heinz‐Georg Jahnke, Bernd Fischer
      Abstract: Di(2‐ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long‐term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in‐vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml–1) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real‐time PCR (qRT‐PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-29T01:04:56.648549-05:
      DOI: 10.1002/jat.3085
  • Impacts of the feed contaminant deoxynivalenol on the intestine of
           monogastric animals: poultry and swine
    • Authors: Khaled Ghareeb; Wageha A. Awad, Josef Böhm, Qendrim Zebeli
      Abstract: Deoxynivalenol (DON) is one of the most prevalent cereal contaminants with major public health concerns owing to its high toxigenic potentials. Once ingested, DON first and foremost targets epithelial cells of the gastrointestinal tract, whose proper functioning, as the first line of defence, is of paramount importance for the host's health. Emerging evidences, summarized in this article, suggest that DON produces its toxicity primarily via activation of the mitogen‐activated protein kinases (MAPKs) signalling pathway and alteration in the expression of genes responsible for key physiological and immunological functions of the intestinal tissue of chickens and pigs. The activation of MAPKs signalling cascade results in disruption of the gut barrier function and an increase in the permeability by reducing expression of the tight junction proteins. Exposure to DON also down‐regulates the expression of multiple transporter systems in the enterocytes with subsequent impairment of the absorption of key nutrients. Other major intestinal cytotoxic effects of DON described herein are modulation of mucosal immune responses, leading to immunosupression or stimulation of local immune cells and cytokine release, and also facilitation of the persistence of intestinal pathogens in the gut. Both of the last events potentiate enteric infections and local inflammation in pigs and poultry, rendering enterocytes and the host more vulnerable to luminal toxic compounds. This review highlights the cytotoxic risks associated with the intake of even low levels of DON and also identifies gaps of knowledge that need to be addressed by future research. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-28T19:58:02.385342-05:
      DOI: 10.1002/jat.3083
  • Cellular uptake and toxicity effects of silver nanoparticles in mammalian
           kidney cells
    • Authors: Mirta Milić; Gerd Leitinger, Ivan Pavičić, Maja Zebić Avdičević, Slaven Dobrović, Walter Goessler, Ivana Vinković Vrček
      Abstract: The rapid progress and early commercial acceptance of silver‐based nanomaterials is owed to their biocidal activity. Besides embracing the antimicrobial potential of silver nanoparticles (AgNPs), it is imperative to give special attention to the potential adverse health effects of nanoparticles owing to prolonged exposure. Here, we report a detailed study on the in vitro interactions of citrate‐coated AgNPs with porcine kidney (Pk15) cells. As uncertainty remains whether biological/cellular responses to AgNPs are solely as a result of the release of silver ions or whether the AgNPs themselves have toxic effects, we investigated the effects of Ag+ on Pk15 cells for comparison. Next, we investigated the cellular uptake of both AgNPs and Ag+ in Pk15 cells at various concentrations applied. The detected Ag contents in cells exposed to 50 mg l−1 AgNPs and 50 mg l−1 Ag+ were 209 and 25 µg of Ag per 106 cells, respectively. Transmission electron microscopy (TEM) images indicated that the Pk15 cells internalized AgNPs by endocytosis. Both forms of silver, nano and ionic, decreased the number of viable Pk15 cells after 24 h in a dose‐dependent manner. In spite of a significant uptake into the cells, AgNPs had only insignificant toxicity at concentrations lower than 25 mg l−1, whereas Ag+ exhibited a significant decrease in cell viability at one‐fifth of this concentration. The Comet assay suggested that a rather high concentration of AgNP (above 25 mg l−1) is able to induce genotoxicity in Pk15 cells. Further studies must seek deeper understanding of AgNP behavior in biological media and their interactions with cellular membranes. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-28T19:21:35.91969-05:0
      DOI: 10.1002/jat.3081
  • Long‐term exposures to di‐n‐butyl phthalate inhibit body
           growth and impair gonad development in juvenile Murray rainbowfish
           (Melanotaenia fluviatilis)
    • Authors: Harpreet Bhatia; Anupama Kumar, John C. Chapman, Mike J. McLaughlin
      Abstract: The aim of the present study was to evaluate whether long‐term exposures to environmentally relevant concentrations of di‐n‐butyl phthalate (DnBP) disrupt the reproduction‐based endpoints in juvenile Murray rainbowfish (Melanotaenia fluviatilis). Fish were exposed to 5, 15 or 50 µg l−1 DnBP for 30, 60 and 90 days each, and the effects on survival, body growth, whole‐body concentrations of sex steroid hormones and gonadal development were investigated. The lowest observed effective concentration to affect the condition factor after 90 days was 5 µg l−1. Complete feminization of the gonad was noted in fish exposed to 5 µg l−1 for 90 days and to 15 and 50 µg l−1 of DnBP for 30 or 60 days. After 90 days of exposure to DnBP, the ovaries were regressed and immature as opposed to the control fish which were in early‐vitellogenic stage. Testes, present only in fish exposed to 5 µg l−1 of DnBP for 30 or 60 days, were immature in comparison to the control fish that contained testes in the mid‐spermatogenic phase. The E2/11‐KT ratio was significantly higher only after exposures to 5 µg l−1 DnBP for 90 days and 50 µg l−1 DnBP for 30 days. Our data suggest that exposures to 5 µg l−1 DnBP for 30 days did not have profound effects on body growth and gonadal differentiation of fish. However, 30 days of exposure to 15 µg l−1 could interfere with the gonad development and to 50 µg l−1 could compromise the hormonal profile of juvenile fish. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-28T01:12:32.140042-05:
      DOI: 10.1002/jat.3076
  • Organ‐specific distribution of gold nanoparticles by their surface
    • Authors: Jong Kwon Lee; Tae Sung Kim, Ji Young Bae, A. Young Jung, Sang Min Lee, Ji Hyun Seok, Hang Sik Roh, Chi Won Song, Mi Jin Choi, Jinyoung Jeong, Bong Hyun Chung, Yun‐Geon Lee, Jayoung Jeong, Wan‐Seob Cho
      Abstract: The behavior and fate of intravenously (i.v.) injected nanoparticles (NPs) can be controlled by several physicochemical factors including size, shape and surface charge. To evaluate the role of surface charge on distribution of NPs, we used neutral‐charged 15‐nm‐sized polyethylene glycol‐coated gold nanoparticles (AuNPPEG) as a core NP and carboxyl or amine groups were conjugated to AuNPPEG to generate negative (AuNPCOOH) or positive AuNP (AuNPNH2), respectively. Each type of AuNP was i.v. injected into mice (1 mg kg–1) and the concentration of Au was measured in different organs at 30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months post‐injection. The organ distribution also showed the higher deposition rate depending on their functional groups: AuNPPEG for mesenteric lymph node, kidney, brain and testis; AuNPCOOH for liver; AuNPNH2 for spleen, lung and heart. The blood circulation time and the major excretion route were different depending on their functional groups. In conclusion, functional groups conjugated on the surface of AuNPs produce differences in blood kinetics, organ distribution and elimination pattern which can be important information for directing NPs to specific organs or improving the kinetic properties. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-28T00:35:02.572138-05:
      DOI: 10.1002/jat.3075
  • Assessment of temperature‐induced hERG channel blockade variation by
    • Authors: Rahul R. Kauthale; Shruta S. Dadarkar, Raghib Husain, Vikas V. Karande, Madhumanjiri M. Gatne
      Abstract: Drug‐induced QT prolongation has been reported in humans and animals. This potentially lethal effect can be induced by drugs interacting with a cardiac potassium channel, namely hERG (human ether‐a go‐go‐related gene) leading to arrhythmia or torsade de pointes (TdP). Hence, in vitro evaluation of therapeutics for their effects on the rapid delayed rectifier current (IKr) mediated by the K+ ion channel encoded by hERG is a valuable tool for identifying potential arrhythmic side effects during drug safety testing. Our objective was to evaluate the temperature‐induced hERG channel blockade variation by human and veterinary drugs using the IonFlux 16 system. A panel of eight drugs was tested for IKr inhibition at both ambient (23 °C) and physiological (37 °C) temperatures at various concentrations using IonFlux 16, an automated patch clamp system. Our results established that both amiodarone (IC50 = 0.56 μM at 23 °C and 0.30 μM at 37 °C) and β‐estradiol (IC50 = 24.72 μM at 23 °C and 8.17 μM at 37 °C) showed a dose‐dependent IKr blockade with a higher blockade at 37 °C. Whereas, blockade of IKr by both ivermectin (IC50 = 12.52 μM at 23 °C and 24.41 μM at 37 °C) and frusemide (IC50 = 12.58 μM at 23 °C and 25.55 μM at 37 °C) showed a dose‐dependent IKr blockade with a lower blockade at 37 °C. Gentamicin, enrofloxacin, xylazine and albendazole did not block IKr at both the assessed temperatures. Collectively, these results demonstrate that the effect of temperature variation should be taken into consideration during the evaluation of test drugs for their hERG channel blockade potential. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-28T00:14:26.431173-05:
      DOI: 10.1002/jat.3074
  • d‐α‐tocopheryl polyethylene glycol 1000
           succinate‐containing vehicles provide no detectable chemoprotection
           from oxidative damage
    • Authors: Bethany R. Baumgart; Terry R. Van Vleet, Damir Simic, Theodora W. Salcedo, Kimberley Lentz, Michael Donegan, Marc H. Davies, Roderick T. Bunch, Thomas P. Sanderson, Robert W. Lange
      Abstract: The objective of this study was to evaluate potential protective effects of vehicles containing d‐α‐tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d‐α‐tocopheryl succinate (TS) and d‐α‐tocopherol as well as oxidative status of plasma following oral dosing of TPGS‐containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2O2. Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg–1 day–1 TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol‐400 (PEG‐400; no vitamin E) and positive control animals received a single 100 mg kg–1 day–1 IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H2O2, with or without TS (0.5, 5, 50 or 500 μm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d‐α‐tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2O2‐treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-27T23:54:17.450279-05:
      DOI: 10.1002/jat.3072
  • Comparison of the kinetics of various biomarkers of benzo[a]pyrene
           exposure following different routes of entry in rats
    • Authors: Marjory Moreau; Michèle Bouchard
      Abstract: The effect of route of exposure on the kinetics of key biomarkers of exposure to benzo[a]pyrene (BaP), a known human carcinogen, was studied. Rats were exposed to an intravenous, intratracheal, oral and cutaneous dose of 40 µmol kg–1 BaP. BaP and several metabolites were measured in blood, urine and feces collected at frequent intervals over 72 h post‐treatment, using high‐performance liquid chromatography/fluorescence. Only BaP and 3‐hydroxyBaP (3‐OHBaP) were detectable in blood at all time points. There were route‐to‐route differences in the excreted amounts (% dose) of metabolites but the observed time courses of the excretion rate were quite similar. In urine, total amounts of BaP metabolites excreted over the 0–72 h period followed the order: trans‐4,5‐dihydrodiolBaP (4,5‐diolBaP) ≥ 3‐OHBaP > 7‐OHBaP ≥ 7,8‐diolBaP after intravenous injection and intratracheal instillation; 3‐OHBaP ≈ 7‐OHBaP ≥ 4,5‐diolBaP > 7,8‐diolBaP after cutaneous application; 3‐OHBaP ≥ 4,5‐diolBaP ≈ 7‐OHBaP > 7,8‐diolBaP following oral administration. In feces, total amounts of BaP metabolites recovered were: 7‐OHBaP ≈ 3‐OHBaP > 4,5‐diolBaP > 7,8‐diolBaP > BaP‐7,8,9,10‐tetrol following all administration routes. For all exposure routes, excretion of 4,5‐ and 7,8‐diolBaP was almost complete over the 0–24 h period in contrast with that of 3‐ and 7‐OHBaP. This study confirms the interest of measuring multiple metabolites due to route‐to‐route differences in the relative excretion of the different biomarkers and in the time courses of diolBaPs versus OHBaPs. Concentration ratios of the different metabolites may help indicate time and main route of exposure. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-27T20:01:41.409244-05:
      DOI: 10.1002/jat.3070
  • Tween‐80 and impurity induce anaphylactoid reaction in zebrafish
    • Authors: Rui Yang; Qiao‐Cong Lao, Hang‐Ping Yu, Yong Zhang, Hong‐Cui Liu, Lin Luan, Hui‐Min Sun, Chun‐Qi Li
      Abstract: A number of recent reports suspected that Tween‐80 in injectable medicines, including traditional Chinese medicine injections could cause life‐threatening anaphylactoid reaction, but no sound conclusion was drawn. A drug‐induced anaphylactoid reaction is hard to be assayed in vitro and in conventional animal models. In this study, we developed a microplate‐based quantitative in vivo zebrafish assay for assessing anaphylactoid reaction and live whole zebrafish mast cell tryptase activity was quantitatively measured at a wavelength of 405 nm using N‐benzoyl‐dl‐arginine p‐nitroanilide as a substrate. We assessed 10 batches of Tween‐80 solutions from various national and international suppliers and three Tween‐80 impurities (ethylene glycol, 2‐chloroethanol and hydrogen peroxide) in this model and found that three batches of Tween‐80 (nos 2, 20080709 and 20080616) and one Tween‐80 impurity, hydrogen peroxide (H2O2), induced anaphylactoid reactions in zebrafish. Furthermore, we found that H2O2 residue and peroxide value were much higher in Tween‐80 samples 2, 20080709 and 20080616. These findings suggest that H2O2 residue in combination with oxidized fatty acid residues (measured as peroxide value) or more likely the oxidized fatty acid residues in Tween‐80 samples, but not Tween‐80 itself, may induce anaphylactoid reaction. High‐throughput zebrafish tryptase assay developed in this report could be used for assessing safety of Tween‐80‐containing injectable medicines and potentially for screening novel mast cell‐modulating drugs. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-27T02:05:30.018625-05:
      DOI: 10.1002/jat.3069
  • Mini review on blood–brain barrier penetration of pyridinium
    • Authors: H. Kalász; S. M. Nurulain, G. Veress, S. Antus, F. Darvas, E. Adeghate, A. Adem, F. Hashemi, K. Tekes
      Pages: n/a - n/a
      Abstract: This paper reviews the blood–brain barrier (BBB) penetration of newly developed pyridinium aldoximes. Pyridinium aldoximes are highly charged hydrophilic compounds used in the treatment of subjects exposed to organophosphonates because they are effective as acetylcholinesterase reactivators. Pyridinium aldoximes have antidotal effects against poisoning with cholinesterase inhibitors, a frequent problem affecting people working with organophosphate‐based insecticides and pesticides. Toxic organophosphonate products such as sarin and tabun can be used by terrorists as chemical warfare agents. This poses a severe challenge to all innocent and peace‐loving people worldwide. This review gives a brief summary of BBB transporters and description of the current in vitro and in vivo methods for the characterization of BBB penetration of established and novel pyridinium aldoximes. The authors provide a putative mechanism of penetration, outline some future ways of formulation and discuss the possible advantages and disadvantages of increasing BBB penetration. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-07T05:21:33.385324-05:
      DOI: 10.1002/jat.3048
  • Neurotoxic effects of ochratoxin A on the subventricular zone of adult
           mouse brain
    • Authors: Sara Paradells; Brenda Rocamonde, Cristina Llinares, Vicente Herranz‐Pérez, Misericordia Jimenez, Jose Manuel Garcia‐Verdugo, Ivan Zipancic, Jose Miguel Soria, Ma. Angeles Garcia‐Esparza
      Abstract: Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5‐bromo‐2‐deoxyuridine‐positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose‐dependent manner. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-25T07:57:47.345347-05:
      DOI: 10.1002/jat.3061
  • Oral cadmium exposure during rat pregnancy: assessment of transplacental
           micronutrient transport and steroidogenesis at term
    • Authors: Anja Mikolić; Martina Piasek, Antonija Sulimanec Grgec, Veda M. Varnai, Sandra Stasenko, Saša Kralik Oguić
      Abstract: Diet is the main source of cadmium (Cd) exposure. Gastrointestinal absorption increases during pregnancy. Cadmium accumulated in the placenta may interfere with nutrient transport to the foetus. Data on the potential of Cd to act as a steroid disruptor of pregnancy are limited. We evaluated the effects of oral Cd exposure during pregnancy on placental function in micronutrient transfer to the foetus and steroidogenesis in Wistar rats (regular 4‐day cyclers) that mated with unexposed males. Pregnant rats were randomly assigned to a Cd group exposed orally to 50 mg Cd l–1 (CdCl2xH2O dissolved in demineralized water), ≈7.5 mg Cd kg–1 a day, during 20 days of gestation and control (supplied with demineralized water). Non‐pregnant rats were treated under the same experimental conditions. On day 20, all of the rats were killed and samples were taken for element analyses (by electrothermal atomic absorption spectrometry). Progesterone and testosterone were measured in serum and placenta‐derived samples (by immunoenzymometric assay and/or enzyme‐linked immunosorbent assay). In the exposed rats, Cd increased in blood and organs, more in pregnant rats, and in placenta and foetus whereas zinc increased in liver. Iron decreased in maternal organs and in foetus, whereas zinc decreased in maternal kidney and placenta. Liver copper was lower and kidney copper higher in all pregnant vs. non‐pregnant rats. Steroids in serum and placenta did not change. In conclusion, oral Cd exposure during rat pregnancy does not affect progesterone and testosterone at term. Transplacental iron and zinc handover are disrupted, which may put at risk the maintenance of foetal nutrition and viability. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-25T07:52:07.664676-05:
      DOI: 10.1002/jat.3055
  • Evaluation and refinement of a field‐portable drinking water
           toxicity sensor utilizing electric cell–substrate impedance sensing
           and a fluidic biochip
    • Authors: Mark W. Widder; Linda M. Brennan, Elizabeth A. Hanft, Mary E. Schrock, Ryan R. James, William H. Schalie
      Abstract: The US Army's need for a reliable and field‐portable drinking water toxicity sensor was the catalyst for the development and evaluation of an electric cell–substrate impedance sensing (ECIS) device. Water testing technologies currently available to soldiers in the field are analyte‐specific and have limited capabilities to detect broad‐based water toxicity. The ECIS sensor described here uses rainbow trout gill epithelial cells seeded on fluidic biochips to measure changes in impedance for the detection of possible chemical contamination of drinking water supplies. Chemicals selected for testing were chosen as representatives of a broad spectrum of toxic industrial compounds. Results of a US Environmental Protection Agency (USEPA)‐sponsored evaluation of the field portable device were similar to previously published US Army testing results of a laboratory‐based version of the same technology. Twelve of the 18 chemicals tested following USEPA Technology Testing and Evaluation Program procedures were detected by the ECIS sensor within 1 h at USEPA‐derived human lethal concentrations. To simplify field‐testing methods further, elimination of a procedural step that acclimated cells to serum‐free media streamlined the test process with only a slight loss of chemical sensitivity. For field use, the ECIS sensor will be used in conjunction with an enzyme‐based sensor that is responsive to carbamate and organophosphorus pesticides. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-18T03:31:57.64557-05:0
      DOI: 10.1002/jat.3017
  • Characteristic molecular and proteomic signatures of drug‐induced
           liver injury in a rat model
    • Authors: Jung Woo Eun; Hyun Jin Bae, Qingyu Shen, Se Jin Park, Hyung Seok Kim, Woo Chan Shin, Hee Doo Yang, Chan Young Jin, Jueng Soo You, Hyun Joo Kang, Hoguen Kim, Young Min Ahn, Won Sang Park, Jung Young Lee, Suk Woo Nam
      Abstract: Drug‐induced liver injury (DILI) is a major safety concern during drug development and remains one of the main reasons for withdrawal of drugs from the market. Although it is crucial to develop methods that will detect potential hepatotoxicity of drug candidates as early and as quickly as possible, there is still a lack of sensitive and specific biomarkers for DILI that consequently leads to a scarcity of reliable hepatotoxic data. Hence, in this study, we assessed characteristic molecular signatures in rat liver treated with drugs (pyrazinamide, ranitidine, enalapril, carbamazepine and chlorpromazine) that are known to cause DILI in humans. Unsupervised hierarchical clustering analysis of transcriptome changes induced by DILI‐causing drugs resulted in three different subclusters on dendrogram, i.e., hepatocellular, cholestatic and mixed type of DILI at early time points (2 days), and multiclassification analysis suggested 31 genes as discernible markers for each DILI pattern. Further analysis for characteristic molecular signature of each DILI pattern provided a molecular basis for different modes of DILI action. A proteomics study of the same rat livers was used to confirm the results, and the two sets of data showed 60 matching classifiers. In conclusion, the data of different DILI‐causing drug treatments from genomic analysis in a rat model suggest that DILI‐specific molecular signatures can discriminate different patterns of DILI at an early exposure time point, and that they provide useful information for mechanistic studies that may lead to a better understanding of the molecular basis of DILI. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-18T03:11:37.586599-05:
      DOI: 10.1002/jat.3062
  • Chronic trimethyltin chloride exposure and the development of kidney
           stones in rats
    • Authors: Xuefeng Ren; Xin Wu, Gang Sui, Zhihong Gong, Emmanuel Yawson, Banghua Wu, Guanchao Lai, Xiaolin Ruan, Hongbin Gao, Feng Zhou, Bing Su, James R. Olson, Xiaojiang Tang
      Abstract: We recently reported that occupational exposure to trimethyltin (TMT) is a risk factor for developing kidney stones. To further examine the association between TMT exposure and the formation of kidney stones, we conducted a 180‐day animal study and exposed the randomly grouped Sprague–Dawley (SD) rats to TMT in the drinking water at doses of 0, 8.2, 32.8 and 131.3 µg kg–1 day–1. Transient behavioral changes were observed in the high‐dose group during the first 2 weeks of exposure. TMT exposure led to a significant dose‐dependent inhibition of renal H+/K+‐ATPase and an increase in urinary pH. In comparison to no kidney stones being identified in the control and the lowest dose group, 1 rat in the 32.8 µg kg–1 day–1 dose group and 3 out of 9 rats in the 131.3 µg kg–1 day–1 dose group were found to have stones in the kidney/urinary tract. Pathological analysis showed that more wide spread calcium disposition was observed in kidneys of rats with TMT exposure compared with the rats in the control group. However, X‐ray diffraction (XRD) analysis found that the kidney stones were mainly composed of struvite with the formula: NH4MgPO4 6H2O, while calcium‐containing components were also detected. Together, this study further demonstrates through animal studies that chronic exposure to a relatively low level of TMT induces nephrotoxicity and increases the risk for developing kidney stones. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-16T05:47:12.006379-05:
      DOI: 10.1002/jat.3054
  • Non‐clinical safety evaluation of single and repeated intramuscular
           administrations of MAGE‐A3 Cancer Immunotherapeutic in rabbits and
           cynomolgus monkeys
    • Authors: Eric Destexhe; Emilie Grosdidier, Nathalie Baudson, Roy Forster, Catherine Gerard, Nathalie Garçon, Lawrence Segal
      Abstract: The MAGE‐A3 recombinant protein combined with AS15 immunostimulant (MAGE‐A3 Cancer Immunotherapeutic) is under development by GlaxoSmithKline for the treatment of lung cancer and melanoma. We performed non‐clinical safety studies evaluating potential local and systemic toxic effects induced by MAGE‐A3 Cancer Immunotherapeutic in rabbits (study 1) and cynomolgus monkeys (study 2). Animals were allocated to two groups to receive a single (rabbits) or 25 repeated (every 2 weeks) injections (monkeys) of MAGE‐A3 Cancer Immunotherapeutic (treatment groups) or saline (control groups). All rabbits were sacrificed 3 days post‐injection and monkeys 3 days following last injection (3/5 per gender per group) or after a 3‐month treatment‐free period (2/5 per gender per group). Local and systemic reactions and MAGE‐A3‐specific immune responses (monkeys) were assessed. Macroscopic and microscopic (for rabbits, injection site only) post‐mortem examinations were performed on all animals. No systemic toxicity or unscheduled mortalities were recorded. Single (rabbits) and repeated (monkeys; up to four times at the same site) injections were well tolerated. Following five to seven repeated injections, limb circumferences increased up to 26% (5 h post‐injection), but returned to normal after 1–8 days. Three days after the last injection, enlargements of iliac, popliteal, axillary and inguinal lymph nodes, and increased incidence or severity of mononuclear inflammatory cell infiltrates was observed in injected muscles of treated monkeys. No treatment‐related macroscopic findings were recorded after the treatment‐free period. MAGE‐A3‐specific antibody and T‐cell responses were raised in all treated monkeys, confirming test item exposure. Single or repeated intramuscular injections of MAGE‐A3 Cancer Immunotherapeutic were well tolerated in rabbits and monkeys. Copyright © 2014 GlaxoSmithKline Vaccines. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.
      PubDate: 2014-09-12T22:50:49.939421-05:
      DOI: 10.1002/jat.3025
  • Graphene supports in vitro proliferation and osteogenic differentiation of
           goat adult mesenchymal stem cells: potential for bone tissue engineering
    • Authors: Hoda Elkhenany; Lisa Amelse, Andersen Lafont, Shawn Bourdo, Marc Caldwell, Nancy Neilsen, Enkeleda Dervishi, Oshin Derek, Alexandru S. Biris, David Anderson, Madhu Dhar
      Abstract: Current treatments for bone loss injuries involve autologous and allogenic bone grafts, metal alloys and ceramics. Although these therapies have proved useful, they suffer from inherent challenges, and hence, an adequate bone replacement therapy has not yet been found. We hypothesize that graphene may be a useful nanoscaffold for mesenchymal stem cells and will promote proliferation and differentiation into bone progenitor cells. In this study, we evaluate graphene, a biocompatible inert nanomaterial, for its effect on in vitro growth and differentiation of goat adult mesenchymal stem cells. Cell proliferation and differentiation are compared between polystyrene‐coated tissue culture plates and graphene‐coated plates. Graphitic materials are cytocompatible and support cell adhesion and proliferation. Importantly, cells seeded on to oxidized graphene films undergo osteogenic differentiation in fetal bovine serum‐containing medium without the addition of any glucocorticoid or specific growth factors. These findings support graphene's potential to act as an osteoinducer and a vehicle to deliver mesenchymal stem cells, and suggest that the combination of graphene and goat mesenchymal stem cells provides a promising construct for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-12T22:12:52.239351-05:
      DOI: 10.1002/jat.3024
  • Exposure to MnCl2 · 4H2O during development induces
           activation of microglial and perivascular macrophage populations in the
           hippocampal dentate gyrus of rats
    • Authors: Hajime Abe; Takumi Ohishi, Fumiyuki Nakane, Ayako Shiraki, Takeshi Tanaka, Toshinori Yoshida, Makoto Shibutani
      Abstract: Developmental exposure to Mn caused Mn accumulation in the brain tissue and transient disruption of granule cell neurogenesis, targeting the late stage differentiation of progenitor cells in the subgranular zone of the hippocampal dentate gyrus of rats. Because neurogenesis is influenced by proinflammatory responses, this study was performed to determine whether Mn exposure causes microglial activation in the dentate hilus, a region anatomically close to the subgranular zone of the dentate gyrus. Pregnant rats were treated with dietary MnCl2 · 4H2O at 32, 160 or 800 ppm from gestational day 10 to day 21 after delivery. An immunohistochemical analysis revealed increases in Iba1+ microglia in the hilus on postnatal day 21 following exposure to MnCl2 · 4H2O in a dose‐unrelated manner at 32 and at 800 ppm and an increase in CD163+ macrophage at 800 ppm in the hilus. Real‐time reverse transcription–polymerase chain reaction analysis revealed increases in the mRNA levels of Il1α, Il6, Nos2 and Tnf after 800 ppm MnCl2 · 4H2O. These results suggest that activation of microglia and perivascular macrophages occurs in the hilus after developmental exposure to MnCl2 · 4H2O at 800 ppm, and probably involves the disruption of neurogenesis through the accumulation of Mn in the brain tissue. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-12T22:06:33.032918-05:
      DOI: 10.1002/jat.3059
  • Cylindrospermopsin induces oxidative stress and genotoxic effects in the
           fish CLC cell line
    • Authors: Anna Sieroslawska; Anna Rymuszka
      Abstract: Cylindrospermopsin (CYN) is a cyanotoxin detected in water reservoirs worldwide. The toxin is a potent protein synthesis inhibitor capable of adversely influencing a wide range of cell functions. While data on the prooxidative potency of CYN are inconsistent, genotoxic effects towards certain mammalian cell types have been described. However, such a potential on fish cells has not yet been investigated. Hence, the aim of the study was to elucidate the prooxidative and genotoxic impact of CYN on a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 0.1, 0.5 or 1 µg ml–1. After 24 h, cytotoxic activity of CYN at the highest used concentration was confirmed by decreased cell membrane integrity and inhibited cell proliferation. Additionally, CYN at 0.5 and 1 µg ml–1 increased intracellular ATP levels and decreased the reduced to oxidized glutathione ratio. Furthermore, a significant increase in the reactive oxygen species (ROS) production with concomitant changes in superoxide dismutase activity was observed after a 3.5‐h exposure of the cells to the toxin. Genotoxic activity of CYN, manifested as oxidative DNA damage and elevated number of micronuclei, was also detected in exposed cells. The obtained results indicate that CYN is able to exert a wide range of adverse effects, including oxidative stress and genotoxicity in fish leucocytes. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-12T22:06:25.237034-05:
      DOI: 10.1002/jat.3040
  • Development of haemostatic decontaminants for the treatment of wounds
           contaminated with chemical warfare agents. 2: Evaluation of in vitro
           topical decontamination efficacy using undamaged skin
    • Authors: Christopher H. Dalton; Charlotte A. Hall, Helen L. Lydon, J. K. Chipman, John S. Graham, John Jenner, Robert P. Chilcott
      Abstract: The risk of penetrating, traumatic injury occurring in a chemically contaminated environment cannot be discounted. Should a traumatic injury be contaminated with a chemical warfare (CW) agent, it is likely that standard haemostatic treatment options would be complicated by the need to decontaminate the wound milieu. Thus, there is a need to develop haemostatic products that can simultaneously arrest haemorrhage and decontaminate CW agents. The purpose of this study was to evaluate a number of candidate haemostats for efficacy as skin decontaminants against three CW agents (soman, VX and sulphur mustard) using an in vitro diffusion cell containing undamaged pig skin. One haemostatic product (WoundStat™) was shown to be as effective as the standard military decontaminants Fuller's earth and M291 for the decontamination of all three CW agents. The most effective haemostatic agents were powder‐based and use fluid absorption as a mechanism of action to sequester CW agent (akin to the decontaminant Fuller's earth). The envisaged use of haemostatic decontaminants would be to decontaminate from within wounds and from damaged skin. Therefore, WoundStat™ should be subject to further evaluation using an in vitro model of damaged skin. Copyright © 2014 Crown copyright. Journal of Applied Toxicology © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-12T21:56:06.175164-05:
      DOI: 10.1002/jat.3060
  • Early chronic lead exposure reduces exploratory activity in young C57BL/6J
    • Authors: Mayra Gisel Flores‐Montoya; Christina Sobin
      Abstract: Research has suggested that chronic low‐level lead exposure diminishes neurocognitive function in children. Tests that are sensitive to behavioral effects at lowest levels of lead exposure are needed for the development of animal models. In this study we investigated the effects of chronic low‐level lead exposure on exploratory activity (unbaited nose poke task), exploratory ambulation (open field task) and motor coordination (Rotarod task) in pre‐adolescent mice. C57BL/6J pups were exposed to 0 ppm (controls), 30 ppm (low‐dose) or 230 ppm (high‐dose) lead acetate via dams’ drinking water administered from birth to postnatal day 28, to achieve a range of blood lead levels (BLLs) from not detectable to 14.84 µg dl–1). At postnatal day 28, mice completed behavioral testing and were killed (n = 61). BLLs were determined by inductively coupled plasma mass spectrometry. The effects of lead exposure on behavior were tested using generalized linear mixed model analyses with BLL, sex and the interaction as fixed effects, and litter as the random effect. BLL predicted decreased exploratory activity and no threshold of effect was apparent. As BLL increased, nose pokes decreased. The C57BL/6J mouse is a useful model for examining effects of early chronic low‐level lead exposure on behavior. In the C57BL/6J mouse, the unbaited nose poke task is sensitive to the effects of early chronic low‐level lead exposure. This is the first animal study to show behavioral effects in pre‐adolescent lead‐exposed mice with BLL below 5 µg dl–1. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-12T21:41:49.427385-05:
      DOI: 10.1002/jat.3064
  • Diagnostic and predictive performance and standardized threshold of
           traditional biomarkers for drug‐induced liver injury in rats
    • Authors: Yutaka Tonomura; Yuki Kato, Hiroyuki Hanafusa, Yuji Morikawa, Keigo Matsuyama, Takeki Uehara, Motonobu Ueno, Mikinori Torii
      Pages: n/a - n/a
      Abstract: Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug‐induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver‐operating characteristic (ROC) curve, using a free‐access database where 142 hepatotoxic or non‐hepatotoxic compounds were administrated to male rats (n = 5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in‐house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-04T05:02:30.374456-05:
      DOI: 10.1002/jat.3053
  • Perturbation of cytosolic calcium by 2‐aminoethoxydiphenyl borate
           and caffeine affects zebrafish myofibril alignment
    • Authors: Hsin‐Ju Wu; Tsorng‐Harn Fong, Shen‐Liang Chen, Jen‐Cheng Wei, I‐Jong Wang, Chi‐Chung Wen, Chao‐Yuan Chang, Xing‐Guang Chen, Wei‐Yu Chen, Hui‐Min Chen, Juin‐Lin Horng, Yun‐Hsin Wang, Yau‐Hung Chen
      Pages: n/a - n/a
      Abstract: The objective of the current study was to investigate the effects of Ca2+ levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca2+ levels affect myofibril alignment, we exposed zebrafish embryos to 2‐aminothoxyldiphenyl borate (2‐APB; an inositol 1,4,5‐trisphosphate receptor inhibitor that reduces cytosolic Ca2+ levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca2+ levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2‐APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine‐treated embryos. Subtle morphological changes, including changes in muscle fibers, F‐actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α‐laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2‐APB and/or caffeine led to myofibril misalignment; (2) 2‐APB‐treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine‐treated embryos were twisted and wavy; and (3) zebrafish embryos co‐exposed to 2‐APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca2+ is important for myogenesis, particularly for myofibril alignment. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-04T04:56:51.010578-05:
      DOI: 10.1002/jat.3057
  • Skin absorption of six performance amines used in metalworking fluids
    • Authors: Lauriane N. Roux; James D. Brooks, James L. Yeatts, Ronald E. Baynes
      Pages: n/a - n/a
      Abstract: Every year, 10 million workers are exposed to metalworking fluids (MWFs) that may be toxic. There are four types of MWFs: neat oils and three water‐based MWFs (soluble oil, semisynthetic and synthetic), which are diluted with water and whose composition varies according to the mineral oils ratio. MWFs also contain various additives. To determine the absorption of six amines used as corrosion inhibitors and biocides in MWFs, porcine skin flow‐through diffusion cell experiments were conducted with hydrophilic ethanolamines (mono‐, di‐ and triethanolamine, MEA, DEA and TEA respectively) and a mixture of lipophilic amines (dibutylethanolamine, dicyclohexylamine and diphenylamine). The six amines were dosed in four vehicles (water and three generic water‐based MWF formulations) and analyzed using a scintillation counter or gas chromatography/mass spectrometry. These 24 h studies showed that dermal absorption significantly (P 
      PubDate: 2014-09-04T04:56:30.081537-05:
      DOI: 10.1002/jat.3056
  • A representative retinoid X receptor antagonist UVI3003 induced
           teratogenesis in zebrafish embryos
    • Authors: Liang Zheng; Ting Xu, Daoji Li, Junliang Zhou
      Pages: n/a - n/a
      Abstract: Retinoid X receptor (RXR) interfering activity has been detected in different water resources. To study RXR disruptor‐induced toxicological effects on vertebrates, embryos of zebrafish (Danio rerio) were exposed to a representative RXR antagonist UVI3003. Results showed that the teratogenic index (LC50/EC50) of UVI3003 was as high as 5.4. UVI3003 induced multiple malformations of embryos, including deformed fins, reduced brains, small jaws, bent tails and edema in hearts, the degree of which became more severe with increasing exposure concentration. Although no significant difference was observed in the hatching rates between the exposure group and control, the whole body length was significantly reduced by 6.5% and 8.9% when exposed to 200 and 300 µg l−1 of UVI3003, respectively. The heart rate also significantly decreased by 8.8–50.2% during exposure. Further experiments revealed that the pharyngula stage was the most sensitive development phase in terms of embryo response to UVI3003. The results demonstrated severe teratogenicity of RXR antagonist in zebrafish embryos and provided important data for ecotoxicological evaluation of RXR antagonists. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-03T03:07:21.862962-05:
      DOI: 10.1002/jat.3051
  • Immunophenotyping does not improve predictivity of the local lymph node
           assay in mice
    • Authors: Volker Strauss; Susanne N. Kolle, Naveed Honarvar, Martina Dammann, Sibylle Groeters, Frank Faulhammer, Robert Landsiedel, Bennard Ravenzwaay
      Pages: n/a - n/a
      Abstract: The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry‐based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I‐Aκ and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non‐irritating sensitizers and non‐sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-03T03:06:12.16689-05:0
      DOI: 10.1002/jat.3042
  • Reversible cholinesterase inhibitors as pre‐treatment for exposure
           to organophosphates: assessment using azinphos‐methyl
    • Authors: Georg A. Petroianu; Syed M. Nurulain, Mohamed Y. Hasan, Kamil Kuča, Dietrich E. Lorke
      Pages: n/a - n/a
      Abstract: Pre‐treatment with reversible acetylcholinesterase (AChE) inhibitors before organophosphorous compound (OPC) exposure can reduce OPC‐induced mortality. However, pyridostigmine, the only substance employed for such prophylaxis, is merely efficacious against a limited number of OPCs. In search of more efficacious and broad‐range alternatives, we have compared in vivo the ability of five reversible AChE inhibitors (pyridostigmine, physostigmine, ranitidine, tacrine and K‐27) to reduce mortality induced by the OPC azinphos‐methyl. Protection was quantified using Cox analysis by determining the relative risk (RR) of death in rats that were administered these AChE inhibitors in equitoxic dosage (25% of LD01) 30 min before azinphos‐methyl exposure. Azinphos‐methyl‐induced mortality was significantly reduced by all five tested compounds as compared with the reference group that was only exposed to azinphos‐methyl without prior pre‐treatment (RR = 1). The most efficacious prophylactic agents were K‐27 (RR = 0.15) and physostigmine (RR = 0.21), being significantly more efficacious than ranitidine (RR = 0.62) and pyridostigmine (RR = 0.37). Pre‐treatment with tacrine (RR = 0.29) was significantly more efficacious than pre‐treatment with ranitidine, but the difference between tacrine and pyridostigmine was not significant. Our results indicate that prophylactic administration of the oxime K‐27 may be a promising alternative in cases of imminent OPC exposure. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-03T03:04:57.769676-05:
      DOI: 10.1002/jat.3052
  • Carnosic acid induces autophagic cell death through inhibition of the
           Akt/mTOR pathway in human hepatoma cells
    • Authors: Qilong Gao; Huaimin Liu, Yamin Yao, Liang Geng, Xinfeng Zhang, Lifeng Jiang, Bian Shi, Feng Yang
      Abstract: The therapeutic goal of cancer treatment is now geared towards triggering tumour‐selective cell death with autophagic cell death being required for the chemotherapy of apoptosis‐resistant cancer. In this study, Carnosic acid (CA), a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), significantly induced autophagic cell death in HepG2 cells. Ca treatment caused the formation of autophagic vacuoles produced an increasing ratio of LC3‐II to LC3‐I in a time‐ and dose‐dependent manner but had no effect on the levels of autophagy‐related protein ATG6 and ATG13 expression. Autophagy inhibitors, 3‐methyladenine (3‐MA), chloroquine and bafilomycin A1, or ATG genes silencing in HepG2 cells significantly inhibited CA‐induced autophagic cell death. The CA treatment decreased the levels of phosphorylated Akt and mTOR without any effects on PI3K or PTEN. Most importantly, overexpression of Akt and knockdown of PTEN attenuated autophagy induction in CA‐treated cells. Taken together, our results indicated that CA induced autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells. These findings suggest that CA has a great potential for the treatment of hepatoma via autophagic induction. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-09-01T07:01:21.780556-05:
      DOI: 10.1002/jat.3049
  • Protective effects of ascorbic acid against the genetic and epigenetic
           alterations induced by 3,5‐dimethylaminophenol in AA8 cells
    • Authors: Ming‐Wei Chao; P𝚤nar Erkekoglu, Chia‐Yi Tseng, Wenjie Ye, Laura J. Trudel, Paul L. Skipper, Steven R. Tannenbaum, Gerald N. Wogan
      Abstract: Exposure to monocyclic aromatic alkylanilines (MAAs), namely 2,6‐dimethylaniline (2,6‐DMA), 3,5‐dimethylaniline (3,5‐DMA) and 3‐ethylaniline (3‐EA), was significantly and independently associated with bladder cancer incidence. 3,5‐DMAP (3,5‐dimethylaminophenol), a metabolite of 3,5‐DMA, was shown to induce an imbalance in cytotoxicity cellular antioxidant/oxidant status, and DNA damage in mammalian cell lines. This study was designed to evaluate the protective effect of ascorbic acid (Asc) against the cytotoxicity, reactive oxygen species (ROS) production, genotoxicity and epigenetic changes induced by 3,5‐DMAP in AA8 Chinese Hamster Ovary (CHO) cells. In different cellular fractions, 3,5‐DMAP caused alterations in the enzyme activities orchestrating a cellular antioxidant balance, decreases in reduced glutathione levels and a cellular redox ratio as well as increases in lipid peroxidation and protein oxidation. We also suggest that the cellular stress caused by this particular alkylaniline leads to both genetic (Aprt mutagenesis) and epigenetic changes in histones 3 and 4 (H3 and H4). This may further cause molecular events triggering different pathological conditions and eventually cancer. In both cytoplasm and nucleus, Asc provided increases in 3,5‐DMAP‐reduced glutathione levels and cellular redox ratio and decreases in the lipid peroxidation and protein oxidation. Asc was also found to be protective against the genotoxic and epigenetic effects initiated by 3,5‐DMAP. In addition, Asc supplied protection against the cell cycle (G1 phase) arrest induced by this particular alkylaniline metabolite.
      PubDate: 2014-09-01T06:51:00.294209-05:
      DOI: 10.1002/jat.3046
  • In utero and early childhood exposure to arsenic decreases lung function
           in children
    • Authors: Rogelio Recio‐Vega; Tania Gonzalez‐Cortes, Edgar Olivas‐Calderon, R. Clark Lantz, A. Jay Gandolfi, Cesar Gonzalez‐De Alba
      Abstract: The lung is a target organ for adverse health outcomes following exposure to As. Several studies have reported a high prevalence of respiratory symptoms and diseases in subjects highly exposed to As through drinking water; however, most studies to date has been performed in exposed adults, with little information on respiratory effects in children. The objective of the study was to evaluate the association between urinary levels of As and its metabolites with lung function in children exposed in utero and in early childhood to high As levels through drinking water. A total of 358 healthy children were included in our study. Individual exposure was assessed based on urinary concentration of inorganic As. Lung function was assessed by spirometry. Participants were exposed since pregnancy until early childhood to an average water As concentration of 152.13 µg l–1. The mean urinary As level registered in the studied subjects was 141.2 µg l–1 and only 16.7% had a urinary concentration below the national concern level. Forced vital capacity was significantly decreased in the studied population and it was negatively associated with the percentage of inorganic As. More than 57% of the subjects had a restrictive spirometric pattern. The urinary As level was higher in those children with restrictive lung patterns when compared with the levels registered in subjects with normal spirometric patterns. Exposure to As through drinking water during in utero and early life was associated with a decrease in forced vital capacity and with a restrictive spirometric pattern in the children evaluated. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-15T08:01:31.55848-05:0
      DOI: 10.1002/jat.3023
  • Development of haemostatic decontaminants for the treatment of wounds
           contaminated with chemical warfare agents. 1: Evaluation of in vitro
           clotting efficacy in the presence of certain contaminants
    • Authors: Charlotte A. Hall; Helen L. Lydon, Christopher H. Dalton, J. K. Chipman, John S. Graham, Robert P. Chilcott
      Abstract: The treatment of penetrating, haemorrhaging injuries sustained within a hazardous environment may be complicated by contamination with toxic chemicals. There are currently no specific medical countermeasures for such injuries. Haemostats with an absorbent mechanism of action have the potential to simultaneously stop bleeding and decontaminate wounds. However, a primary requirement of a ‘haemostatic decontaminant’ is the retention of clotting function in the presence of chemical contaminants. Thus, the aim of this study was to investigate the haemostatic efficacy of seven commercially available haemostats in the presence of toxic chemicals (soman, VX, sulphur mustard, petrol, aviation fuel and motor oil). Clot viscosity was assessed ex vivo using thrombelastography following treatment of pig blood with: (i) toxic chemical; (ii) haemostat; or (iii) haemostat in combination with toxic chemical. Several contaminants (VX, petrol and GD) were found to be pro‐haemostatic and none had an adverse effect on the rate with which the test products attained haemostasis. However, the total clot strength for blood treated with certain haemostats in the presence of sulphur mustard, soman and petrol was significantly decreased. Three test products failed to demonstrate haemostatic function in this ex vivo (thrombelastography) model; this was tentatively ascribed to the products achieving haemostasis through a tamponade mechanism of action, which can only be replicated using in vivo models. Overall, this study has identified a number of commercial products that may have potential as haemostatic decontaminants and warrant further investigation to establish their decontaminant efficacy. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-15T07:46:27.370339-05:
      DOI: 10.1002/jat.3019
  • Systemic drugs inducing non‐immediate cutaneous adverse reactions
           and contact sensitizers evoke similar responses in THP‐1 cells
    • Authors: Margarida Gonçalo; João Martins, Ana Silva, Bruno Neves, Américo Figueiredo, Teresa Cruz, Celeste Lopes
      Abstract: Contact sensitizers induce phenotypic and functional changes in dendritic cells (DC) that enhance their antigen‐presenting capacity and, ultimately, modulate the T cell response. To evaluate if there is a similar effect of drugs causing T‐cell‐mediated cutaneous adverse drug reactions (CADR), we studied the in vitro effect of drugs on THP‐1 cells, a cell line widely used to evaluate the early molecular and cellular events triggered by contact sensitizers. The effect of allopurinol, oxypurinol, ampicillin, amoxicillin, carbamazepine and sodium valproate, at EC30 concentrations, was evaluated on p38 MAPK activation, by Western Blot, and on the expression of genes coding for DC maturation markers, pro‐inflammatory cytokine/chemokines and hemeoxygenase 1 (HMOX1), by real‐time RT‐PCR. Results were compared with lipopolysaccharide (LPS), a DC maturation stimulus, and the strong contact sensitizer, 1‐fluoro‐2,4‐dinitrobenzene (DNFB). All drugs studied significantly upregulated HMOX1 gene transcription and all, except the anticonvulsants, also upregulated IL8. Allopurinol and oxypurinol showed the most intense effect, in a magnitude similar to DNFB and superior to betalactams. Transcription of CD40, IL12B and CXCL10 genes by drugs was more irregular. Moreover, like DNFB, all drugs activated p38 MAPK, although significantly only for oxypurinol. Like contact sensitizers, drugs that cause non‐immediate CADR activate THP‐1 cells in vitro, using different signalling pathways and affecting gene transcription with an intensity that may reflect the frequency and severity of the CADR they cause. Direct activation of antigen‐presenting DC by systemic drugs may be an important early step in the pathophysiology of non‐immediate CADR. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-04T09:46:53.569007-05:
      DOI: 10.1002/jat.3033
  • Analysis of drugs of abuse in human plasma by dispersive
           liquid–liquid microextraction and high‐performance liquid
    • Authors: P. Fernández; M. Regenjo, A. M. Bermejo, A. M. Fernández, R. A. Lorenzo, A. M. Carro
      Abstract: Opioids and cocaine are widely used at present, both for recreational purposes and as drugs of abuse. This raises the need to develop new analytical methods specifically designed for the simultaneous detection of several drugs of abuse in biological samples. In this work, dispersive liquid–liquid microextraction (DLLME) was assessed as a new sample treatment for the simultaneous extraction of morphine (MOR), 6‐acetylmorphine (6AM), cocaine (COC), benzoylecgonine (BZE) and methadone (MET) from human plasma. Preliminary assays were done before developing an experimental design based on a Uniform Network Doehlert which allowed the optimum extraction conditions to be identified, namely: a volume of extractant solvent (chloroform) and dispersant solvent (acetonitrile) of 220 µl and 3.2 ml, respectively; 0.2 g of NaCl as a salting‐out additive; pH 10.6 and ultrasound stirring for 3.5 min. The resulting extracts were analyzed by high‐performance liquid chromatography with photodiode array detection (HPLC‐PDA), using an XBridge® RP18 column (250 × 4.6 mm i.d., 5 µm particle size). Calibration graphs were linear over the concentration range 0.1–10 µg ml–1, and detection limits ranged from 13.9 to 28.5 ng ml–1. Precision calculated at three different concentration levels in plasma was included in the range 0.1–6.8% RSD. Recoveries of the five drugs were all higher than 84% on average. Finally the proposed method was successfully applied to 22 plasma samples from heroin, cocaine and/or methadone users, and the most frequently detected drug was benzoylecgonine, followed by methadone, cocaine and morphine. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-04T09:42:27.742954-05:
      DOI: 10.1002/jat.3035
  • The relationship between chemical‐induced kidney weight increases
           and kidney histopathology in rats
    • Authors: Evisabel A. Craig; Zhongyu Yan, Q. Jay Zhao
      Abstract: The kidney is a major site of chemical excretion, which results in its propensity to exhibit chemically‐induced toxicological effects at a higher rate than most other organs. Although the kidneys are often weighed in animal toxicity studies, the manner in which these kidney weight measurements are interpreted and the value of this information in predicting renal damage remains controversial. In this study we sought to determine whether a relationship exists between chemically‐induced kidney weight changes and renal histopathological alterations. We also examined the relative utility of absolute and relative (kidney‐to‐body weight ratio) kidney weight in the prediction of renal toxicity. For this, data extracted from oral chemical exposure studies in rats performed by the National Toxicology Program were qualitatively and quantitatively evaluated. Our analysis showed a statistically significant correlation between absolute, but not relative, kidney weight and renal histopathology in chemically‐treated rats. This positive correlation between absolute kidney weight and histopathology was observed even with compounds that statistically decreased terminal body weight. Also, changes in absolute kidney weight, which occurred at subchronic exposures, were able to predict the presence or absence of kidney histopathology at both subchronic and chronic exposures. Furthermore, most increases in absolute kidney weight reaching statistical significance (irrespective of the magnitude of change) were found to be relevant for the prediction of histopathological changes. Hence, our findings demonstrate that the evaluation of absolute kidney weight is a useful method for identifying potential renal toxicants. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-04T09:42:22.00032-05:0
      DOI: 10.1002/jat.3036
  • Protective role of L‐ascorbic acid, N‐acetylcysteine and
           apocynin on neomycin‐induced hair cell loss in Zebrafish
    • Authors: Chia‐Yen Wu; Han‐Jung Lee, Chi‐Fang Liu, Mallikarjuna Korivi, Hwei‐Hsien Chen, Ming‐Huan Chan
      Abstract: Hair cells are highly sensitive to environmental insults and other therapeutic drugs. The adverse effects of drugs such as aminoglycosides can cause hair cell death and lead to hearing loss and imbalance. The objective of the present study was to evaluate the protective activity of L‐ascorbic acid, N‐acetylcysteine (NAC) and apocynin on neomycin‐induced hair cell damage in zebrafish (Danio rerio) larvae at 5 days post fertilization (dpf). Results showed that the loss of hair cells within the neuromasts of the lateral lines after neomycin exposure was evidenced by a significantly lower number of neuromasts labeled with fluorescent dye FM1‐43FX observed under a microscope. Co‐administration with L‐ascorbic acid, NAC and apocynin protected neomycin‐induced hair cell loss within the neuromasts. Moreover, these three compounds reduced the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin, indicating that their antioxidant action is involved. In contrast, the neuromasts were labeled with specific fluorescent dye Texas‐red conjugated with neomycin to detect neomycin uptake. Interestingly, the uptake of neomycin into hair cells was not influenced by these three antioxidant compounds. These data imply that prevention of hair cell damage against neomycin by L‐ascorbic acid, NAC and apocynin might be associated with inhibition of excessive ROS production, but not related to modulating neomycin uptake. Our findings conclude that L‐ascorbic acid, NAC and apocynin could be used as therapeutic drugs to protect aminoglycoside‐induced listening impairment after further confirmatory studies. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-04T09:42:16.707002-05:
      DOI: 10.1002/jat.3043
  • Plasma miR‐208 as a useful biomarker for drug‐induced
           cardiotoxicity in rats
    • Authors: Yoko Nishimura; Chiaki Kondo, Yuji Morikawa, Yutaka Tonomura, Mikinori Torii, Jyoji Yamate, Takeki Uehara
      Abstract: Cardiotoxicity is one of the major safety concerns in drug development. Therefore, detecting and monitoring cardiotoxicity throughout preclinical and clinical studies is important for pharmaceutical companies. The present study was conducted in order to explore a plasma miRNA biomarker for cardiotoxicity in rats. As organ specificity is an important factor for a biomarker, we analyzed the miRNA microarray dataset in 55 organs/tissues in normal male rats. Based on this analysis, 5 miRNAs consisting of miR‐208 (heart‐specific), miR‐1, miR‐133a, miR‐133b (heart and skeletal muscle‐specific) and miR‐206 (skeletal muscle‐specific) were selected. Next, we evaluated the usefulness of those 5 miRNAs as circulating biomarkers in rats administered with single‐dose isoproterenol or doxorubicin. Plasma miR‐208 was consistently increased through 24 h after dosing in rats administered with isoproterenol, whereas plasma concentrations of cardiac troponin (cTn) showed transient elevation. In contrast, the plasma levels of miR‐1, miR‐133a, miR‐133a and miR‐206 were elevated after treatment with doxorubicin, probably as a result of skeletal muscle toxicity. Additionally, the plasma miR‐208 level was elevated even after repeat‐dose administration (once daily for 7 days) of isoproterenol under which the pathological condition proceeded to the sub‐chronic phase such as fibrosis. Thus, our data suggest that miR‐208 is a promising plasma biomarker for cardiotoxicity in rats. Monitoring of plasma miR‐208 levels in rats may lead to more accurate evaluation of cardiotoxicity in preclinical studies. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-08-04T09:03:05.796088-05:
      DOI: 10.1002/jat.3044
  • In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS)
           and perfluorooctanoic acid (PFOA) on IL‐2 production in human
    • Authors: Kristin Midgett; Margie M. Peden‐Adams, Gary S. Gilkeson, Diane L. Kamen
      Abstract: Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)‐2 production in the human Jurkat T‐cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD‐3/anti CD‐28, or anti CD‐3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml−1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml−1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD‐3 exhibited decreased IL‐2 production beginning at 50 µg PFOS ml−1 and 5 µg PFOS ml−1 respectively, but stimulation with anti‐CD3/anti‐CD28 resulted in no changes compared with the control. Addition of the PPAR‐alpha antagonist GW6471 to PFOS‐dosed cells stimulated with PHA/PMA resulted in decreases in IL‐2 production starting at 50 µg PFOS ml−1, which suggests PFOS affected T‐cell IL‐2 production via PPAR‐alpha‐independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL‐2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL‐2 production, but PFOS suppressed IL‐2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL‐2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-23T08:44:08.47006-05:0
      DOI: 10.1002/jat.3037
  • Characterization of Oryzias latipes glucocorticoid receptors and their
           unique response to progestins
    • Authors: Shinichi Miyagawa; Anke Lange, Saki Tohyama, Yukiko Ogino, Takeshi Mizutani, Tohru Kobayashi, Norihisa Tatarazako, Charles R. Tyler, Taisen Iguchi
      Abstract: Various receptor bioassays, including estrogens, androgens and thyroid hormones, have been developed and applied successfully for assessing hormone function in a wide range of animal species, including fish. In fish, corticosteroids play a pivotal role in physiology as they do in mammals, but far less is known about the corticosteroid receptor system in fish compared with in mammals. Here we established a transient transactivation assay using the Japanese medaka, Oryzias latipes, glucocorticoid receptors (olGRs) and mineralocorticoid receptor to analyse their functional properties in a fish. We found that olGR2 was highly responsive to glucocorticoids, similar to the human GR, whereas the olGR1 subtype was minimally responsive. Thus, olGR2 most likely mediates glucocorticoid signaling in medaka. We further tested crosstalk between GRs and other steroid hormones, and found that progestins could activate or inactivate olGR2‐mediating transcription, depending on the presence or absence of cortisol. The transactivation assays developed for medaka GRs provide tools to gain useful insights into corticosteroid signaling in fish and for in vitro screening of environmental substances activating GRs. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-23T08:42:52.17801-05:0
      DOI: 10.1002/jat.3020
  • Cytotoxic and apoptotic activities of the plancitoxin I from the venom of
           crown‐of‐thorns starfish (Acanthaster planci) on A375.S2 cells
    • Authors: Chi‐Chiu Lee; Hernyi Justin Hsieh, Deng‐Fwu Hwang
      Abstract: This study reports on a cytotoxic toxin derived from the venom of the crown‐of‐thorns starfish Acanthaster planci (CAV). The protein toxin was isolated through both ion‐exchange and gel‐filtration chromatography, and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and mass spectrum analyzes. The CAV was identified as plancitoxin I protein. The mechanistic role of the CAV toxin was explored in human malignant melanoma A375.S2 cell death. The results indicated that after incubation with CAV toxin, cells significantly decreased in A375.S2 cell viability and increased in the lactate dehydrogenase (LDH) level in a dose‐dependent manner. The assays indicated that CAV toxin promoted reactive oxygen species (ROS) production, induced nitric oxide (NO) formation, lost mitochondrial membrane potential (ΔΨm) and induced inter‐nucleosomal DNA fragmentation in A375.S2 cells. The molecular cytotoxicity of the CAV toxin was tested through evaluation of the apoptosis/necrosis ratio by double staining with annexin V‐FITC and a propidium iodide (PI) assay. The results suggested that CAV toxin induced a cytotoxic effect in A375.S2 cells via the apoptotic procedure, and may be associated with the regulation of the p38 pathways. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-21T09:24:19.994647-05:
      DOI: 10.1002/jat.3034
  • Elevated levels of antibodies against xenobiotics in a subgroup of healthy
    • Authors: Aristo Vojdani; Datis Kharrazian, Partha Sarathi Mukherjee
      Abstract: In spite of numerous research efforts, the exact etiology of autoimmune diseases remains largely unknown. Genetics and environmental factors, including xenobiotics, are believed to be involved in the induction of autoimmune disease. Some environmental chemicals, acting as haptens, can bind to a high‐molecular‐weight carrier protein such as human serum albumin (HSA), causing the immune system to misidentify self‐tissue as an invader and launch an immune response against it, leading to autoimmunity. This study aimed to examine the percentage of blood samples from healthy donors in which chemical agents mounted immune challenges and produced antibodies against HSA‐bound chemicals. The levels of specific antibodies against 12 different chemicals bound to HSA were measured by ELISA in serum from 400 blood donors. We found that 10% (IgG) and 17% (IgM) of tested individuals showed significant antibody elevation against aflatoxin‐HSA adduct. The percentage of elevation against the other 11 chemicals ranged from 8% to 22% (IgG) and 13% to 18% (IgM). Performance of serial dilution and inhibition of the chemical–antibody reaction by specific antigens but not by non‐specific antigens were indicative of the specificity of these antibodies. Although we lack information about chemical exposure in the tested individuals, detection of antibodies against various protein adducts may indicate chronic exposure to these chemical haptens in about 20% of the tested individuals. Currently the pathological significance of these antibodies in human blood is still unclear, and this protein adduct formation could be one of the mechanisms by which environmental chemicals induce autoimmune reactivity in a significant percentage of the population. Copyright © 2014. The
      Authors . Journal of Applied Toxicology Published by John Wiley & Sons Ltd.
      PubDate: 2014-07-18T04:30:19.876942-05:
      DOI: 10.1002/jat.3031
  • Reactive oxygen species‐dependent JNK downregulated
           olaquindox‐induced autophagy in HepG2 cells
    • Authors: Dongxu Zhao; Congcong Wang, Shusheng Tang, Chaoming Zhang, Shen Zhang, Yan Zhou, Xilong Xiao
      Abstract: Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase‐9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox‐treated HepG2 cells. Olaquindox‐induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy‐related MAP‐LC3 and Beclin 1 proteins. The autophagy inhibitor 3‐methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl‐2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N‐acetyl‐L‐cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c‐Jun N‐terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox‐induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox‐induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox‐induced apoptosis can be enhanced by 3‐methyladenine. Olaquindox‐induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS‐dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-18T04:30:06.618358-05:
      DOI: 10.1002/jat.3022
  • Inhibitory effect of apocynin on methylglyoxal‐mediated glycation in
           osteoblastic MC3T3‐E1 cells
    • Authors: Kwang Sik Suh; Sang Youl Rhee, Young Seol Kim, Eun Mi. Choi
      Abstract: Methylglyoxal (MG), a highly reactive metabolite of hyperglycemia, can enhance protein glycation, oxidative stress or inflammation. The present study investigated the effects of apocynin on the mechanisms associated with MG toxicity in osteoblastic MC3T3‐E1 cells. Pretreatment of MC3T3‐E1 cells with apocynin prevented the MG‐induced protein glycation and formation of intracellular reactive oxygen species and mitochondrial superoxide in MC3T3‐E1 cells. In addition, apocynin increased glutathione levels and restored the activity of glyoxalase I inhibited by MG. These findings suggest that apocynin provide a protective action against MG‐induced cell damage by reducing oxidative stress and by increasing the MG detoxification system. Apocynin treatment decreased the levels of proinflammatory cytokines such as tumor necrosis factor‐α and interleukin‐6 induced by MG. Additionally, the nitric oxide level reduced by MG was significantly increased by apocynin. These findings indicate that apocynin might exert its therapeutic effects via upregulation of glyoxalase system and antioxidant activity. Taken together, apocynin may prove to be an effective treatment for diabetic osteopathy. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-18T04:30:03.532821-05:
      DOI: 10.1002/jat.3016
  • Renal cells exposed to cadmium in vitro and in vivo: normalizing gene
           expression data
    • Authors: A. R. Nair; K. Smeets, E. Keunen, W.‐K. Lee, F. Thévenod, E. Van Kerkhove, A. Cuypers
      Abstract: Cadmium (Cd) is a toxic metal with a long half‐life in biological systems. This half‐life is partly as a result of metallothioneins (MTs), metal‐binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd‐MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT–metal interactions using various techniques including quantitative real‐time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5‐monooxygenase activation‐protein, zeta polypeptide (Ywhaz) and beta‐actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2. A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-18T04:29:56.735907-05:
      DOI: 10.1002/jat.3047
  • Toxicity of new emerging pollutant tris‐(2,3‐dibromopropyl)
           isocyanurate on BALB/c mice
    • Authors: Juan Li; Xu Zhang, Jieqing Bao, Yuchen Liu, Junfeng Li, Jia Li, Yong Liang, Jie Zhang, Aiqian Zhang
      Abstract: The emerging heterocyclic brominated flame retardant tris‐(2,3‐dibromopropyl) isocyanurate (TBC), widely used in reinforced plastics, has demonstrated toxicity to fish. However, little is known about its toxicity in rodents. This study aims to determine the effect of TBC on growth, biochemical parameters in serum, organs and related gene expression of both male and female BALB/c mice after gastro‐gavage administration of 0, 2, 10 and 50 mg kg−1 TBC for 28 days. Results indicated that exposure to TBC had no effects on basic growth and food intake of mice, but significantly increased serum alanine aminotransferase levels in male mice. Histopathological analyses showed that focal necrosis (2, 10 and 50 mg kg−1 TBC‐exposed groups) and ballooning degeneration (10 and 50 mg kg−1 TBC‐exposed groups) were found in mouse liver, whereas transmission electron microscopy revealed dose‐dependent hepatocyte apoptosis, mitochondrial degeneration and endoplasmic reticulum dilation. Histopathological and ultrastructural assessments in the lung showed dose‐dependent hyperplasia of pulmonary alveolar epithelium, bronchial congestion, infiltration of inflammatory cells and mitochondrial swelling following TBC exposure. Our results also indicated that mitochondria are one of the major target cytoplasmic organelles for TBC, suggesting that damage in mitochondria is one of the pathways that led to toxic effects in the liver and lung of TBC‐treated groups. Moreover, TBC effectively activated the gene expression of p53 in mice liver. Our findings provide strong evidence that TBC induces significant toxicity in mice organs, especially in liver and lung, which play vital roles in detoxification and gas exchange, respectively. This research will contribute to characterize the toxic effects of TBC, which was introduced as one of the candidates for brominated flame retardant replacement. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-07-17T23:01:48.126209-05:
      DOI: 10.1002/jat.3026
  • Toxicity profiles and solvent–toxicant interference in the planarian
           Schmidtea mediterranea after dimethylsulfoxide (DMSO) exposure
    • Authors: An‐Sofie Stevens; Nicky Pirotte, Michelle Plusquin, Maxime Willems, Thomas Neyens, Tom Artois, Karen Smeets
      Abstract: To investigate hydrophobic test compounds in toxicological studies, solvents like dimethylsulfoxide (DMSO) are inevitable. However, using these solvents, the interpretation of test compound‐induced responses can be biased. DMSO concentration guidelines are available, but are mostly based on acute exposures involving one specific toxicity endpoint. Hence, to avoid solvent–toxicant interference, we use multiple chronic test endpoints for additional interpretation of DMSO concentrations and propose a statistical model to assess possible synergistic, antagonistic or additive effects of test compounds and their solvents. In this study, the effects of both short‐ (1 day) and long‐term (2 weeks) exposures to low DMSO concentrations (up to 1000 µl l−1) were studied in the planarian Schmidtea mediterranea. We measured different biological levels in both fully developed and developing animals. In a long‐term exposure set‐up, a concentration of 500 µl l−1 DMSO interfered with processes on different biological levels, e.g. behaviour, stem cell proliferation and gene expression profiles. After short exposure times, 500 µl l−1 DMSO only affected motility, whereas the most significant changes on different parameters were observed at a concentration of 1000 µl l−1 DMSO. As small sensitivity differences exist between biological levels and developmental stages, we advise the use of this solvent in concentrations below 500 µl l−1 in this organism. In the second part of our study, we propose a statistical approach to account for solvent–toxicant interactions and discuss full‐scale solvent toxicity studies. In conclusion, we reassessed DMSO concentration limits for different experimental endpoints in the planarian S. mediterranea. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-06-25T22:54:19.05537-05:0
      DOI: 10.1002/jat.3011
  • Vascular endothelial growth factor mRNA levels as a biomarker for
           short‐term N‐butyl‐N‐(4‐hydroxybutyl)
           nitrosamine‐induced rat bladder carcinogenesis bioassay
    • Authors: Shin Wakui; Tomoko Mutou, Hiroyuki Takahashi, Masahiro Ikegami, Hideki Wanibuchi, Shoji Fukushima
      Abstract: Generically, carcinogenic effects of chemicals in bladder carcinogenesis are judged by induction of papillary or nodular (PN) hyperplasia in rats given N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN) for 4 weeks and the test chemical for 22–28 weeks. However, upregulation of vascular endothelial growth factor (VEGF) begins early in rat BBN bladder carcinogenesis. To establish a short‐term rat bladder carcinogenic bioassay, we analyzed the correlations between VEGF, VEGF mRNA and bladder lesions inductions at 10 and 26 weeks after BBN treatment. Six‐week‐old male Wistar (slc) rats were given 0.05% BBN for 4, 10 or 26 weeks. To avoid individual rat bias, the bladders were investigated by partial cystectomy at 10 weeks and total cystectomy at 26 weeks. After induction, PN hyperplasia and carcinoma in rats increased with the length of BBN treatment and immunohistochemical VEGF expression also increased following carcinogenesis, but the immunoreactivity of individual lesions was quite variable. Moreover, induction of PN hyperplasia at 10 weeks’ BBN treatment was not significantly correlated with that at 26 weeks' treatment; thus, it was not possible to predict the carcinogenic effect due to the induction of PN hyperplasia at 26 weeks' BBN treatment by that at 10 weeks' treatment. However, VEGF mRNA levels of rat bladders at 10 weeks' BBN treatment revealed a strong significant correlation with the incidence of bladder lesions at 26 weeks' treatment. Here, we suggest that quantitative VEGF mRNA levels are a good biomarker for a short‐term BBN‐induced bioassay for rat bladder carcinogenesis. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-05-28T09:25:42.146279-05:
      DOI: 10.1002/jat.3021
  • Developmental toxicity assay using high content screening of zebrafish
    • Authors: Susan Lantz‐McPeak; Xiaoqing Guo, Elvis Cuevas, Melanie Dumas, Glenn D. Newport, Syed F. Ali, Merle G. Paule, Jyotshna Kanungo
      Abstract: Typically, time‐consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post‐fertilization. Here we describe an automated image‐based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post‐acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth‐retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
      PubDate: 2014-05-28T09:20:58.316539-05:
      DOI: 10.1002/jat.3029
  • Cardiotoxicity evaluation of anthracyclines in zebrafish (Danio rerio)
    • Authors: Ying Han; Jing‐pu Zhang, Jian‐qin Qian, Chang‐qin Hu
      Abstract: Drug‐induced cardiotoxicity is a leading factor for drug withdrawals, and limits drug efficacy and clinical use. Therefore, new alternative animal models and methods for drug safety evaluation have been given great attention. Anthracyclines (ANTs) are widely prescribed anticancer agents that have a cumulative dose relationship with cardiotoxicity. We performed experiments to study the toxicity of ANTs in early developing zebrafish embryos, especially their effects on the heart. LC50 values for daunorubicin, pirarubicin, doxorubicin (DOX), epirubicin and DOX‐liposome at 72 h post‐fertilization were 122.7 μM, 111.9 μM, 31.2 μM, 108.3 μM and 55.8 μM, respectively. At the same time, zebrafish embryos were exposed to ANTs in three exposure stages and induced incomplete looping of the heart tube, pericardia edema and bradycardia in a dose‐dependent manner, eventually leading to death. DOX caused the greatest heart defects in the treatment stages and its liposome reduced the effects on the heart, while daunorubicin produced the least toxicity. Genes and proteins related to heart development were also identified to be sensitive to ANT exposure and downregulated by ANTs. It revealed ANTs could disturb the heart formation and development. ANTs induced cardiotoxicity in zebrafish has similar effects in mammalian models, indicating that zebrafish may have a potential value for assessment of drug‐induced developmental cardiotoxicity. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-05-22T16:22:52.444139-05:
      DOI: 10.1002/jat.3007
  • A trivalent approach for determining in vitro toxicology: Examination of
           oxime K027
    • Authors: Adriana Prado; Georg A. Petroianu, Dietrich E. Lorke, Jeremy W. Chambers
      Abstract: Unforeseen toxic effects contribute to compound attrition during preclinical evaluation and clinical trials. Consequently, there is a need to correlate in vitro toxicity to in vivo and clinical outcomes quickly and effectively. We propose an expedited evaluation of physiological parameters in vitro that will improve the ability to predict in vivo toxicity of potential therapeutics. By monitoring metabolism, mitochondrial physiology and cell viability, our approach provides insight to the extent of drug toxicity in vitro. To implement our approach, we used human hepatocellular carcinoma cells (HepG2) and neuroblastoma cells (SH‐SY5Y) to monitor hepato‐ and neurotoxicity of the experimental oxime K027. We utilized a trivalent approach to measure metabolism, mitochondrial stress and induction of apoptosis in 96‐well formats. Any change in these three areas may suggest drug‐induced toxicity in vivo. K027 and pralidoxime, an oxime currently in clinical use, had no effect on glycolysis or oxygen consumption in HepG2 and SH‐SY5Y cells. Similarly, these oximes did not induce oxidant generation nor alter mitochondrial membrane potential. Further, K027 and pralidoxime failed to activate effector caspases, and these oximes did not alter viability. The chemotherapeutic agent, docetaxel, negatively affected metabolism, mitochondrial physiology and viability. Our studies present a streamlined high‐throughput trivalent approach for predicting toxicity in vitro, and this approach reveals that K027 has no measurable hepatotoxicity or neurotoxicity in vitro, which correlates with their in vivo data. This approach could eliminate toxic drugs from consideration for in vivo preclinical evaluation faster than existing toxicity prediction panels and ultimately prevent unnecessary experimentation. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-05-22T16:22:49.472421-05:
      DOI: 10.1002/jat.3013
  • Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA
           exposed to uranyl acetate and ultraviolet radiation
    • Authors: Janice Wilson; Mary C. Zuniga, Filbert Yazzie, Diane M. Stearns
      Abstract: Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB‐ or UVA‐activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB‐activated uranyl ion was measured in repair‐proficient and repair‐deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co‐exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co‐exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non‐photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-05-15T09:03:01.061665-05:
      DOI: 10.1002/jat.3015
  • Animal models for percutaneous absorption
    • Authors: Eui Chang Jung; Howard I. Maibach
      First page: 1
      Abstract: Animal models are important tools to predict human in vivo percutaneous absorption/penetration. Monkey, pig, rat, rabbit, guinea pig, hairless rodents, such as hairless rat, hairless mouse, hairless guinea pig and hairless dog, are among the most used animals for this purpose. Each animal model has its own advantages and weakness or limitation. To better correlate animal data with human skin absorption, we need to be familiar with each animal model's characteristics as well as experimental method and condition. We reviewed the original papers published after 1993 that described permeability of both animal skin and human skin. It showed that monkey, pig and hairless guinea pig are more predictive of human skin absorption/penetration and common laboratory animals, such as rat, rabbit, guinea pig, generally overestimate human skin absorption/penetration. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-10-27T00:23:47.87766-05:0
      DOI: 10.1002/jat.3004
  • Fish multigeneration test with preliminary short‐term reproduction
           assay for estrone using Japanese medaka (Oryzias latipes)
    • Authors: Ataru Nakamura; Ikumi Tamura, Hitomi Takanobu, Masumi Yamamuro, Taisen Iguchi, Norihisa Tatarazako
      First page: 11
      Abstract: The most potent chemicals potentially causing adverse effects on fish species are estrogens in human waste. Sewage is a source of these estrogens and it is difficult to reduce. In particular, although the bioactivity of estrone is estimated to be about half of that of estradiol, multiple studies report that more than 100 ng l–1 of estrone can be detected in urban rivers, including discharges from sewage treatment works; approximately two times as high as estradiol. Few studies have been conducted to investigate the long‐term effects of estrone on wildlife; therefore, we conducted fish multigeneration test using Japanese medaka (Oryzias latipes). Medaka were exposed to estrone for 27 weeks across three generations in environmentally relevant concentrations, being 5.74, 11.4, 24.0, 47.1 and 91.4 ng l–1. No effects on reproduction were observed in the first generation; however, a decline in egg production and fertility was observed in the second generation exposed to 91.4 ng l–1 estrone, which is lower than some known environmental concentrations in urban environments. Furthermore, histopathological abnormalities were observed in the third generation exposed to both 47.1 and 91.4 ng l–1, suggesting that estrone possibly exerts severe effects on the third or later generations. However, appearances of testis–ova were observed in the second and third generation they were not consistent with actual effects on reproduction, notwithstanding the testis‐ova is regarded as the key evidence for endocrine disruption. Accordingly, we consider that qualitative measurement of abnormalities using histopathological observations is required for appropriate evaluation of endocrine disruption. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-02-12T05:32:04.421935-05:
      DOI: 10.1002/jat.2981
  • Benchmark dose of cadmium concentration in rice for renal effects in a
           cadmium‐polluted area in Japan
    • Authors: Kazuhiro Nogawa; Teruhiko Kido, Muneko Nishijo, Hideaki Nakagawa, Yasushi Suwazono
      First page: 24
      Abstract: The aim of this study was to estimate the reference level of cadmium in rice as the benchmark doses (BMD) and their 95% lower confidence limits (BMDL) for various renal effects by applying an updated hybrid approach. The participants were 1120 men and 1274 women aged 50 years or older who lived in the environmentally exposed Kakehashi river basin for at least 30 years. As indicators of renal dysfunction, glucose, protein, aminonitrogen, metallothionein and β2‐microgrobulin in urine were measured. Cadmium concentration was determined for rice samples stored in warehouses of the farmers in all of the polluted hamlets. The BMD and BMDL that corresponded to an additional risk of 5% were calculated with background risk at a zero exposure set at 5%. The obtained BMDLs were 0.39 (aminonitrogen), 0.26 (metallothionein), 0.25 (β2‐microgrobulin) mg kg–1 in men and 0.44 (glucose), 0.32 (protein), 0.33 (aminonitrogen), 0.28 (metallothionein) and 0.24 (β2‐microgrobulin) mg kg–1 in women. The lowest BMDL was 0.25 and 0.24 mg kg–1 (β2‐microgrobulin) in men and women respectively. These values were lower than the maximum level (0.4 mg kg–1) determined by the Codex Alimentarius Commission, indicating that these BMDLs may contribute to further discussion on the health risk assessment of cadmium exposure. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-01-30T02:58:16.248401-05:
      DOI: 10.1002/jat.2982
  • Development and utilization of an ex vivo bromodeoxyuridine local lymph
           node assay protocol for assessing potential chemical sensitizers
    • Authors: W. C. Williams; C. Copeland, E. Boykin, S. J. Quell, D. M. Lehmann
      First page: 29
      Abstract: The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [3H]methyl thymidine. A more recent non‐isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non‐sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU‐enzyme‐linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis‐diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC2 values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
      PubDate: 2014-02-14T07:42:07.38687-05:0
      DOI: 10.1002/jat.2983
  • The effect of diesel exhaust exposure on blood–brain barrier
           integrity and function in a murine model
    • Authors: Sayeh Heidari Nejad; Ryusuke Takechi, Benjamin J. Mullins, Corey Giles, Alexander N. Larcombe, Dean Bertolatti, Krassi Rumchev, Satvinder Dhaliwal, John Mamo
      First page: 41
      Abstract: Epidemiological studies indicate that exposure to diesel exhaust (DE) is associated with vascular‐based disorders. To investigate the effect of DE on blood–brain barrier (BBB) function and integrity, 8‐week‐old BALB/c mice were randomized to DE in a cyclical treatment regimen over a 2‐week period. Functional integrity of BBB was determined by considering brain parenchymal abundance of IgG within the hippocampal formation and cortex at 6 h and 24 h intervals following final exposure treatment. Neurovascular inflammation was expressed as the abundance of glial fibrillar acidic protein. Two doses of DE were studied and compared to air‐only treated mice. Mice exposed to DE had substantially greater abundance of parenchymal IgG compared to control mice not exposed to DE. Increased parenchymal glial fibrillar acidic protein at 24 h post‐DE exposure suggested heightened neurovascular inflammation. Our findings are proof‐of‐concept that inhalation of DE can compromise BBB function and support the broader contention that DE exposure may contribute to neurovascular disease risk. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-01-30T02:55:52.631084-05:
      DOI: 10.1002/jat.2985
  • Development of a multiparametric in vitro model of skin sensitization
    • Authors: Muriel Guyard‐Nicodème; Eloise Gerault, Marion Platteel, Olivier Peschard, Wilfried Veron, Philippe Mondon, Svinareff Pascal, Marc G. J. Feuilloley
      First page: 48
      Abstract: Most animal experiments on cosmetics safety are prohibited and since March 2013, this obligation includes sensitization tests. However, until now there has been no validated alternative in vitro method. In this work, 400 compounds used in the cosmetic industry were selected to cover the greatest diversity of structures, biological activities and sensitizing potential. These molecules were submitted to a series of tests aimed at reproducing essential steps in sensitization and to distinguish between sensitization and irritations, i.e., transcutaneous permeation (factor A), haptenation (factor B), sensitization cytokines production (factor C) and acute toxicity (factor D). The transcutaneous diffusion was measured on human skin explants using Franz cells. Haptenation was tested in solution on human serum albumin. Sensitization cytokine production was investigated by measurement of interleukin‐18 release by keratinocytes. Acute toxicity was determined using an 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide75 cell viability test. As only sufficiently stable, soluble and detectable compounds are usable, 33, 72, 68 and 68 molecules were finally tested on factors A, B, C and D, respectively, and 32 were completely screened by the four factors. The individual correlation of the four factors with the reference in vivo tests was limited but the combination of these factors led to a correlation between in vivo and in vitro assays of 81.2% and the safety of the test (risk of false negative) reached 96.8%. The techniques employed are simple and inexpensive and this model of four tests appears as a promising technique to evaluate in vitro the skin sensitization potential of unknown molecules. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-02-03T20:58:18.785567-05:
      DOI: 10.1002/jat.2986
  • Microvesicle‐associated microRNA expression is altered upon
           particulate matter exposure in healthy workers and in A549 cells
    • Authors: Valentina Bollati; Laura Angelici, Giovanna Rizzo, Laura Pergoli, Federica Rota, Mirjam Hoxha, Francesco Nordio, Matteo Bonzini, Letizia Tarantini, Laura Cantone, Angela C. Pesatori, Pietro Apostoli, Andrea A. Baccarelli, Pier Alberto Bertazzi
      First page: 59
      Abstract: Cardiovascular disease risk has been consistently linked with particulate matter (PM) exposure. Cell‐derived microvesicles (MVs) are released into plasma and transfer microRNAs (miRNAs) between tissues. MVs can be produced by the respiratory system in response to proinflammatory triggers, enter the circulatory system and remotely modify gene expression in cardiovascular tissues. However, whether PM affects MV signaling has never been investigated. In this study, we evaluated expression of microRNAs contained within plasma MVs upon PM exposure both in vivo and in vitro. In the in vivo study, we isolated plasma MVs from healthy steel plant workers before and after workplace PM exposure. We measured the expression of 88 MV‐associated miRNAs by real‐time polymerase chain reaction. To assess a possible source of the MV miRNAs identified in vivo, we measured their miRNA expression in PM‐treated A549 pulmonary cell lines in vitro. MiRNA profiling of plasma MVs showed 5.62‐ and 13.95‐fold increased expression of miR‐128 and miR‐302c, respectively, after 3 days of workplace PM exposure (P 
      PubDate: 2014-02-07T06:08:22.481934-05:
      DOI: 10.1002/jat.2987
  • Growth evaluation method by live imaging of Daphnia magna and its
           application to the estimation of an insect growth regulator
    • Authors: Akiko Suzuki; Yasuhiko Kato, Tomoaki Matsuura, Hajime Watanabe
      First page: 68
      Abstract: The zooplankton Daphnia magna has been widely used as a test organism to assess the toxicity of chemical substances because of its important position in aquatic ecology and its ease of handling. Among the various endpoints for toxicity evaluation, growth rate is one of the most critical and many studies have been conducted. However, measurement of growth rate was time‐consuming and not an ideal endpoint in terms of screening. In this study, we demonstrated a live imaging method to monitor the growth of daphnids by area measurement. In this method, daphnid images were directly obtained from a swimming chamber and these images were processed for the evaluation of growth. The reliability of this method was confirmed by comparison with the conventional dry weight method of the same animals. The body area of daphnids using this method showed a strong correlation with the dry weight method, with R2 = 0.930. In addition, we quantified the effect of a toxicant, fenoxycarb, on the growth of the animal. Fenoxycarb concentrations of 0, 0.027, 0.27 and 2.7 µg l–1 were tested and their effects on growth were estimated by the live imaging method. In the toxicity test, the area of daphnids decreased significantly with increasing fenoxycarb concentration. These results indicate that the present live imaging method is a reliable approach for daphnid toxicity testing. This method is promising for high‐throughput Daphnia toxicity tests and real‐time individual observations. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-02-12T05:35:54.135943-05:
      DOI: 10.1002/jat.2988
  • Establishment of a short‐term, in vivo screening method for
           detecting chemicals with juvenile hormone activity using adult Daphnia
    • Authors: Ryoko Abe; Haruna Watanabe, Masumi Yamamuro, Taisen Iguchi, Norihisa Tatarazako
      First page: 75
      Abstract: Juvenile hormone (JH) and JH agonists have been shown to induce male offspring production in various daphnids, including Daphnia magna using OECD TG211. The critical period (about 1h) for JH action on ova in the parent's ovary to induce male offspring is existing at 7‐8h later from ovulation. Therefore, we considered that adult D. magna could be used to produce a short‐term screening method for detecting JH analogs. Using this method, we successfully demonstrated male offspring induction in the second broods after exposure to JH or JH agonists. After investigating the exposure time, the number of repetitions and the exposure concentration, we established a short‐term, in vivo screening method for detecting JH analogs using adult D. magna. We examined positive and negative control chemicals using a previously developed method and verified the validity of our new testing method. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-01-30T02:15:26.429675-05:
      DOI: 10.1002/jat.2989
  • Effects of cylindrospermopsin on a common carp leucocyte cell line
    • Authors: Anna Sieroslawska; Anna Rymuszka
      First page: 83
      Abstract: Cylindrospermopsin (CYN) is a cytotoxin produced by different cyanobacterial species, increasingly detected in water reservoirs worldwide. There is very little information available concerning the effects of the toxin on fish immune cells. The aim of the study was to elucidate the potential impact of cylindrospermopsin on the selected parameters of a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 10, 1 or 0.1 µg ml–1 for up to 48 h. Cell viability and proliferation, apoptosis/necrosis induction, cell morphology and phagocytic activity were determined. The two higher toxin concentrations occurred to be evidently cytotoxic in a time‐dependent manner and influenced all studied parameters. The lowest used concentration had no effects on cell viability and cell number; however, a strong reduction of bacteria uptake after 24‐h exposure was detected. The obtained results indicate that cylindrospermopsin may interfere with the basic functions of fish phagocytic cells and as a consequence influence the fish immunity. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-01-30T02:59:12.516049-05:
      DOI: 10.1002/jat.2990
  • Molecular biomarkers of phospholipidosis in rat blood and heart after
           amiodarone treatment
    • Authors: Nicola Bocchini; Mery Giantin, Federica Crivellente, Serena Ferraresso, Ivo Faustinelli, Mauro Dacasto, Patrizia Cristofori
      First page: 90
      Abstract: Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypothetically applicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg–1 per day–1 amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real‐Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-02-18T00:25:23.937611-05:
      DOI: 10.1002/jat.2992
  • Preliminary safety evaluation of a taurocholate‐conjugated
           low‐molecular‐weight heparin derivative (LHT7): a potent
           angiogenesis inhibitor
    • Authors: Farzana Alam; Seung Woo Chung, Seung Rim Hwang, Ji‐young Kim, Jooho Park, Hyun Tae Moon, Youngro Byun
      First page: 104
      Abstract: In our previous studies, taurocholic acid (TA)‐conjugated low‐molecular‐weight heparin derivative (LHT7) has been proven to be a potent anti‐angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism‐related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg–1 doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg–1 doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism‐related studies showed that blood pressure at 50 mg kg–1 did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg–1 repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material. Copyright © 2014 John Wiley & Sons, Ltd.
      PubDate: 2014-02-16T21:58:07.934802-05:
      DOI: 10.1002/jat.2995
  • HepaRG culture in tethered spheroids as an in vitro
           three‐dimensional model for drug safety screening
    • Abstract: Conventional two‐dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver‐specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg–Gly–Asp (RGD) and galactose‐conjugated substratum with an optimized hybrid ratio as an in vitro three‐dimensional (3D) human hepatocyte model. The liver‐specific gene expression level and drug metabolizing enzyme activities in HepaRG‐tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug‐induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG‐tethered spheroid would be an alternative in vitro model suitable for drug safety screening. Copyright © 2014 John Wiley & Sons, Ltd.
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