The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Volume 45 , Issue 12
Showing 1-8 articles out of 8 articles from the selected issue
Original Article
  • Fabíola de Azevedo Mello, Beatriz Bertholace Magalhaes Silva, Emmanuel ...
    2020 Volume 45 Issue 12 Pages 737-750
    Published: 2020
    Released: December 01, 2020
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    Supplementary material

    2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most widely used herbicides in the world, but its mutagenic and carcinogenic potential is still controversial. We simulated environmental exposure to 2,4-D, with the objective of evaluating the genotoxic effect of acute and chronic exposure to 2,4-D in rodents. We also evaluated the performance of machine learning algorithms in detecting differences in exposure groups through recognition performed from genotoxic characteristics. In the acute phase, 88 Swiss mice were used, distributed in five groups and exposed to nebulizations at different time intervals (24, 48, 72 and 192 hr). In the chronic phase, 88 Wistar rats were used, distributed in two groups (inhaled and oral) and exposed for six months. Femoral bone marrow cells were collected for a micronucleus test and comet assay. Data were evaluated by pattern recognition algorithms. In acute exposure, medium and high concentrations induced DNA damage in the comet assay, but these concentrations did not increase micronucleated cells. In the chronic exposure, there was an increase in micronuclei and DNA damage in the comet assay in all exposed groups regardless of the exposure route. The data showed a robust pattern of distinction between exposed and nonexposed groups to 2,4-D. Our data showed that both acute inhalation exposure and chronic oral and inhalation exposure to 2,4-D can cause genotoxic effects regardless of concentration. Machine learning showed a clear distinction between the control groups and those exposed to 2,4-D, and the effects of exposure are not concentration-dependent.

Original Article
  • Suhaibee Kuno, Kanitta Srinoun, Tipparat Penglong
    2020 Volume 45 Issue 12 Pages 751-761
    Published: 2020
    Released: December 01, 2020
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    The phorbol 12-myristate 13-acetate (PMA)-induced U937 cell line has been widely used as an in vitro model for studying the functions of human macrophages. However, there are several concentrations of PMA commonly used to drive the differentiation of monocytic cell line to macrophage. Also, the expression of microRNA-155 (miR-155) and miR-125b in PMA-treated human monocytic cell line has not yet been reported. The five usual concentrations of PMA for stimulating macrophage differentiation are 10, 25, 50, 100, and 200 nM. In this study we compared the expression levels of miR-155, miR-125b and their related genes involved in macrophage functions in U937-derived cells after treatment with those five concentrations. The morphological study results showed that the five concentrations of PMA could induce macrophage differentiation in a similar manner. Moreover, cell proliferation and viability were not significantly different among these five conditions excepted the lower cell viability at 200 nM of PMA treatment. The five concentrations of PMA could upregulate the expression of miR-155 and miR-125b and increase the phagocytic activity of U937-derived cells in dose-reversal manner. The upregulation of miR-155 was correlated with increased expression levels of TNFα and decreased expression levels of BACH1 and CEBPβ, while the reduction of IRF4 was correlated with increased expression levels of miR-125b. Our study found that PMA could stimulate macrophage differentiation in a broad range of concentrations, however, the lower concentration could upregulate the higher expression of both miR-155 and miR-125b, and that correlated with the phagocytic functional activity of U937-derived macrophages.

Letter
  • Yusuke Kamiya, Mayu Yanagi, Shiori Hina, Kazuki Shigeta, Tomonori Miur ...
    2020 Volume 45 Issue 12 Pages 763-767
    Published: 2020
    Released: December 01, 2020
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    A simplified physiologically based pharmacokinetic (PBPK) model consisting of chemical receptor, metabolizing and/or excreting, and central compartments was recently proposed. In the current study, this type of PBPK model was set up for perfluorooctane sulfonate, an environmental toxicant with liver effects, as a model compound; the model was then used to estimate tissue concentrations. The pharmacokinetic parameter input values for the model were calculated to give the best fit to reported/measured blood substrate concentrations in rats. The maximum concentrations and areas under the concentration versus time curves in plasma, liver, and kidney extrapolated using PBPK models for perfluorobutane sulfonic acid, perfluorohexane sulfonic acid, and perfluorooctane sulfonic acid were consistent with the reported mean values in rats. Using the rat models and scaled-up human PBPK models, some accumulation of perfluorooctane sulfonic acid in plasma and liver was seen after repeated doses. The reported 50th and 95th percentile concentrations of perfluorooctane sulfonic acid in human blood (0.0048 and 0.0183 ng/mL, respectively) in the general population underwent reverse dosimetry analysis using our PBPK models. These human blood concentrations potentially imply exposures of 0.041 and 0.16 µg/kg/day, respectively, for 90 days, values that are roughly similar to the reference dose (0.02 μg/kg/day) with an uncertainty factor of 30. These results indicate the relatively good estimates for tissue and blood exposures of chemical substrates after oral doses generated using the latest PBPK models.

Original Article
  • Yuichiro Takanami, Nobumasa Kitamura, Shigeaki Ito
    2020 Volume 45 Issue 12 Pages 769-782
    Published: 2020
    Released: December 01, 2020
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    A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

Original Article
  • Yukino Kobayashi, Ami Oguro, Erina Yagi, Akira Mitani, Suguru N. Kudoh ...
    2020 Volume 45 Issue 12 Pages 783-794
    Published: 2020
    Released: December 01, 2020
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    Supplementary material

    Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer’s disease and Parkinson’s disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.

Letter
  • Takato Hara, Misato Saeki, Yuichi Negishi, Toshiyuki Kaji, Chika Yamam ...
    2020 Volume 45 Issue 12 Pages 795-800
    Published: 2020
    Released: December 01, 2020
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    We have previously reported the cytotoxicity and various biological responses of organic-inorganic hybrid molecules. However, because all the molecules used were electrophilic, the effect of the hybrid molecule without electrophilicity remains unclear. The glutathione-protected gold nanocluster, Au25(SG)18, is an organic-inorganic hybrid molecule that shows a low intramolecular polarity and high stability. In this study, we examined the cytotoxicity and intracellular accumulation of Au25(SG)18 in cultured vascular endothelial cells and compared these characteristics with those of negatively charged gold nanoparticles (AuNPs). Both Au25(SG)18 and AuNPs accumulated in vascular endothelial cells in a dose-dependent manner without cytotoxicity and more accumulation was observed at low cell densities. However, Au25(SG)18 accumulated significantly less than AuNPs in the cells. These results suggest that the intramolecular polarity of organic-inorganic hybrid molecules could regulate intracellular accumulation.

Letter
  • Tomoya Fujie, Yusuke Ozaki, Fukuta Takenaka, Misaki Nishio, Takato Har ...
    2020 Volume 45 Issue 12 Pages 801-806
    Published: 2020
    Released: December 01, 2020
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    Metallothionein (MT) is an inducible protein with cytoprotective activity against heavy metals such as cadmium, zinc, and copper. MT-1 and MT-2 are the isoforms of MT induced by and bind the heavy metals. Bovine aortic endothelial cells contain three types of MT genes, namely, MT-1A, MT-1E, and MT-2A; however, the associated protein expression of these MT isoforms has not been identified. In the present study, the expression of MT subisoform proteins in cells treated with cadmium chloride was identified using a high-performance liquid chromatography-inductively coupled plasma-mass spectrometry system. It was revealed that: (1) transcriptional induction of MT-1A by cadmium was markedly more sensitive than that of MT-1E/2A; (2) MT-1A and MT-2A proteins were the predominant MT subisoforms induced by cadmium; and (3) there might be differentiation in the functions of MT-1 and MT-2 against cadmium cytotoxicity, although the actual roles of the MT isoforms in the cells were not distinct. This is the first study to show the differential induction of isoforms of MT proteins in vascular endothelial cells by cadmium.

Erratum
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