Neurotoxicity Research

, Volume 35, Issue 3, pp 668–683 | Cite as

Developmental Exposure of Mice to T-2 Toxin Increases Astrocytes and Hippocampal Neural Stem Cells Expressing Metallothionein

  • Kota Nakajima
  • Takeshi Tanaka
  • Yasunori Masubuchi
  • Yuko Ito
  • Satomi Kikuchi
  • Gye-Hyeong Woo
  • Toshinori Yoshida
  • Makoto ShibutaniEmail author
Original Article


We previously reported that developmental exposure to T-2 toxin caused transient disruption of the hippocampal neurogenesis targeting neural stem cells (NSCs) and early-stage progenitor cells involving oxidative stress on weaning in mouse offspring. The present study examined metallothionein (MT) expression changes and their cellular identity in brain regions of these animals. T-2 toxin at 0, 1, 3, and 9 mg/kg was given in the diet of maternal mice from gestational day 6 to postnatal day (PND) 21 on weaning. Offspring were maintained through PND 77 without T-2 toxin exposure. Male offspring were analyzed. Immunohistochemically, MT-I/II+ cells increased in the subgranular zone (SGZ) of the dentate gyrus and cerebral cortex at ≥ 3 mg/kg and in the hilus of the dentate gyrus, corpus callosum, and cerebellum at 9 mg/kg on PND 21, suggestive of operation of cytoprotective function against oxidative stress throughout the brain. Double immunohistochemistry analysis revealed MT-I/II+ SGZ cells to be NSCs and MT-I/II+ cells in other brain regions to be astrocytes as toxicity targets of T-2 toxin. Phosphorylated STAT3+ cell numbers increased only in the cerebellum in parallel with the increase of GFAP+ astrocytes at 9 mg/kg, suggesting a STAT3-mediated transcriptional GFAP upregulation in cerebellar astrocytes. In the dentate gyrus, Il1a, Il1r1, and Mt2 increased transcripts at 9 mg/kg, suggesting activation of the IL-1 signaling cascade, possibly causing MT-II upregulation. The increase of MT-I/II+ cells in all brain regions disappeared or was suppressed below the control level on PND 77, suggesting a recovery from the T-2 toxin-induced oxidative stress.


T-2 toxin Metallothionein Developmental neurotoxicity Oxidative stress Mouse 



The authors thank Shigeko Suzuki for her technical assistance in preparing the histological specimens and Yosuke Watanabe for his technical advice in this study.


This work was supported by Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and Welfare of Japan (Grant No. H28-shokuhin-ippan-004) and by a Research Fund from Institute of Global Innovation Research, Tokyo University of Agriculture and Technology.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Research Involving Human Participants and/or Animals

All procedures in this study were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, 1 June 2006) and according to the protocol approved by the Animal Care and Use Committee of The Tokyo University of Agriculture and Technology. All efforts were made to minimize animal suffering.

Supplementary material

12640_2018_9981_MOESM1_ESM.docx (2.2 mb)
ESM 1 (DOCX 2.19 mb)


  1. Bauman JW, Liu J, Liu YP, Klaassen CD (1991) Increase in metallothionein produced by chemicals that induce oxidative stress. Toxicol Appl Pharmacol 110:347–354. CrossRefPubMedGoogle Scholar
  2. Braun W, Vasák M, Robbins AH, Stout CD, Wagner G, Kägi JH, Wüthrich K (1992) Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2. Proc Natl Acad Sci U S A 89:10124–10128CrossRefPubMedPubMedCentralGoogle Scholar
  3. Buniatian GH, Hartmann HJ, Traub P, Weser U, Wiesinger H, Gebhardt R (2001) Acquisition of blood–tissue barrier–supporting features by hepatic stellate cells and astrocytes of myofibroblastic phenotype. Inverse dynamics of metallothionein and glial fibrillary acidic protein expression. Neurochem Int 38:373–383. CrossRefPubMedGoogle Scholar
  4. Buniatian GH, Hartmann HJ, Traub P, Wiesinger H, Albinus M, Nagel W, Shoeman R, Mecke D, Weser U (2002) Glial fibrillary acidic protein-positive cells of the kidney are capable of raising a protective biochemical barrier similar to astrocytes: expression of metallothionein in podocytes. Anat Rec 267:296–306. CrossRefPubMedGoogle Scholar
  5. Chaudhary M, Bhaskar AS, Rao PV (2015) Differential effects of route of T-2 toxin exposure on hepatic oxidative damage in mice. Environ Toxicol 30:64–73. CrossRefPubMedGoogle Scholar
  6. Chaudhary M, Rao PV (2010) Brain oxidative stress after dermal and subcutaneous exposure of T-2 toxin in mice. Food Chem Toxicol 48:3436–3442. CrossRefPubMedGoogle Scholar
  7. Damiani CL, O’Callaghan JP (2007) Recapitulation of cell signaling events associated with astrogliosis using the brain slice preparation. J Neurochem 100:720–726. CrossRefPubMedGoogle Scholar
  8. DeSilva TM, Borenstein NS, Volpe JJ, Kinney HC, Rosenberg PA (2012) Expression of EAAT2 in neurons and protoplasmic astrocytes during human cortical development. J Comp Neurol 520:3912–3932. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Doi K, Uetsuka K (2011) Mechanisms of mycotoxin-induced neurotoxicity through oxidative stress-associated pathways. Int J Mol Sci 12(8):5213-5237CrossRefPubMedPubMedCentralGoogle Scholar
  10. Foo LC, Dougherty JD (2013) Aldh1L1 is expressed by postnatal neural stem cells in vivo. Glia 61:1533–1541. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Guanizo AC, Fernando CD, Garama DJ, Gough DJ (2018) STAT3: a multifaceted oncoprotein. Growth Factors 6:1–14CrossRefGoogle Scholar
  12. Herrera F, Chen Q, Schubert D (2010) Synergistic effect of retinoic acid and cytokines on the regulation of glial fibrillary acidic protein expression. J Biol Chem 285:38915–38922. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hidalgo J, Aschner M, Zatta P, Vasák M (2001) Roles of the metallothionein family of proteins in the central nervous system. Brain Res Bull 55:133–145. CrossRefPubMedGoogle Scholar
  14. Hodge RD, Kowalczyk TD, Wolf SA, Encinas JM, Rippey C, Enikolopov G, Kempermann G, Hevner RF (2008) Intermediate progenitors in adult hippocampal neurogenesis: Tbr2 expression and coordinate regulation of neuronal output. J Neurosci 28:3707–3717. CrossRefPubMedGoogle Scholar
  15. Holloway AF, Stennard FA, Dziegielewska KM, Weller L, West AK (1997) Localisation and expression of metallothionein immunoreactivity in the developing sheep brain. Int J Dev Neurosci 15:195–203. CrossRefPubMedGoogle Scholar
  16. Hsu IC, Smalley EB, Strong FM, Ribelin WE (1972) Identification of T-2 toxin in moldy corn associated with a lethal toxicosis in dairy cattle. Appl Microbiol 24:684–690PubMedPubMedCentralGoogle Scholar
  17. Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S (1998) Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res 57:1–9. CrossRefPubMedGoogle Scholar
  18. Karki P, Kim C, Smith K, Son DS, Aschner M, Lee E (2015) Transcriptional regulation of the astrocytic excitatory amino acid transporter 1 (EAAT1) via NF-κB and yin yang 1 (YY1). J Biol Chem 290:23725–23737. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kruczek C, Görg B, Keitel V, Bidmon HJ, Schliess F, Häussinger D (2011) Ammonia increases nitric oxide, free Zn2+, and metallothionein mRNA expression in cultured rat astrocytes. Biol Chem 392:1155–1165. CrossRefPubMedGoogle Scholar
  20. Lazo JS, Pitt BR (1995) Metallothioneins and cell death by anticancer drugs. Annu Rev Pharmacol Toxicol 35:635–653. CrossRefPubMedGoogle Scholar
  21. Lee DK, Carrasco J, Hidalgo J, Andrews GK (1999) Identification of a signal transducer and activator of transcription (STAT) binding site in the mouse metallothionein-I promoter involved in interleukin-6-induced gene expression. Biochem J 337:59–65. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Leung YK, Pankhurst M, Dunlop SA, Ray S, Dittmann J, Eaton ED, Palumaa P, Sillard R, Chuah MI, West AK, Chung RS (2010) Metallothionein induces a regenerative reactive astrocyte phenotype via JAK/STAT and RhoA signalling pathways. Exp Neurol 221:98–106. CrossRefPubMedGoogle Scholar
  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔC T method. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  24. Masiulis I, Yun S, Eisch AJ (2011) The interesting interplay between interneurons and adult hippocampal neurogenesis. Mol Neurobiol 44:287–302. CrossRefPubMedPubMedCentralGoogle Scholar
  25. McDonald HY, Wojtowicz JM (2005) Dynamics of neurogenesis in the dentate gyrus of adult rats. Neurosci Lett 385:70–75. CrossRefPubMedGoogle Scholar
  26. Montaron MF, Koehl M, Lemaire V, Drapeau E, Abrous DN, Le Moal M (2004) Environmentally induced long-term structural changes: cues for functional orientation and vulnerabilities. Neurotox Res 6:571–580. CrossRefPubMedGoogle Scholar
  27. O’Callaghan JP, Sriram K, Miller DB (2008) Defining “neuroinflammation”. Ann N Y Acad Sci 1139:318–330CrossRefPubMedGoogle Scholar
  28. Pedersen MØ, Jensen R, Pedersen DS, Skjolding AD, Hempel C, Maretty L, Penkowa M (2009) Metallothionein-I+II in neuroprotection. Biofactors 35:315–325. CrossRefPubMedGoogle Scholar
  29. Sato M, Bremner I (1993) Oxygen free radicals and metallothionein. Free Radic Biol Med 14:325–337. CrossRefPubMedGoogle Scholar
  30. Sehata S, Kiyosawa N, Makino T, Atsumi F, Ito K, Yamoto T, Teranishi M, Baba Y, Uetsuka K, Nakayama H, Doi K (2004) Morphological and microarray analysis of T-2 toxin-induced rat fetal brain lesion. Food Chem Toxicol 42:1727–1736. CrossRefPubMedGoogle Scholar
  31. Sibbe M, Kulik A (2017) GABAergic regulation of adult hippocampal neurogenesis. Mol Neurobiol 54:5497–5510. CrossRefPubMedGoogle Scholar
  32. Takano H, Satoh M, Shimada A, Sagai M, Yoshikawa T, Tohyama C (2000) Cytoprotection by metallothionein against gastroduodenal mucosal injury caused by ethanol in mice. Lab Investig 80:371–377. CrossRefPubMedGoogle Scholar
  33. Takano H, Inoue K, Yanagisawa R, Sato M, Shimada A, Morita T, Sawada M, Nakamura K, Sanbongi C, Yoshikawa T (2004) Protective role of metallothionein in acute lung injury induced by bacterial endotoxin. Thorax 59:1057–1062. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Tanaka T, Abe H, Kimura M, Onda N, Mizukami S, Yoshida T, Shibutani M (2016) Developmental exposure to T-2 toxin reversibly affects postnatal hippocampal neurogenesis and reduces neural stem cells and progenitor cells in mice. Arch Toxicol 90:2009–2024. CrossRefPubMedGoogle Scholar
  35. Teoh SL, Ogawa S, Parhar IS (2015) Localization of genes encoding metallothionein-like protein (mt2 and smtb) in the brain of zebrafish. J Chem Neuroanat 70:20–32. CrossRefPubMedGoogle Scholar
  36. Varró P, Béldi M, Kovács M, Világi I (2018) T-2 mycotoxin treatment of newborn rat pups does not significantly affect nervous system functions in adulthood. Acta Biol Hung 69(1):29–41CrossRefPubMedGoogle Scholar
  37. Walton NM, Shin R, Tajinda K, Heusner CL, Kogan JH, Miyake S, Chen Q, Tamura K, Matsumoto M (2012) Adult neurogenesis transiently generates oxidative stress. PLoS One 7:e35264. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Walz W, Lang MK (1998) Immunocytochemical evidence for a distinct GFAP-negative subpopulation of astrocytes in the adult rat hippocampus. Neurosci Lett 257:127–130. CrossRefPubMedGoogle Scholar
  39. Wang J, Fitzpatrick DW, Wilson JR (1993) Effect of dietary T-2 toxin on biogenic monoamines in discrete areas of the rat brain. Food Chem Toxicol 31:191–197. CrossRefPubMedGoogle Scholar
  40. Weidner M, Lenczyk M, Schwerdt G, Gekle M, Humpf HU (2013) Neurotoxic potential and cellular uptake of T-2 toxin in human astrocytes in primary culture. Chem Res Toxicol 26:347–355. CrossRefPubMedGoogle Scholar
  41. Wu QH, Wang X, Yang W, Nüssler AK, Xiong LY, Kuča K, Dohnal V, Zhang XJ, Yuan ZH (2014) Oxidative stress-mediated cytotoxicity and metabolism of T-2 toxin and deoxynivalenol in animals and humans: an update. Arch Toxicol 88:1309–1326. CrossRefPubMedGoogle Scholar
  42. Yang L, Yu Z, Hou J, Deng Y, Zhou Z, Zhao Z, Cui J (2016) Toxicity and oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler hepatocytes. Food Chem Toxicol 87:128–137. CrossRefPubMedGoogle Scholar
  43. Yang JY, Zhang YF, Li YX, Meng XP, Bao JF (2018) L-arginine protects against oxidative damage induced by T-2 toxin in mouse Leydig cells. J Biochem Mol Toxicol 32:e22209. CrossRefPubMedGoogle Scholar
  44. Zhang YF, Yang JY, Li YK, Zhou W (2017) Toxicity and oxidative stress induced by T-2 toxin in cultured mouse Leydig cells. Toxicol Mech Methods 27:100–106CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Veterinary Pathology, Division of Animal Life Science, Institute of AgricultureTokyo University of Agriculture and TechnologyFuchu-shiJapan
  2. 2.Pathogenetic Veterinary Science, United Graduate School of Veterinary SciencesGifu UniversityGifu-shiJapan
  3. 3.Cooperative Division of Veterinary Sciences, Graduate School of AgricultureTokyo University of Agriculture and TechnologyFuchu-shiJapan
  4. 4.Laboratory of Histopathology, Department of Clinical Laboratory ScienceSemyung UniversityJecheon-siRepublic of Korea
  5. 5.Institute of Global Innovation ResearchTokyo University of Agriculture and TechnologyFuchu-shiJapan

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