Late Effect of Developmental Exposure to 3,3 -Iminodipropionitrile on Neurogenesis In

Late effect of developmental exposure to 3,3′-iminodipropionitrile on neurogenesis in the hippocampal dentate gyrus of mice

Neurotoxicity Research

Yasuko Hasegawa-Baba 1 ･ Takeshi Tanaka 1,2 ･ Yousuke Watanabe 1,2 ･ Liyun Wang 1 ･ Megu Itahashi 1 ･ Toshinori Yoshida 1 ･ Makoto Shibutani 1,*

1 Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan

2 Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan

*Corresponding to: Prof. Makoto Shibutani, D.V.M., Ph.D.

Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan.

Telephone/Fax: +81-42-367-5771

E-mail address:

Supplementary Figure 1

Overview of the hippocampal formation in a male mouse on postnatal day (PND) 21 stained with hematoxylin and eosin. (Inset) Higher magnification of the granule cell layer (GCL) and subgranular zone (SGZ). The distribution of immunoreactive cells for neuron-specific nuclear protein (NeuN), activity-regulated cytoskeleton-associated protein (ARC), FBJ osteosarcoma oncogene (FOS), glial fibrillary acidic protein (GFAP), brain lipid-binding protein (BLBP), T-box brain 2 (TBR2), doublecortin (DCX), calbindin-D-29K (CALB2), proliferating cells and apoptotic cells were measured in the GCL or SGZ. The numbers of cells in the hilus of the dentate gyrus (as enclosed by the dotted line) displaying immunoreactivity for reelin (RELN), parvalbumin (PVALB), calbindin-D-28K (CALB1), CALB2 and NeuN were counted. Cornu ammonis (CA) 3 neurons were excluded. The immunoreactivity of phosphorylated neurofilament, heavy polypeptide (phospho-NF-H) was evaluated in the stratum radiatum and molecular layer of the dentate gyrus. Magnification ×100 (Inset: ×400); bar = 200 µm (Inset: 50 µm).

Supplementary Figure 2

Body weight of dams exposed to 3,3'-iminodipropionitrile (IDPN) from gestation day (GD) 6 to postnatal day (PND) 21 on weaning of offspring. Mean values are plotted. N = 8–12/group (0 ppm controls, 11; 600 ppm IDPN, 12; 1200 ppm IDPN, 8). * P < 0.05, ** P < 0.01, significantly different from 0 ppm controls by Dunnett’s test or Steel’s test.

Supplementary Figure 3

Distribution and number of immunoreactive cells for markers of interneurons, excitatory mossy cells and mature neurons in the hilus of the hippocampal dentate gyrus in male offspring at postnatal day (PND) 21 and PND 77. (a) reelin (RELN). (b) parvalbumin (PVALB). (c) calbindin-D-28K (CALB1). (d) calbindin-D-29K (CALB2). (e) NeuN. Graphs show the number of immunoreactive cells/unit area (mm2) of the hilus of bilateral hemispheres. Values are expressed as the mean + SD. N = 12/group.

Supplementary Figure 4

Immunoreactivity of phosphorylated neurofilament, heavy polypeptide (phospho-NF-H) in the molecular layer of the dentate gyrus and stratum radiatum of male offspring at postnatal day (PND) 21 and 77. Representative images from the 0 ppm controls (left) and 1200 ppm 3,3'-iminodipropionitrile (IDPN) group (right) at PND 21 (a) and PND 77 (b) are shown. Magnification ×40; bar = 500 µm. No changes were observed in any portions between the 0 ppm controls and any of the IDPN groups on PND 21 and PND 77 as examined in male offspring.

Supplementary Table 1

Antibodies used in this study

Antigen / Abbreviated name / Host species / Clonality / Clone name / Dilution / Antigen retrieval / Manufacturer (City, State, Country)
Activity-regulated cytoskeleton-associated protein / ARC / Rabbit / Polyclonal IgG / n.a. / 1:2000 / Microwaving a / Synaptic System (Göttingen, Germany)
Brain lipid-binding protein / BLBP / Rabbit / Polyclonal IgG / n.a. / 1:300 / None / Abcam Inc. (Cambridge, UK)
Calbindin-D-28K / CALB1 / Mouse / Monoclonal
IgG1 / CB-955 / 1:500 / Microwaving a / Sigma-Aldrich Chemical Co. (St. Louis, MO, USA)
Calbindin-D-29K
(Calretinin) / CALB2 / Mouse / Monoclonal
IgG1 / CRT01/O.N.40 / 1:100 / Microwaving a / LifeSpan Biosciences, Inc. (Seattle, WA, USA)
Doublecortin / DCX / Rabbit / Polyclonal IgG / n.a. / 1:1000 / None / Abcam Inc.
FBL osteosarcoma oncogene / FOS / Rabbit / Polyclonal IgG / n.a. / 1:500 / None / Santa Cruz Biotechnology, Inc. (Dallas, TX, USA)
Glial fibrillary acidic protein / GFAP / Mouse / Monoclonal
IgG1 / GA5 / 1:200 / None / EMD Millipore (Billerica, MA, USA)
Neuron-specific nuclear protein / NeuN / Mouse / Monoclonal
IgG1 / A60 / 1:100 / None / EMD Millipore
Parvalbumin / PVALB / Mouse / Monoclonal
IgG1 / PARV-19 / 1:1000 / Microwaving a / EMD Millipore
Phosphorylated neurofilament, heavy polypeptide / phospho- NF-H / Mouse / Monoclonal
IgG1 / RT97 / 1:100 / Microwaving a / EMD Millipore
Proliferating cell nuclear antigen / PCNA / Mouse / Monoclonal IgG2a / PC10 / 1:200 / None / Dako (Glostrup, Denmark)
Reelin / RELN / Mouse / Monoclonal
IgG1 / G10 / 1:1000 / None / Novus Biologicals, Inc. (Littleton, CO, USA)
T-box brain 2 / TBR2 / Rabbit / Polyclonal IgG / n.a. / 1:500 / Autoclaving b / Abcam Inc.

Abbreviation: n.a., not applicable.

a 90°C for 10 min in 10 mM citrate buffer (pH 6.0).

b 121°C for 10 min in 10 mM citrate buffer (pH 6.0).

Supplementary Table 2

Primer sequence for real-time RT-PCR analysis

Gene / Accession No. / Forward primer (5' → 3') / Reverse primer (5' → 3')
Neuronal stage-defining markers of neuronal cell lineages
Gfap / NM_010277.3 / GAAACCAGCCTGGACACCAA / CTTTACCACGATGTTCCTCTTGAG
Pax6 / NM_013627 / TCCCCAGTCAGACCTCCTCATA / TCATAACTCCGCCCATTCACT
Eomes / NM_010136 / CTCCCCCCTCCATCAAGTCT / TCTCTTGCAAGCGCTGTTGTA
Dcx / NM_001110222 / CTCAGGGAGTGCGCTACAATTTA / GCAGTTCATCCATGCTTCCAAT
Neurotrophic factor
Bdnf / NM_007540.4 / CAAGACACATTACCTTCCTGCATCT / ACCGAAGTATGAAATAACCATAGTAAG
Stem cell regulators
Kitl / NM_013598 / GCAGTAATAGGAAAGCCGCA AA / CAATGCCATGGCTGTCCAT
Kit / NM_001122733 / CTTCAGGACCCAAGACGTAACAG / GTGGCGGCTTTGAGTAAATTCTAG
Glutamate receptors
Gria1 / NM_008165 / TGCTTCATCACTCCAAGTTTTCC / GGCGCAGCTGAAGGACAA
Gria2 / NM_013540 / AGCCAACAGTTTCGCAGTCA / TCGCATAGACGCCTCTTGAA
Grin2a / NM_008170 / TCAAACCTGCCCACCTACTCA / CGCAGACAGGCATCACACTT
Grin2d / NM_008172 / AGAGCAACAGCTGCAGGTCAT / CGCGTAGTCACTGCCACAA
Housekeeping genes for normalization
Gapdh / NM_008084 / TGTCAAGCTCATTTCCTGGTATGA / TCTTACTCCTTGGAGGCCATGTA
Hprt / NM_013556 / TTGTATACCTAATCATTATGCCGAGG / CAGAGGGCCACAATGTGATG

Abbreviations: Bdnf, brain-derived neurotrophic factor; Dcx, doublecortin; Eomes (Tbr2), eomesodermin homolog; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Gfap, grial fibrillary acidic protein; Gria1, glutamate receptor, ionotropic, AMPA1 (alpha 1); Gria2, glutamate receptor, ionotropic, AMPA2 (alpha 2); Grin2a, glutamate receptor, ionotropic, NMDA2A (epsilon 1); Grin2d, glutamate receptor, ionotropic, NMDA2D (epsilon 4); Hprt, hypoxanthine guanine phosphoribosyl transferase; Kit, kit oncogene; Kitl, kit ligand; RT-PCR, reverse-transcription polymerase chain reaction.