Megu Itahashi

Aberration in Epigenetic Gene Regulation in Hippocampal Neurogenesis by Developmental Exposure to Manganese Chloride in Mice

We have shown that maternal manganese (Mn) exposure caused sustained disruption of hippocampal neurogenesis of mouse offspring. To clarify the effects of maternal Mn exposure on epigenetic gene regulation contributing to the sustained disruption of hippocampal neurogenesis, we treated pregnant ICR mice with MnCl₂ in diet from gestational day 10 through day 21 after delivery on weaning and searched epigenetically downregulated genes by global promoter methylation analysis in the hippocampal dentate gyrus of male offspring on postnatal day (PND) 21 and PND 77. By CpG promoter microarray analysis on PND 21 following 800-ppm Mn exposure, sustained promoter hypermethylation and transcript downregulation through PND 77 were confirmed with Mid1, Atp1a3, and Nr2f1, whereas Pvalb showed a transient hypermethylation only on weaning. The numbers of Pvalb⁺ and ATP1a3⁺ neurons suggestive of γ-aminobutyric acid (GABA)ergic interneurons, Mid1⁺ cells suggestive of late-stage granule cell lineage and GABAergic interneurons, and COUP-TF1⁺ cells suggestive of early-stage granule cell lineage were all reduced on PND 21, and reductions were sustained on PND 77 except for no change in Pvalb⁺ cells. Mid1⁺ cells showed asymmetric distribution with right-side predominance, and Mn exposure abolished it by promoter hypermethylation of the right side. These findings indicate epigenetic mechanisms as mediators, through which Mn exposure modulates neurogenesis involving both granule cell lineage and GABAergic interneurons with long-lasting and stable repercussions. Disruption of asymmetric cellular distribution of Mid1 suggests that higher brain functions specialized in the left or right side of the brain were affected.

Glycidol Induces Axonopathy by Adult-Stage Exposure and Aberration of Hippocampal Neurogenesis Affecting Late-Stage Differentiation by Developmental Exposure in Rats

To investigate the neurotoxicity profile of glycidol and its effect on developmental hippocampal neurogenesis, pregnant Sprague Dawley rats were given drinking water containing 0, 100, 300, or 1000 ppm glycidol from gestational day 6 until weaning on day 21 after delivery. At 1000 ppm, dams showed progressively worsening gait abnormalities, and histopathological examination showed generation of neurofilament-L(+) spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells, and axonal degeneration in the sciatic nerves. Decreased dihydropyrimidinase-like 3(+) immature granule cells in the subgranular zone (SGZ) and increased immature reelin(+) or calbindin-2(+) γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein (NeuN)(+) mature neurons were found in the dentate hilus of the offspring of the 1000 ppm group on weaning. Hilar changes remained until postnatal day 77, with the increases in reelin(+) and NeuN(+) cells being present at ≥ 300 ppm, although the SGZ change disappeared. Thus, glycidol caused axon injury in the central and peripheral nervous systems of adult rats, suggesting that glycidol targets the newly generating nerve terminals of immature granule cells, resulting in the suppression of late-stage hippocampal neurogenesis. The sustained hilar changes may be a sign of continued aberrations in neurogenesis and migration. The no-observed-adverse-effect level was determined to be 300 ppm (48.8mg/kg body weight/day) for dams and 100 ppm (18.5mg/kg body weight/day) for offspring. The sustained developmental exposure effect on offspring neurogenesis was more sensitive than the adult axonal injury.

Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells of the outer stripe of the outer medulla (OSOM). This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA in the OSOM. Rats were treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or α-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1, Hmox1, Aldh1a1, Por, Prdx1 and Txn1. OTA also did not change the levels of thiobarbituric acid-reactive substances, glutathione disulfide/reduced glutathione, and the immunoreactive tubular cell distribution of nuclear factor erythroid 2-related factor 2 in the OSOM. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without relation to induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.

Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells of the hippocampal neurogenesis in rat offspring via dysfunction of cholinergic inputs by myelin vacuolation.

Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2(+) progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling(+) apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction of cholinergic inputs into granule cell lineages and/or GABAergic interneurons as indicated by decreased transcript levels of Chrnb2 and numbers of Chrnb2(+) interneurons caused by myelin vacuolation in the septal-hippocampal pathway.

Expression alterations of genes on both neuronal and glial development in rats after developmental exposure to 6-propyl-2-thiouracil

The present study was performed to determine target gene profiles associated with pathological mechanisms of developmental neurotoxicity. For this purpose, we selected a rat developmental hypothyroidism model because thyroid hormones play an essential role in both neuronal and glial development. Region-specific global gene expression analysis was performed at postnatal day (PND) 21 on four brain regions representing different structures and functions, i.e., the cerebral cortex, corpus callosum, dentate gyrus and cerebellar vermis of rats exposed to 6-propyl-2-thiouracil in the drinking water at 3 and 10ppm from gestational day 6 to PND 21. Expression changes of gene clusters of neuron differentiation and development, cell migration, synaptic function, and axonogenesis were detected in all four regions. Characteristically, gene expression profiles suggestive of affection of ephrin signaling and glutamate transmission were obtained in multiple brain regions. Gene clusters suggestive of suppression of myelination and glial development were specifically detected in the corpus callosum and cerebral cortex. Immunohistochemically, immature astrocytes immunoreactive for vimentin and glial fibrillary acidic protein were increased, and oligodendrocytes immunoreactive for oligodendrocyte lineage transcription factor 2 were decreased in the corpus callosum. Immunoreactive intensity of myelin basic protein was also decreased in the corpus callosum and cerebral cortex. The hippocampal dentate gyrus showed downregulation of Ptgs2, which is related to synaptic activity and neurogenesis, as well as a decrease of cyclooxygenase-2-immunoreactive granule cells, suggesting an impaired synaptic function related to neurogenesis. These results suggest that multifocal brain region-specific microarray analysis can determine the affection of neuronal or glial development.

Onset of hepatocarcinogen-specific cell proliferation and cell cycle aberration during the early stage of repeated hepatocarcinogen administration in rats

We have previously reported that a 28‐day treatment of carcinogens evoking target cell proliferation activates G1/S checkpoint function and apoptosis, as well as induction of aberrant ubiquitin D (Ubd) expression, suggesting disruptive spindle checkpoint function, in rats. The present study aimed to determine the onset time of rat liver cells to undergo carcinogen‐specific cell cycle aberration and proliferation. Animals were treated orally with a hepatocarcinogenic dose of methyleugenol or thioacetamide for 3, 7 or 28 days. For comparison, some animals were subjected to partial hepatectomy or treated with noncarcinogenic hepatotoxicants (acetaminophen, α‐naphthyl isothiocyanate or promethazine). Carcinogen‐specific liver cell kinetics appeared at day 28 as evident by increases of cell proliferation, p21Cip1+ cells, phosphorylated‐Mdm2+ cells and cleaved caspase 3+ cells, and upregulation of DNA damage‐related genes. Hepatocarcinogens also downregulated Rbl2 and upregulated Cdkn1a and Mdm2, and decreased Ubd+ cells co‐expressing phosphorylated‐histone H3 (p‐Histone H3) and p‐Histone H3+ cell ratio within the Ki‐67+ proliferating population. These results suggest that it takes 28 days to induce hepatocarcinogen‐specific early withdrawal of proliferating cells from M phase due to disruptive spindle checkpoint function as evidenced by reduction of Ubd+ cells staying at M phase. Disruption of G1/S checkpoint function reflected by downregulation of Rbl2 as well as upregulation of Mdm2 suggestive of sequestration of retinoblastoma protein is responsible for the facilitation of carcinogen‐induced cell proliferation at day 28. Accumulation of DNA damage probably in association with facilitation of p53 degradation by activation of Mdm2 may be a prerequisite for aberrant p21Cip1 activation, which is responsible for apoptosis. Copyright

Glycidol induces axonopathy and aberrations of hippocampal neurogenesis affecting late-stage differentiation by exposure to rats in a framework of 28-day toxicity study

Developmental exposure to glycidol induces aberrations of late-stage neurogenesis in the hippocampal dentate gyrus of rat offspring, whereas maternal animals develop axonopathy. To investigate the possibility whether similar effects on adult neurogenesis could be induced by exposure in a framework of 28-day toxicity study, glycidol was orally administered to 5-week-old male Sprague-Dawley rats by gavage at 0, 30 or 200 mg/kg for 28 days. At 200 mg/kg, animals revealed progressively worsening gait abnormalities as well as histopathological and immunohistochemical changes suggestive of axonal injury as evidenced by generation of neurofilament-L(+) spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells and axonal degeneration in the sciatic nerves. At the same dose, animals revealed aberrations in neurogenesis at late-stage differentiation as evidenced by decreases of both doublecortin(+) and dihydropyrimidinase-like 3(+) cells in the subgranular zone (SGZ) and increased reelin(+) or calbindin-2(+) γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein(+) mature neurons in the dentate hilus. These effects were essentially similar to that observed in offspring after maternal exposure to glycidol. These results suggest that glycidol causes aberrations in adult neurogenesis in the SGZ at the late stage involving the process of neurite extension similar to the developmental exposure study in a standard 28-day toxicity study.

Downregulation of immediate-early genes linking to suppression of neuronal plasticity in rats after 28-day exposure to glycidol.

We previously found that the 28-day oral toxicity study of glycidol at 200mg/kg/day in rats resulted in axonopathy in both the central and peripheral nervous systems and aberrations in the late-stage of hippocampal neurogenesis targeting the process of neurite extension. To capture the neuronal parameters in response to glycidol toxicity, these animals were subjected to region-specific global gene expression profiling in four regions of cerebral and cerebellar architectures, followed by immunohistochemical analysis of selected gene products. Expression changes of genes related to axonogenesis and synaptic transmission were observed in the hippocampal dentate gyrus, cingulate cortex and cerebellar vermis at 200mg/kg showing downregulation in most genes. In the corpus callosum, genes related to growth, survival and functions of glial cells fluctuated their expression. Immunohistochemically, neurons expressing gene products of immediate-early genes, i.e., Arc, Fos and Jun, decreased in their number in the dentate granule cell layer, cingulate cortex and cerebellar vermis. We also applied immunohistochemical analysis in rat offspring after developmental exposure to glycidol through maternal drinking water. The results revealed increases of Arc(+) neurons at 1000ppm and Fos(+) neurons at ≥300ppm in the dentate granule cell layer of offspring only at the adult stage. These results suggest that glycidol suppressed neuronal plasticity in the brain after 28-day exposure to young adult animals, in contrast to the operation of restoration mechanism to increase neuronal plasticity at the adult stage in response to aberrations in neurogenesis after developmental exposure.

N-methyl-N-nitrosourea during late gestation results in concomitant but reversible progenitor cell reduction and delayed neurogenesis in the hippocampus of rats.

N-Methyl-N-nitrosourea (MNU) is an alkylating agent having genotoxic potential to cause gene mutations and antiproliferative cytotoxic activity on developing brains to cause microcephaly by mid-gestational exposure in rodents. This study investigated the transient genotoxic and cytocidal effect of MNU at the beginning of the subgranular zone (SGZ) formation in the hippocampal dentate gyrus on neurogenesis in later life using rats. Pregnant rats were injected with MNU at 0 (vehicle controls), 1 or 3mg/kg body weight intraperitoneally from gestational day (GD) 18 to GD 20 once a day. In offspring, effects were observed at 3mg/kg. Fetal brains on GD 21 after the last MNU injection increased TUNEL(+) apoptotic cells in the tertiary germinal matrices. At postnatal day (PND) 21 on weaning, offspring displayed decrease of doublecortin (Dcx)(+) cells and cell proliferation in the SGZ and increase of calbindin (Calb1)(+) interneurons in the dentate hilus. Postnatal single bromodeoxyuridine (BrdU) injection on PND 3 resulted in an increase of BrdU(+)/Dcx(+) cells. On PND 77, Dcx(+) cells recovered in number, but cell proliferation increased in the SGZ. Thus, late-gestational maternal MNU exposure may induce reversible reductions of type-3 progenitor and/or immature granule cells and cell proliferation on weaning in response to progenitor cell apoptosis, as well as delayed neurogenesis due to cell cycle arrest. Increases of Calb1(+) interneurons on weaning and SGZ cell proliferation later on may reflect compensatory mechanism for MNU-induced aberrant neurogenesis. Considering the lack of effects on PND 77, MNU may mainly target transient populations of highly proliferative progenitor cells without affecting their stem cells to undergo progenitor production. Protective and plasticity mechanism may be operated against genotoxic agents on hippocampal neurogenesis.

Reversible effect of maternal exposure to chlorpyrifos on the intermediate granule cell progenitors in the hippocampal dentate gyrus of rat offspring.

To examine the effects of developmental exposure to chlorpyrifos (CPF) on neurogenesis in the hippocampal dentate gyrus, pregnant rats were treated with 2.8, 14 or 70 ppm CPF in the diet from gestational day 10 to day 21 after delivery. Dams had decreased cholinesterase (ChE) activities in red blood cells (RBC) at intakes of ≥2.8 ppm and in brain at 70 ppm. Offspring on postnatal day (PND) 21 had decreased ChE activities in the RBC and brain at 70 ppm. There were no behavioral abnormalities in the offspring. Immunohistochemical analysis showed decreases in the numbers of cells positive for proliferating cell nuclear antigen and T box brain 2 in the subgranular zone (SGZ) of the dentate gyrus on PND 21 at 70 ppm, while other progenitor cell populations and the apoptotic cell number were unaffected in this zone. However, on PND 77 all changes had disappeared. The distribution of the progenitor cell population expressing nicotinic acetylcholine receptor α7 and lacking expression of postmitotic neuron-specific nuclear protein was unchanged by CPF-exposure, suggesting no effect of cholinergic stimulation on neurogenesis. These results suggest that developmental exposure to CPF directly but transiently affect the proliferation of type-2 progenitor cell populations in the hippocampal neurogenesis. The lowest-observed-adverse-effect level (LOAEL) of CPF was determined to be 2.8 ppm (0.36 mg/kg body weight/day) for dams by the inhibition of ChE activity in the RBC at this dose. As for offspring, no-observed-adverse-effect level (NOAEL) was determined to be 14 ppm (1.86 mg/kg body weight/day) by the decrease of type-2 progenitor cell proliferation in the SGZ and the inhibition of ChE activity in the RBC and brain at 70 ppm. The NOAEL of dams based on the offsprings effects was approximately 2800 times higher than the estimated consumption of CPF through food in the general population and in pregnant women as examined in Japan.