Hajime Abe

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.

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.

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.

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.

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats.

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.

Developmental exposure to cuprizone reduces intermediate-stage progenitor cells and cholinergic signals in the hippocampal neurogenesis in rat offspring.

The exposure to cuprizone (CPZ) leads to demyelination in the central nervous system in rodents. To examine the developmental effects of CPZ exposure on hippocampal neurogenesis, pregnant rats were treated with 0, 0.1 or 0.4% CPZ in the diet from gestational day 6 to day 21 after delivery. On postnatal day 21, male offspring had a decreased density of new glue2(+) oligodendrocyte progenitor cells in the dentate hilus and in the area of the cerebellar medulla in the presence of 0.4% CPZ. With regard to neurogenesis-related parameters, offspring had decreased T box brain 2(+) progenitor cells and increased apoptotic cells, as detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling, which was accompanied by the up-regulation of Casp12 and Bcl2l11 in the subgranular zone, and increased reelin(+) interneurons in the dentate hilus. In addition, the density of phosphorylated TrkB(+) interneurons decreased in the dentate hilus, which was accompanied by transcript down-regulation of Bdnf and Chrna7 in the dentate gyrus. Moreover, granule cells expressing gene products of immediate-early genes, i.e., Arc and Fos, decreased. These results suggest that maternal exposure to 0.4% CPZ decreases proliferative type-2 progenitor cells via endoplasmic reticulum stress-mediated apoptosis and inhibition of cholinergic signals to intermediate-stage progenitor cells following reduced oligodendrocyte production and suppression of the brain-derived neurotrophic factor signaling cascade. Increases in reelin-expressing interneurons may compensate for impaired granule cell migration and/or correct positioning due to decreased immediate-early gene-mediated neuronal plasticity. However, all observed fluctuations disappeared at the adult stage, suggesting that CPZ-induced developmental neurotoxicity was reversible.

Cuprizone decreases intermediate and late-stage progenitor cells in hippocampal neurogenesis of rats in a framework of 28-day oral dose toxicity study

Developmental exposure to cuprizone (CPZ), a demyelinating agent, impairs intermediate-stage neurogenesis in the hippocampal dentate gyrus of rat offspring. To investigate the possibility of alterations in adult neurogenesis following postpubertal exposure to CPZ in a framework of general toxicity studies, CPZ was orally administered to 5-week-old male rats at 0, 120, or 600mg/kg body weight/day for 28days. In the subgranular zone (SGZ), 600mg/kg CPZ increased the number of cleaved caspase-3(+) apoptotic cells. At ≥120mg/kg, the number of SGZ cells immunoreactive for TBR2, doublecortin, or PCNA was decreased, while that for SOX2 was increased. In the granule cell layer, CPZ at ≥120mg/kg decreased the number of postmitotic granule cells immunoreactive for NEUN, CHRNA7, ARC or FOS. In the dentate hilus, CPZ at ≥120mg/kg decreased phosphorylated TRKB(+) interneurons, although the number of reelin(+) interneurons was unchanged. At 600mg/kg, mRNA levels of Bdnf and Chrna7 were decreased, while those of Casp4, Casp12 and Trib3 were increased in the dentate gyrus. These data suggest that CPZ in a scheme of 28-day toxicity study causes endoplasmic reticulum stress-mediated apoptosis of granule cell lineages, resulting in aberrations of intermediate neurogenesis and late-stage neurogenesis and following suppression of immediate early gene-mediated neuronal plasticity. Suppression of BDNF signals to interneurons caused by decreased cholinergic signaling may play a role in these effects of CPZ. The effects of postpubertal CPZ on neurogenesis were similar to those observed with developmental exposure, except for the lack of reelin response, which may contribute to a greater decrease in SGZ cells.

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

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

Promoter-region hypermethylation and expression downregulation of Yy1 (Yin yang 1) in preneoplastic liver lesions in a thioacetamide rat hepatocarcinogenesis model.

Thioacetamide (TAA) has been used to develop a rodent model for hepatocarcinogenesis. To determine the genes with epigenetic modifications in early hepatocarcinogenesis, we did a genome-wide scan for hypermethylated promoter regions using CpG island microarrays in TAA-promoted rat liver tissue. Eight genes were selected based on the microarray profile; of these, Yy1 and Wdr45b were confirmed to be hypermethylated by methylation-specific polymerase chain reaction (PCR) and pyrosequencing and downregulated by real-time reverse transcription PCR. Non-neoplastic liver cells had nuclear Yy1 immunoreactivity, while preneoplastic foci with glutathione S-transferase placental form (GST-P) immunoreactivity had decreased Yy1 immunoreactivity. The incidence of these foci was proportional to the dose of TAA administered. Co-expression analysis of gene products downstream of Yy1 revealed increased nuclear phospho-c-Myc(+) foci as well as nuclear and cytoplasmic p21(Cip1+) foci in Yy1(-) or GST-P(+) foci in response to TAA-promotion dose. Although the absolute number of cells was low, the incidence of death receptor 5(-) foci was increased in Yy1(-) foci in proportion to the TAA dose. Yy1(-)/GST-P(+) foci revealed a higher number of proliferating cell nuclear antigen (PCNA)-immunoreactive cells than Yy1(+)/GST-P(+) foci, while cleaved caspase-3(+) cells were unchanged between Yy1(-)/GST-P(+) and Yy1(+)/GST-P(+) foci. In the case of Wdr45b, most GST-P(+) foci were Wdr45b(-) and were not increased by TAA promotion. These results suggest involvement of Yy1 in the epigenetic gene regulation at the early stages of TAA promoted cell proliferation and concomitant cell cycle arrest in preneoplastic lesions.