DNA methylation-altered genes in the rat hippocampal neurogenic niche after continuous exposure to amorphous curcumin.

Rat offspring who are exposed to an amorphous formula of curcumin (CUR) from the embryonic stage have anti-anxiety-like behaviors, enhanced fear extinction learning, and increased synaptic plasticity in the hippocampal dentate gyrus (DG). In the present study, we investigated the links between genes with altered methylation status in the neurogenic niche and enhanced neural functions after CUR exposure. We conducted methylation and RNA sequencing analyses of the DG of CUR-exposed rat offspring on day 77 after delivery. Methylation status and transcript levels of candidate genes were validated using methylation-sensitive high-resolution melting and real-time reverse-transcription PCR, respectively. In the CUR group, we confirmed the hypermethylation and downregulation of Gpr150, Mmp23, Rprml, and Pcdh8 as well as the hypomethylation and upregulation of Ppm1j, Fam222a, and Opn3. Immunohistochemically, reprimo-like+ hilar cells and protocadherin-8+ granule cells were decreased and opsin-3+ hilar cells were increased by CUR exposure. Both reprimo-like and opsin-3 were partially expressed on subpopulations of glutamic acid decarboxylase 67+ γ-aminobutyric acid-ergic interneurons. Furthermore, the transcript levels of genes involved in protocadherin-8-mediated N-cadherin endocytosis were altered with CUR exposure; this was accompanied by Ctnnb1 and Syp upregulation and Mapk14, Map2k3, and Grip1 downregulation, suggesting that CUR-induced enhanced synaptic plasticity is associated with cell adhesion. Together, our results indicate that functionally different genes have altered methylation and expression in different neuronal populations of the hippocampal neurogenic niche, thus enhancing synaptic plasticity after CUR exposure.

In vitro and in vivo induction of ochratoxin A exposure-related micronucleus formation in rat proximal tubular epithelial cells and expression profiling of chromosomal instability-related genes.

Ochratoxin A (OTA) is a renal carcinogen in rats, and repeated administration induces karyomegaly in proximal tubular epithelial cells (PTECs) of the outer stripe of the outer medulla (OSOM) before inducing proliferative lesions. To investigate whether OTA induces micronuclei (MN) in PTECs, we performed an in vitro MN assay using rat renal NRK-52E PTECs after treatment for ≤21 days, and an in vivo OSOM MN assay in rats treated with OTA, other renal carcinogens, or non-carcinogenic renal toxicants for 4 or 13 weeks. The in vitro assay revealed an increased frequency of micronucleated cells from the acceptable dose level for cell viability, even after 21 days of treatment. The in vivo assay also revealed a dose- and treatment period-dependent increase in PTECs with γ-H2AX+ MN. OTA-specific gene expression profiling by OSOM RNA sequencing after week 13 revealed the altered expression of genes related to microtubule-kinetochore binding, the kinesin superfamily, centriole assembly, DNA damage repair, and cell cycle regulation. MN formation was also observed with other renal carcinogens that induce karyomegaly similarly to OTA. These results imply that γ-H2AX+ MN formation by OTA treatment is related to the induction of chromosomal instability accompanying karyomegaly formation before proliferative lesions form, providing a new insight into the carcinogenic mechanism that may be relevant to humans.

Adipose Stem Cell-Seeded Decellularized Porcine Pericardium: A Promising Functional Biomaterial to Synergistically Restore the Cardiac Functions Post-Myocardial Infarction.

Myocardial infarction (MI) is a serious cardiovascular disease as the leading cause of death globally. Hence, reconstruction of the cardiac tissue comes at the forefront of strategies adopted to restore heart functions following MI. In this investigation, we studied the capacity of rat adipose-derived mesenchymal stem cells (r-AdMSCs) and decellularized porcine pericardium (DPP) to restore heart functions in MI animals. MI was induced in four different groups, three of which were treated either using DPP (MI-DPP group), stem cells (MI-SC group), or both (MI-SC/DPP group). Cardiac functions of these groups and the Sham group were evaluated using echocardiography, the intraventricular pressure gradient (IVPG) on weeks 2 and 4, and intraventricular hemodynamics on week 4. On day 31, the animals were euthanized for histological analysis. Echocardiographic, IVPG and hemodynamic findings indicated that the three treatment strategies shared effectively in the regeneration process. However, the MI-SC/DPP group had a unique synergistic ability to restore heart functions superior to the other treatment protocols. Histology showed that the MI-SC/DPP group presented the lowest (p < 0.05) degeneration score and fibrosis % compared to the other groups. Conclusively, stem cell-seeded DPP is a promising platform for the delivery of stem cells and restoration of heart functions post-MI.

Suppression of neurogranin expression by disruption of epigenetic DNA methylation in hippocampal mature granule cells after developmental exposure to neurotoxicants in rats.

We previously performed comprehensive analyses of genes hypermethylated promoter regions and downregulated transcripts in the hippocampal dentate gyrus (DG) of rats upon weaning at postnatal day (PND) 21 after developmental exposure to 6-propyl-2-thiouracil (PTU), valproic acid, and glycidol (GLY), all of which are known to show irreversible effects on hippocampal neurogenesis in adulthood on PND 77. Here, we selected neurotransmitter and neurogenesis-related genes for validation analysis of methylation and expression. As a result, Nrgn by GLY and Shisa7, Agtpbp1, and Cyp46a1 by PTU underwent DNA hypermethylation and sustained downregulation. Immunohistochemical analysis of candidate gene products revealed that the number of neurogranin (NRGN)+ granule cells was decreased in the ventral DG by GLY on PND 21 and 77 and by PTU on PND 21. Among the samples of developmental or 28-day young adult-age exposure to known developmental neurotoxicants in humans, i.e., lead acetate, ethanol, and aluminum chloride, a decrease of NRGN+ cells by ethanol was also observed on PND 77 after developmental exposure. Double immunohistochemistry analysis revealed that NRGN was expressed in mature granule cells, and a similar immunoreactive cell distribution was found for phosphorylated calcium/calmodulin-activated protein kinase, a NRGN downstream molecule. After developmental PTU exposure, the number of activity-regulated cytoskeleton-associated protein+ granule cells was also profoundly decreased in the ventral DG in parallel with the decrease in NRGN+ cells on PND 21. These results suggest that NRGN is a potential marker for suppression of synaptic plasticity in mature granule cells in the ventral DG.

Exploring the effects of embryonic and neonatal exposure to lipopolysaccharides on oligodendrocyte differentiation in the rat hippocampus and the protective effect of alpha-glycosyl isoquercitrin.

This study compared the effects of embryonic and neonatal lipopolysaccharides (LPS) exposure (E-LPS and N-LPS) on oligodendrocyte (OL) differentiation in the hippocampus of male rats and explored the protective effect of the antioxidant alpha-glycosyl isoquercitrin (AGIQ). Using SD rats, LPS exposure occurred either intraperitoneally in dams between gestational days 15 and 16 (50 µg/kg body weight/time) or in male pups on postnatal day (PND) 3 (1 mg/kg body weight). Under both regimens, AGIQ at 0.5% (w/w) was supplemented, to dams from the gestation period (before LPS exposure) until weaning on PND 21 and to male offspring from weaning until PND 77 (adulthood). Compared with a control treatment, E-LPS treatment resulted in fewer NG2+ OL progenitor cells (OPCs) and an upregulation of Tcf4 at PND 6; by PND 21, low NG2+ OPC number persisted, but OLIG2+ OL lineage cells increased, while CNPase+ mature OLs counts were unchanged. By contrast, N-LPS treatment resulted in fewer OLIG2+ cells and an upregulation of Bmp4 at PND 6; by PND 21, NG2+ OPCs decreased, while GFAP+ astrocytes increased at both PND 6 and 21. After N-LPS treatment, Kl and Yy1 were downregulated and there were fewer Klotho+ and CNPase+ cells at PND 21. Results suggest that E-LPS treatment facilitates OPC differentiation into pre- and immature OLs until weaning, while N-LPS treatment suppresses OPC differentiation into mature OLs but facilitates astrocyte generation; however, these changes spontaneously recovered by adulthood under both regimens. AGIQ treatment ameliorated the effects of LPS treatment of both regimens, suggesting that LPS-induced disruption of OPC/OL differentiation occurs via neuroinflammation.

Identification of genes showing altered DNA methylation and gene expression in the renal proximal tubular cells of rats treated with ochratoxin A for 13 weeks.

Ochratoxin A (OTA) is a mycotoxin that causes renal carcinogenicity following the induction of karyomegaly in proximal tubular cells after repeated administration to rats. Here, we performed gene profiling regarding altered DNA methylation and gene expression in the renal tubules focusing on the mechanism of OTA-induced carcinogenesis. For this purpose, OTA or 3-chloro-1,2-propanediol (3-MCPD), a renal carcinogen not inducing karyomegaly, was administered to rats for 13 weeks, and DNA methylation array and RNA sequencing analyses were performed on proximal tubular cells. Genes for which OTA altered the methylation status and gene expression level, after excluding genes showing similar expression changes by 3-MCPD, were subjected to confirmation analysis of the transcript level by real-time reverse-transcription PCR. Gene Ontology (GO)-based functional annotation analysis of validated genes revealed a cluster of hypermethylated and downregulated genes enriched under the GO term "mitochondrion," such as those associated with metabolic reprogramming in carcinogenic process (Clpx, Mrpl54, Mrps34, and Slc25a23). GO terms enriched for hypomethylated and upregulated genes included "response to arsenic-containing substance," represented by Cdkn1a involved in cell cycle arrest, and "positive regulation of IL-17 production," represented by Osm potentiating cell proliferation promotion. Other genes that did not cluster under any GO term included Lrrc14 involved in NF-κB-mediated inflammation, Gen1 linked to DNA repair, Has1 related to chromosomal aberration, and Anxa3 involved in tumor development and progression. In conclusion, a variety of genes engaged in carcinogenic processes were obtained by epigenetic gene profiling in rat renal tubular cells specific to OTA treatment for 13 weeks.

Natural antioxidant formula ameliorates lipopolysaccharide-induced impairment of hippocampal neurogenesis and contextual fear memory through suppression of neuroinflammation in rats.

This study investigated the ameliorating effects of a natural antioxidant formula (NAF) consisting of Ginkgo biloba leaf extract, docosahexaenoic acid/eicosapentaenoic acid, ferulic acid, flaxseed oil, vitamin E, and vitamin B12 on a lipopolysaccharide (LPS)-induced cognitive dysfunction model in rats. Six-week-old rats received a diet containing 0.5% (w/w) NAF for 38 days from Day 1, and LPS (1 mg/kg body weight) was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased interleukin-1ß and tumor necrosis factor-α in the hippocampus and cerebral cortex and the numbers of M1-type microglia/macrophages and GFAP+ reactive astrocytes in the hilus of the hippocampal dentate gyrus. NAF treatment decreased brain proinflammatory cytokine levels and increased the number of M2-type microglia/macrophages. During Days 34-38, LPS alone impaired fear memory acquisition and the extinction learning process, and NAF facilitated fear extinction learning. On Day 38, LPS alone decreased the number of type-3 neural progenitor cells in the hippocampal neurogenic niche, and NAF restored the number of type-3 neural progenitor cells and increased the numbers of both immature granule cells in the neurogenic niche and reelin+ hilar interneurons. Thus, NAF exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects on hippocampal neurogenesis and fear memory learning, possibly through amplification of reelin signaling by hilar interneurons. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory learning, and supplementation with NAF in the present study helped to prevent hippocampal neurogenesis and disruptive neurobehaviors caused by neuroinflammation.

Pharmacokinetics and 28-day repeated-dose toxicity of enniatin B after oral administration in mice.

Enniatins are emerging mycotoxins that contaminate foods. The present study investigated the oral pharmacokinetics and 28-day repeated-dose oral toxicity of enniatin B (ENNB) in CD1 (ICR) mice. In the pharmacokinetic study, male mice received a single oral or intravenous dose of ENNB [30 mg/kg body weight (BW) and 1 mg/kg BW, respectively]. After oral dosing, ENNB exhibited 139.9% bioavailability, a 5.1-h elimination half-life, 5.26% fecal excretion from 4 to 24 h post-dose, and upregulation of Cyp7a1, Cyp2a12, Cyp2b10, and Cyp26a1 in the liver 2 h post-dosing. In the 28-day toxicity study, ENNB was administered to male and female mice by oral gavage at 0, 7.5, 15, and 30 mg/kg BW/day. Females (7.5 and 30 mg/kg) showed dose-unrelated decreased food consumption without accompanying changes in clinical parameters. Males (30 mg/kg) showed low red blood cell counts and high blood urea nitrogen levels and absolute kidney weights; however, other related parameters including the histopathology of systemic organs/tissues were unchanged. These results suggest that ENNB may not induce toxicity after 28 days of oral administration in mice, despite high absorption. The no-observed-adverse-effect level of ENNB after 28 days of repeated oral doses was 30 mg/kg BW/day for both sexes of mice.

Amelioration of lipopolysaccharides-induced impairment of fear memory acquisition by alpha-glycosyl isoquercitrin through suppression of neuroinflammation in rats.

This study investigated the role of neuroinflammation in a lipopolysaccharides (LPS)-induced cognitive dysfunction model in rats using an antioxidant, α-glycosyl isoquercitrin (AGIQ). Six-week-old rats were dietary treated with 0.5% (w/w) AGIQ for 38 days, and LPS at 1 mg/kg body weight was administered intraperitoneally once daily on Days 8 and 10. On Day 11, LPS alone increased or tended to increase interleukin-1ß and tumor necrosis factor-α in the hippocampus and cerebral cortex. Immunohistochemically, LPS alone increased the number of Iba1+ and CD68+ microglia, and GFAP+ astrocytes in the hilus of the hippocampal dentate gyrus (DG). AGIQ treatment decreased or tended to decrease brain proinflammatory cytokine levels and the number of CD68+ microglia in the DG hilus. In the contextual fear conditioning test during Day 34 and Day 38, LPS alone impaired fear memory acquisition, and AGIQ tended to recover this impairment. On Day 38, LPS alone decreased the number of DCX+ cells in the neurogenic niche, and AGIQ increased the numbers of PCNA+ cells in the subgranular zone and CALB2+ hilar interneurons. Additionally, LPS alone decreased or tended to decrease the number of synaptic plasticity-related FOS+ and COX2+ granule cells and AGIQ recovered them. The results suggest that LPS administration induced acute neuroinflammation and subsequent impairment of fear memory acquisition caused by suppressed synaptic plasticity of newborn granule cells following disruptive neurogenesis. In contrast, AGIQ exhibited anti-inflammatory effects and ameliorated LPS-induced adverse effects. These results suggest that neuroinflammation is a key factor in the development of LPS-induced impairment of fear memory acquisition.

Gene expression analysis of antioxidant and DNA methylation on the rat liver after 4-week wood preservative chromated copper arsenate exposure.

Our previous 4-week repeated dose toxicity study showed that wood preservative chromated copper arsenate (CCA) induced hepatocellular hypertrophy accompanied by biochemical hepatic dysfunction and an increase in oxidative stress marker, 8-hydroxydeoxyguanosine, in female rats. To further explore the molecular mechanisms of CCA hepatotoxicity, we analyzed 10%-buffered formalin-fixed liver samples from female rats for cell proliferation, apoptosis, and protein glutathionylation and conducted microarray analysis on frozen liver samples from female rats treated with 0 or 80 mg/kg/day of CCA. Chemical analysis revealed that dimethylated arsenical was the major metabolite in liver tissues of male and female rats. CCA increase labeling indices of proliferating cell nuclear antigen and decrease terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling accompanied with increased expression of protein glutathionylation, indicating a decrease in glutathione (GSH) in hepatocytes of female rats. Microarray analysis revealed that CCA altered gene expression of antioxidants, glutathione-S-transferase (GST), heat shock proteins and ubiquitin-proteasome pathway, cell proliferation, apoptosis, DNA methylation, cytochrome P450, and glucose and lipid metabolism in female rats. Increased expression of GSTs, including Gsta2, Gsta3, Mgst1, and Cdkn1b (p27), and decreased expression of the antioxidant Mt1, and DNA methylation Dnmt1, Dnmt3a, and Ctcf were confirmed in the liver of female rats in a dose-dependent manner. Methylation status of the promoter region of the Mt1 was not evidently changed between control and treatment groups. The results suggested that CCA decreased GSH and altered the expression of several genes, including antioxidants, GST, and DNA methylation, followed by impaired cell proliferation in the liver of female rats.

Phenobarbital, a hepatic metabolic enzyme inducer, inhibits preneoplastic hepatic lesions with expression of selective autophagy receptor p62 and ER-phagy receptor FAM134B in high-fat diet-fed rats through the inhibition of ER stress.

We investigated the role of endoplasmic reticulum (ER)-phagy in NAFLD-related hepatocarcinogenesis in high-fat diet (HFD)-fed and/or phenobarbital (PB)-treated rats by clustering the expression levels of the selective autophagy receptor p62 and the ER-phagy-specific receptor FAM134B in preneoplastic hepatic lesions. We obtained four clusters with variable expression levels of p62 and FAM134B in preneoplastic lesions, and a variable population of clusters in each group. PB administration increased the clusters with high expression levels of p62 while HFD feeding increased the clusters with high expression levels of both p62 and FAM134B. The areas of preneoplastic lesions of these clusters were significantly increased than those of other clusters with low expression levels of p62 and FAM134B. The combination of HFD feeding with PB counteracted the effects of each other, and the cluster composition was similar to that in the control group. The results were associated with decreased gene expression of ER stress, inflammatory cytokine, autophagy, and increased expression of antioxidant enzyme. The present study demonstrated that clustering analysis is useful for understanding the role of autophagy in each preneoplastic lesion, and that HFD feeding increased preneoplastic lesions through the inhibition of ER-phagy, which was cancelled with PB administration through the induction of ER-phagy.

Continuous exposure to alpha-glycosyl isoquercitrin from mid-gestation ameliorates polyinosinic-polycytidylic acid-disrupted hippocampal neurogenesis in rats.

Polyinosinic-polycytidylic acid (PIC) provides a model of developmental neuropathy by inducing maternal immune activation. We investigated the effects of an antioxidant, alpha-glycosyl isoquercitrin (AGIQ), on PIC-induced developmental neuropathy in rats, focusing on postnatal hippocampal neurogenesis. On gestational day 15, PIC at 4 mg/kg body weight was administered to dams intravenously. AGIQ either at 0.25% or 0.5% was administered through the diet to dams from gestational day 10 until weaning on day 21 post-delivery and, thereafter, to offspring until postnatal day 77 (adult stage). At weaning, the numbers of TBR2+ cells and PCNA+ cells in the subgranular zone and reelin+ cells in the dentate gyrus hilus in offspring of dams treated with PIC only were decreased compared with untreated controls. In contrast, 0.5% AGIQ ameliorated these changes and increased the transcript levels of genes related to signaling of reelin (Reln and Vldlr), growth factors (Bdnf, Cntf, Igf1, and Igf1r), and Wnt/ß-catenin (Wnt5a, Lrp6, Fzd1, and Fzd3). In adults, AGIQ increased the number of FOS+ granule cells at 0.25% and the transcript levels of NMDA-type glutamate receptor genes, Grin2a and Grin2b, at 0.25% and 0.5%, respectively. These results suggest that mid-gestation PIC treatment decreased the abundance of type-2b neural progenitor cells (NPCs) by reducing NPC proliferation in relation with suppression of reelin signaling at weaning. We suggest that AGIQ ameliorated the PIC-induced suppressed neurogenesis by enhancing reelin, growth factor, and Wnt/ß-catenin signaling at weaning to rescue NPC proliferation and increased synaptic plasticity by enhancing glutamatergic signaling via NMDA-type receptors after maturation.

Trastuzumab-based near-infrared photoimmunotherapy in xenograft mouse of breast cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel form of cancer treatment using conjugates of antibody against overexpressed antigens in cancers and photoabsorber IRDye700DX. HER2 is overexpressed in various cancers, for which molecular targeted therapy such as trastuzumab has been developed. The present study investigated the efficacy potential of HER2-targeted NIR-PIT using trastuzumab-IRDye700DX conjugate (Tra-IR700) in HER2-positive breast cancer. We first examined the reactivity of Tra-IR700 and the cytotoxicity of NIR-PIT in vitro. HER2-positive BT-474 and SK-BR-3 cells and HER2-negative BT-20 cells were used. Tra-IR700 fluorescence was only observed in HER2-positive breast cancer cell lines, and the fluorescence was localized to the cell surface. Furthermore, HER2-positive breast cancer cell lines treated with NIR-PIT showed swelling and blebbing shortly after irradiation, and eventually increased PI-positive dead cells. Next, tumor accumulation of Tra-IR700 and tumor damage by NIR-PIT were examined in vivo. Tra-IR700 was administered intravenously to a xenograft model in which BT-474 cells were implanted subcutaneously in BALB/c nude mice. Tra-IR700 fluorescence was the highest in tumor tissue 1 day after administration, and the fluorescence was localized to the cell membrane of tumor cells. At this time point, NIR-PIT resulted in diffuse necrosis of tumor tissues 1 day after irradiation. These results suggest that NIR-PIT with Tra-IR700 induces a highly selective therapeutic effect in a HER2-positive breast cancer model. NIR-PIT using Tra-IR700 is expected to be a novel treatment for HER2-positive cancers, including breast cancer.

Progressive disruption of neurodevelopment by mid-gestation exposure to lipopolysaccharides and the ameliorating effect of continuous alpha-glycosyl isoquercitrin treatment.

We investigated the effect of lipopolysaccharide (LPS)-induced maternal immune activation used as a model for producing neurodevelopmental disorders on hippocampal neurogenesis and behaviors in rat offspring by exploring the antioxidant effects of alpha-glycosyl isoquercitrin (AGIQ). Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS (50 µg/kg body weight) at gestational days 15 and 16. AGIQ was administered in the diet to dams at 0.5% (w/w) from gestational day 10 until weaning at postnatal day 21 and then to offspring until adulthood at postnatal day 77. During postnatal life, offspring of LPS-injected animals did not show neuroinflammation or oxidative stress in the brain. At weaning, LPS decreased the numbers of type-2b neural progenitor cells (NPCs) and PCNA+ proliferating cells in the subgranular zone, FOS-expressing granule cells, and GAD67+ hilar interneurons in the dentate gyrus. In adulthood, LPS decreased type-1 neural stem cells, type-2a NPCs, and GAD67+ hilar interneurons, and downregulated Dpysl3, Sst, Fos, Mapk1, Mapk3, Grin2a, Grin2b, Bdnf, and Ntrk2. In adults, LPS suppressed locomotor activity in the open field test and suppressed fear memory acquisition and fear extinction learning in the contextual fear conditioning test. These results indicate that mid-gestation LPS injections disrupt programming of normal neurodevelopment resulting in progressive suppression of hippocampal neurogenesis and synaptic plasticity of newborn granule cells by suppressing GABAergic and glutamatergic neurotransmitter signals and BDNF/TrkB signaling to result in adult-stage behavioral deficits. AGIQ ameliorated most aberrations in hippocampal neurogenesis and synaptic plasticity, as well as behavioral deficits. Effective amelioration by continuous AGIQ treatment starting before LPS injections may reflect both anti-inflammatory and anti-oxidative stress effects during gestation and neuroprotective effects of continuous exposure through adulthood.

Oral Exposure to Lead Acetate for 28 Days Reduces the Number of Neural Progenitor Cells but Increases the Number and Synaptic Plasticity of Newborn Granule Cells in Adult Hippocampal Neurogenesis of Young-Adult Rats.

Lead (Pb) causes developmental neurotoxicity. Developmental exposure to Pb acetate (PbAc) induces aberrant hippocampal neurogenesis by increasing or decreasing neural progenitor cell (NPC) subpopulations in the dentate gyrus (DG) of rats. To investigate whether hippocampal neurogenesis is similarly affected by PbAc exposure in a general toxicity study, 5-week-old Sprague-Dawley rats were orally administered PbAc at 0, 4000, and 8000 ppm (w/v) in drinking water for 28 days. After exposure to 4000 or 8000 ppm PbAc, Pb had accumulated in the brains. Neurogenesis was suppressed by 8000 ppm PbAc, which was related to decreased number of type-2b NPCs, although number of mature granule cells were increased by both PbAc doses. Gene expression in the 8000 ppm PbAc group suggested suppressed NPC proliferation and increased apoptosis resulting in suppressed neurogenesis. PbAc exposure increased numbers of metallothionein-I/II+ cells and GFAP+ astrocytes in the DG hilus, and upregulated Mt1, antioxidant genes (Hmox1 and Gsta5), and Il6 in the DG, suggesting the induction of oxidative stress and neuroinflammation related to Pb accumulation resulting in suppressed neurogenesis. PbAc at 8000 ppm also upregulated Ntrk2 and increased the number of CALB2+ interneurons, suggesting the activation of BDNF-TrkB signaling and CALB2+ interneuron-mediated signals to ameliorate suppressed neurogenesis resulting in increased number of newborn granule cells. PbAc at both doses increased the number of ARC+ granule cells, suggesting the facilitation of synaptic plasticity of newborn granule cells through the activation of BDNF-TrkB signaling. These results suggest that PbAc exposure during the young-adult stage disrupted hippocampal neurogenesis, which had a different pattern from developmental exposure to PbAc. However, the induction of oxidative stress/neuroinflammation and activation of identical cellular signals occurred irrespective of the life stage at PbAc exposure.

Continuous Exposure to Alpha-Glycosyl Isoquercitrin from Gestation Ameliorates Disrupted Hippocampal Neurogenesis in Rats Induced by Gestational Injection of Valproic Acid.

This study examined the ameliorating effect of alpha-glycosyl isoquercitrin (AGIQ), an antioxidant, on disrupted hippocampal neurogenesis in the dentate gyrus (DG) in a rat model of autism spectrum disorder induced by prenatal valproic acid (VPA) exposure. Dams were intraperitoneally injected with 500 mg/kg VPA on gestational day 12. AGIQ was administered in the diet at 0.25 or 0.5% to dams from gestational day 13 until weaning at postnatal day (PND) 21 and then to pups until PND 63. At PND 21, VPA-exposed offspring showed decreased numbers of type-2a and type-3 neural progenitor cells (NPCs) among granule cell lineage subpopulations. AGIQ treatment at both doses rescued the reduction in type-3 NPCs. AGIQ upregulated Reln and Vldlr transcript levels in the DG at 0.5% and ≥ 0.25%, respectively, and increased the number of reelin+ interneurons in the DG hilus at 0.5%. AGIQ at 0.25% and/or 0.5% also upregulated Ntrk2, Cntf, Igf1, and Chrnb2. At PND 63, there were no changes in the granule cell lineage subpopulations in response to VPA or AGIQ. AGIQ at 0.25% increased the number of FOS+ granule cells, accompanied by Gria2 and Gria3 upregulation and increasing trend in the number of FOS+ granule cells at 0.5%. There was no definitive evidence of VPA-induced oxidative stress in the hippocampus throughout postnatal life. These results indicate that AGIQ ameliorates the VPA-induced disruption of hippocampal neurogenesis at weaning involving reelin, BDNF-TrkB, CNTF, and IGF1 signaling, and enhances FOS-mediated synaptic plasticity in adulthood, potentially through AMPA-receptor upregulation. The ameliorating effects of AGIQ may involve direct interactions with neural signaling cascades rather than antioxidant capacity.

Inhibition of autophagy with expression of NADPH oxidase subunit p22phox in preneoplastic lesions in a high-fat diet and streptozotocin-related hepatocarcinogenesis rat model.

To study the effects of autophagy inducer carbamazepine (CBZ) in a high-fat diet (HFD)/streptozotocin (STZ)-related early hepatocarcinogenesis model, we determined autophagic flux by immunohistochemical analysis of autophagy marker expression in preneoplastic liver foci and compared that with the expression of the NADPH oxidase subunit. Male F344 rats were fed a basal diet or HFD and subjected to two-stage hepatocarcinogenesis; diabetes mellitus was induced via STZ administration. Several STZ-treated, HFD-fed rats were administered CBZ (a total of five doses every one or two days) at week 7 and 8. STZ-treated, HFD-fed rats decreased ß cells in the islet of Langerhans and increased adipophilin-positive lipid droplets in the liver; moreover, they had a larger area of glutathione S-transferase placental form-immunopositive preneoplastic liver foci, which was associated with inhibition of autophagy and induction of the NADPH oxidase subunit, as demonstrated by increased immunohistochemical expression of an autophagosome receptor marker microtubule-associated protein light chain 3 (LC3)-binding protein p62, and of an NADPH oxidase subunit p22phox in the preneoplastic foci. An increased trend of an autophagy phagophore marker LC3 in preneoplastic foci was also detected. CBZ administration could induce autophagy and impair p22phox expression, as shown by altered expression of autophagy regulators (Atg5, Atg6, Lamp1, Lamp2, and Lc3), NADPH oxidase subunits (P22phox and P67phox), and antioxidant enzymes Gpx1 and Gpx2. These results suggest that inhibition of autophagy and induction of p22phox might contribute to HFD/STZ-related early hepatocarcinogenesis in rats; however, the effects of CBZ administration on the STZ/HFD-increased preneoplastic foci were marginal in this study.

The potential of organoids in toxicologic pathology: role of toxicologic pathologists in in vitro chemical hepatotoxicity assessment.

The development of in vitro toxicity assessment methods using cultured cells has gained popularity for promoting animal welfare in animal experiments. Herein, we briefly discuss the current status of hepatoxicity assessment using human- and rat-derived hepatocytes; we focus on the liver organoid method, which has been extensively studied in recent years, and discuss how toxicologic pathologists can use their knowledge and experience to contribute to the development of in vitro chemical hepatotoxicity assessment methods for drugs, pesticides, and chemicals. We also propose how toxicological pathologists should assess toxicity regarding the putative distribution of undifferentiated and differentiated cells in the organoid when liver organoids are observed in hematoxylin and eosin-stained specimens. This was done while considering the usefulness and limitations of in vitro studies for toxicologic pathology assessment.

Oral exposure to high-dose ethanol for 28 days in rats reduces neural stem cells and immediate nascent neural progenitor cells as well as FOS-expressing newborn granule cells in adult hippocampal neurogenesis.

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. In rats, developmental exposure to ethanol (EtOH) at high doses has shown to induce aberrant neurogenesis in neural progenitor cells (NPCs) during weaning and suppress synaptic plasticity of newborn granule cells after maturation; neuroinflammation was even sustained until the adult stage in the hippocampal dentate gyrus (DG). To investigate whether hippocampal neurogenesis is affected by EtOH exposure in a general toxicity study, EtOH was administered orally to 5-week-old Sprague-Dawley rats at 0%, 10%, and 16% (w/v) in drinking water for 28 days. Exposure to 16% EtOH decreased type-1 neural stem cells (NSCs) and type-2a NPCs in the DG subgranular zone. A reduction in reelin-positive (reelin+) interneurons and an increased number of parvalbumin+ interneurons in the DG hilus, as well as downregulation of Mcm6 and Calb2 in the DG, suggested that self-renewal and proliferation of type-1 NSCs were suppressed. Exposure to 16% EtOH also induced M1-type microglia/peripheral macrophages, and upregulated Il1a and Tnf, suggesting that neuroinflammation might be responsible for the suppressed neurogenesis. In contrast, Drd2 and Tgfb3 upregulation might be ameliorating responses against suppressed neurogenesis. EtOH exposure (16%) also decreased the number of FOS+ granule cells, suggesting that synaptic plasticity was suppressed; concurrent upregulation of glutamate receptor/transporter genes may have occurred as a compensatory response against suppressed synaptic plasticity. Thus, high-dose EtOH exposure in young adult rats disrupted hippocampal neurogenesis differently to exposure during development. However, induction of neuroinflammation and suppressed synaptic plasticity occurred at both EtOH exposure stages.

Oral exposure to aluminum chloride for 28 days suppresses neural stem cell proliferation and increases mature granule cells in adult hippocampal neurogenesis of young-adult rats.

Aluminum (Al), a common light metal, affects the developing nervous system. Developmental exposure to Al chloride (AlCl3 ) induces aberrant neurogenesis by targeting neural stem cells (NSCs) and/or neural progenitor cells (NPCs) in the dentate gyrus (DG) of rats and mice. To investigate whether hippocampal neurogenesis is similarly affected by AlCl3 exposure in a general toxicity study, AlCl3 was orally administered to 5-week-old Sprague Dawley rats at dosages of 0, 4000, or 8000 ppm in drinking water for 28 days. AlCl3 downregulated Sox2 transcript level in the DG at the highest dosage and produced a dose-dependent decrease of SOX2+ cells without altering numbers of GFAP+ or TBR2+ cells in the subgranular zone, suggesting that AlCl3 decreases Type 2a NPCs. High-dose exposure downregulated Pcna, upregulated Pvalb, and altered expression of genes suggestive of oxidative stress induction (upregulation of Nos2 and downregulation of antioxidant enzyme genes), indicating suppressed proliferation and differentiation of Type 1 NSCs. AlCl3 doses also increased mature granule cells in the DG. Upregulation of Reln may have contributed to an increase of granule cells to compensate for the decrease of Type 2a NPCs. Moreover, upregulation of Calb2, Gria2, Mapk3, and Tgfb3, as well as increased numbers of activated astrocytes in the DG hilus, may represent ameliorating responses against suppressed neurogenesis. These results suggest that 28-day exposure of young-adult rats to AlCl3 differentially targeted NPCs and mature granule cells in hippocampal neurogenesis, yielding a different pattern of disrupted neurogenesis from developmental exposure.