Similar toxicity potential of glyphosate and glyphosate-based herbicide on cerebellar development after maternal exposure in rats.

Studies on the effects of glyphosate (GlyP) and glyphosate-based herbicides (GBHs) on cerebellar development are extremely limited. This study examined the effects of maternal exposure to GlyP and GBH on rat cerebellar development in male offspring. From day 6 of gestation until day 21 postpartum at weaning, dams were given GlyP at 1.5% or 3.0% in diet or GBH at 1.0% in drinking water (corresponding to 0.36% GlyP). At weaning, GBH exposure was linked to increased numbers of DCX+ migrating granule cells in the cortex and TUNEL+ apoptotic cells in the internal granular layer (IGL), suggesting the disappearance of mismigrated granule cells via apoptosis. GBH also upregulated Nr4a3 and downregulated Cdk5 in the cerebellar vermis, suggesting a causal relation with the impaired granule cell development at this time. GlyP (3.0%) tended to increase in the number of DCX+ migrating granule cells in the IGL and upregulated Nr4a3 at weaning. Both compounds also upregulated genes related to granule cell migration (Astn1, Astn2, Nfia, and/or Nfix) at weaning and in adulthood, which might be an ameliorative response to delayed granule cell migration. Moreover, GBH induced Purkinje cell misalignment at weaning, which could be the result of delayed granule cell migration. In adulthood, GBH was associated with upregulation of the reelin signaling-related genes Reln, Dab1, and Efnb1, suggesting a compensatory response to Purkinje cell misalignment. GlyP induced the same gene expression changes. These results suggest that GBH reversibly disrupts cerebellar development, primarily by targeting granule cell migration and differentiation, whereas GlyP exhibited similar toxic potential as GBH.

Suppression of Hippocampal Neurogenesis and Oligodendrocyte Maturation Similar to Developmental Hypothyroidism by Maternal Exposure of Rats to Ammonium Perchlorate, a Gunpowder Raw Material and Known Environmental Contaminant.

The environmental contaminant perchlorate raises concern for hypothyroidism-related brain disorders in children. This study investigated the effects of developmental perchlorate exposure on hippocampal neurogenesis and oligodendrocyte (OL) development. Pregnant Sprague-Dawley rats were administered with ammonium perchlorate (AP) in drinking water at concentrations of 0 (control), 300, and 1000 ppm from gestation day 6 until weaning [postnatal day (PND) 21]. On PND 21, offspring displayed decreased serum triiodothyronine and thyroxine concentrations at 1000 ppm and thyroid follicular epithelial cell hyperplasia at ≥300 ppm (accompanying increased proliferation activity at 1000 ppm). Hippocampal neurogenesis indicated suppressed proliferation of neurogenic cells at ≥300 ppm, causing decreases in type-1 neural stem cells (NSCs) and type-2a neural progenitor cells. In addition, an increase of SST+ GABAergic interneurons and decreasing trend for ARC+ granule cells were observed at 1000 ppm. CNPase+ mature OLs were decreased in number in the dentate gyrus hilus at ≥300 ppm. At PND 77, thyroid changes had disappeared; however, the decrease of type-1 NSCs and increase of SST+ interneurons persisted, CCK+ interneurons were increased, and white matter tissue area was decreased at 1000 ppm. Obtained results suggest an induction of hypothyroidism causing suppressed hippocampal neurogenesis (targeting early neurogenic processes and decreased synaptic plasticity of granule cells involving ameliorative interneuron responses) and suppressed OL maturation during the weaning period. In adulthood, suppression of neurogenesis continued, and white matter hypoplasia was evident. Observed brain changes were similar to those caused by developmental hypothyroidism, suggesting that AP-induced developmental neurotoxicity was due to hypothyroidism.

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.

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.

Identification of gene targets of developmental neurotoxicity focusing on DNA hypermethylation involved in irreversible disruption of hippocampal neurogenesis in rats.

We have previously found that maternal exposure to 6-propyl-2-thiouracil (PTU), valproic acid (VPA), or glycidol (GLY) has a sustained or late effect on hippocampal neurogenesis at the adult stage in rat offspring. Herein, we searched for genes with hypermethylated promoter region and downregulated transcript level to reveal irreversible markers of developmental neurotoxicity. The hippocampal dentate gyrus of male rat offspring exposed maternally to PTU, VPA, or GLY was subjected to Methyl-Seq and RNA-Seq analyses on postnatal day (PND) 21. Among the genes identified, 170 were selected for further validation analysis of gene expression on PND 21 and PND 77 by real-time reverse transcription-PCR. PTU and GLY downregulated many genes on PND 21, reflecting diverse effects on neurogenesis. Furthermore, genes showing sustained downregulation were found after PTU or VPA exposure, reflecting a sustained or late effect on neurogenesis by these compounds. In contrast, such genes were not observed with GLY, probably because of the reversible nature of the effects. Among the genes showing sustained downregulation, Creb, Arc, and Hes5 were concurrently downregulated by PTU, suggesting an association with neuronal mismigration, suppressed synaptic plasticity, and reduction in neural stem and progenitor cells. Epha7 and Pvalb were also concurrently downregulated by PTU, suggesting an association with the reduction in late-stage progenitor cells. VPA induced sustained downregulation of Vgf and Dpysl4, which may be related to the aberrations in synaptic plasticity. The genes showing sustained downregulation may be irreversible markers of developmental neurotoxicity.

Comparison of microRNA expressions for the identification of chemical hazards in in vivo and in vitro hepatic injury models.

Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.

Intermittent PTH administration improves alveolar bone formation in type 1 diabetic rats with periodontitis.

BACKGROUND: Periodontitis is an infectious disease that manifests as alveolar bone loss surrounding the roots of teeth. Diabetes aggravates periodontitis-induced alveolar bone loss via suppression of bone formation. Intermittent parathyroid hormone (PTH) administration displays an anabolic effect on bone. In this study, we investigated the effect of intermittent PTH administration on alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), periodontitis (P), periodontitis treated with PTH (P + PTH), diabetes with periodontitis (DP), and diabetes with periodontitis treated with PTH (DP + PTH) groups. To induce type 1 diabetes, rats were injected with streptozotocin and periodontitis was induced bilaterally by applying ligatures to the mandibular first molars for 30 days. During the experimental period, the P + PTH and DP + PTH groups were subcutaneously injected with PTH (40 µg/kg) three times per week, whereas the C, P, and DP groups were injected with citrate buffer. To observe the mineralization of the alveolar bone, the DP and DP + PTH groups were injected with calcein on days 10 and 27, and with alizarin red on day 20. Thirty days after ligation, histological findings and fluorescence labeling were analyzed in the furcations of the mandibular first molars. Sclerostin-positive osteocytes were assessed by immunohistochemical analyses. RESULTS: The DP groups had smaller areas of alveolar bone than the other groups, and the DP + PTH group had a larger alveolar bone area than the DP group. The DP group had less osteoid formation than the C group, whereas the DP + PTH had greater osteoid formation than the DP group. Fluorescence labeling results revealed that the DP + PTH group had more mineral deposition on the alveolar bone than the DP group. The DP + PTH group exhibited lower percentage of sclerostin-positive osteocytes in alveolar bone than the DP group. CONCLUSIONS: Intermittent PTH administration diminishes alveolar bone loss and sclerostin expression in osteocytes, but increases osteoid formation and mineralization, suggesting that intermittent PTH administration attenuates diabetes-aggravated alveolar bone loss by the induction of bone formation. PTH-induced bone formation may be related to the regulation of osteocytic sclerostin expression in type 1 diabetic rats with periodontitis.

Detection and phylogenetic analysis of a new adenoviral polymerase gene in reptiles in Korea.

Over a period of 7 years (2004-2011), samples from 34 diseased reptiles provided by local governments, zoos, and pet shops were tested for viral infection. Animals were diagnosed based on clinical signs, including loss of appetite, diarrhea, rhinorrhea, and unexpected sudden death. Most of the exotic animals had gastrointestinal problems, such as mucosal redness and ulcers, while the native animals had no clinical symptoms. Viral sequences were found in seven animals. Retroviral genes were amplified from samples from five Burmese pythons (Python molurus bivittatus), an adenovirus was detected in a panther chameleon (Furcifer pardalis), and an adenovirus and a paramyxovirus were detected in a tropical girdled lizard (Cordylus tropidosternum). Phylogenetic analysis of retroviruses and paramyxoviruses showed the highest sequence identity to both a Python molurus endogenous retrovirus and a Python curtus endogenous retrovirus and to a lizard isolate, respectively. Partial sequencing of an adenoviral DNA polymerase gene from the lizard isolate suggested that the corresponding virus was a novel isolate different from the reference strain (accession no. AY576677.1). The virus was not isolated but was detected, using molecular genetic techniques, in a lizard raised in a pet shop. This animal was also coinfected with a paramyxovirus.

Histopathologic and immunohistological evaluation of anaplastic large cell lymphoma with Epstein-Barr virus in an orangutan (Pongo pygmaeus).

BACKGROUND: An 18-month-old female orangutan (Pongo pygmaeus) died after exhibiting fever, cough, and rapid breathing. METHODS AND RESULTS: Based on serological, virological, histopathological and immunohistochemical examination, anaplastic large cell lymphoma was confirmed. CONCLUSION: To the best of our knowledge, this is the first report of anaplastic large cell lymphoma associated with Epstein-Barr virus (EBV) in an orangutan.

Effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia.

OBJECTIVE: This study examined the effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia in animal models. MATERIALS AND METHODS: Rats were divided into normal, periodontitis, diabetic and diabetic with periodontitis groups. After injection of streptozotocin to induce diabetes, periodontitis was induced by ligation of both lower-side first molars for 30 days. Alveolar bone loss and trabecular bone volume fraction (BVF) of the mandibular condyle and tibia were estimated via hematoxylin and eosin staining and micro-computed tomography, respectively. Osteoclastogenesis of bone marrow cells isolated from tibia and femur was assayed using tartrate-resistant acid phosphatase staining. RESULTS: The cemento-enamel junction to the alveolar bone crest distance and ratio of periodontal ligament area in the diabetic with periodontitis group were significantly increased compared to those of the periodontitis group. Mandibular condyle BVF did not differ among groups. The BVF of tibia in the diabetic and diabetic with periodontitis groups was lower than that of the normal and periodontitis groups. Osteoclastogenesis of bone marrow cells in the diabetic groups was higher than that in the non-diabetic groups. However, the BVF of tibia and osteoclastogenesis in the diabetic with periodontitis group were not significantly different than those in the diabetic group. CONCLUSIONS: Type 1 diabetes mellitus aggravates alveolar bone loss induced by periodontitis, but periodontitis does not alter the mandibular condyle and tibia bone loss induced by diabetes. Alveolar bone, mandibular condyle and tibia may have different responses to bone loss stimuli in the diabetic environment.

Endogenous lipid pneumonia in a ringed seal (Pusa hispida subsp. ochotensis).

An adult female ringed seal died suddenly and was subsequently examined for diagnostic purposes. The animal's lungs demonstrated mild non-collapsibility and multifocal white to yellow patches. Histopathological examination revealed multifocal pulmonary histiocytosis. Alveoli were filled with numerous foamy macrophages cytoplasm and scattered multinucleated giant cells containing cholesterol clefts. The foamy cytoplasm of the macrophages stained with oil red O stain. Further, lipid droplets within the cytoplasm were detected by electron microscopy. To the author's knowledge, this is the first case report describing the histochemical staining and electron microscopic findings associated with endogenous lipid pneumonia in ringed seal.

Gallic acid improves glucose tolerance and triglyceride concentration in diet-induced obesity mice.

Gallic acid, a phenolic phytochemical, has been shown to exert a variety of effects, including anti-oxidative, anti- carcinogenic, anti-allergic, and anti-inflammatory effects. In this study, we attempted to determine whether gallic acid affects metabolic syndrome such as obesity and diabetes. Diet-induced obesity mice were treated intraperitoneally once per day with gallic acid (10 mg/kg/day). After 2 weeks of treatment, the mice were sacrificed to collect the blood for metabolic parameter assessments, and the adipose tissues and liver to weigh and analyze. The triglyceride concentrations were significantly improved in the gallic acid group relative to those measured in the control group. And most importantly, the blood glucose concentrations in the gallic acid group were significantly improved. In the epididymal white adipose tissue of the gallic acid group, adipocyte size was reduced, PPARγ expression was induced, and the Akt signaling pathway was activated. Our results demonstrate that gallic acid improves glucose tolerance and lipid metabolism in the obesity mice, thereby showing evidence of anti-hyperglycemic activity. The findings of an upregulation of PPARγ expression and Akt activation also contribute to our current understanding of the mechanisms underlying the effects of gallic acid on glucose metabolism.

Increased cellular distribution of vimentin and ret in the cingulum of rat offspring after developmental exposure to decabromodiphenyl ether or 1,2,5,6,9,10-hexabromocyclododecane.

To determine effects of developmental exposure to brominated flame retardants (BFRs), weak thyroid hormone disruptors, on white matter development, white matter-specific global gene expression analysis was performed using microdissection techniques and microarrays in male rats exposed maternally to decabromodiphenyl ether (DBDE), one of the representative BFRs, at 10, 100 or 1000 ppm. Based on previous gene expression profiles of developmental hypothyroidism and DBDE-exposed cases, vimentin(+) immature astrocytes and ret proto-oncogene (Ret)(+) oligodendrocytes were immunohistochemically examined after developmental exposure to representative BFRs, i.e., DBDE, 1,2,5,6,9,10-hexabromocyclododecane (HBCD; 100, 1000 or 10,000 ppm) and tetrabromobisphenol A (TBBPA; 100, 1000 or 10,000 ppm). Vimentin(+) and Ret(+) cell populations increased at ≥ 100 ppm and ≥ 10 ppm DBDE, respectively. Vimentin(+) and Ret(+) cells increased at ≥ 1000 ppm HBCD, with no effect of TBBPA. The highest dose of DBDE and HBCD revealed subtle fluctuations in serum thyroid-related hormone concentrations. Thus, DBDE and HBCD may exert direct effects on glial cell development at ≥ middle doses. At high doses, hypothyroidism may additionally be an inducing mechanism, although its contribution is rather minor.

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.

Comparison of the effect of glyphosate and glyphosate-based herbicide on hippocampal neurogenesis after developmental exposure in rats.

Increasing evidence indicates that glyphosate (GlyP)-based herbicides (GBHs) induce developmental neurotoxicity. The present study investigated the developmental exposure effect of GlyP and GBH on hippocampal neurogenesis in rats. Dams were treated from gestational day 6 to day 21 post-delivery on weaning with a diet containing 1.5% or 3.0% GlyP or drinking water with 1.0% GBH (containing 0.36% GlyP). Dams in the 1.5%-GlyP, 3.0%-GlyP, and GBH groups received 1.04, 2.16, and 0.25 g GlyP/kg body weight (BW)/day during gestation, and 2.27, 4.65, and 0.58 g GlyP/kg BW/day during lactation, respectively. On weaning, 3.0% GlyP- and GBH-exposed offspring decreased the BW, and the latter also decreased the brain weight. Both compounds suppressed neural progenitor cell proliferation in the neurogenic niche, and GlyP-exposed offspring showed a decreased number of TUBB3+ immature granule cells. In contrast, both compounds increased the number of ARC+ granule cells, suggesting increased synaptic plasticity. Both compounds downregulated antioxidant genes (Cat and Sod2) in the dentate gyrus, suggestive of increased sensitivity to oxidative stress, which might be related to the suppression of neurogenesis. At the adult age, GBH alone sustained decreases in body and brain weights. Both compounds increased hippocampal malondialdehyde levels and upregulated Cat in the dentate gyrus, suggesting induction of oxidative stress. Both compounds upregulated Casp9, and GBH increased neural progenitor cell apoptosis, suggesting disruption of neurogenesis related to oxidative stress. GBH increased the number of COX2+ granule cells, and both compounds upregulated Arc, suggesting increased synaptic plasticity. These results suggest that GlyP and GBH might cause similar effects on disruption of neurogenesis accompanying compensatory responses and induction of oxidative stress responses through the adult age in the hippocampus. However, effects on adult age were more evident with GBH, suggesting that the surfactants contained in GBH might have contributed to the enhanced neurotoxicity of GlyP, similar to the enhanced general toxicity.

Transient aberration of neuronal development in the hippocampal dentate gyrus after developmental exposure to brominated flame retardants in rats.

We immunohistochemically investigated the impact and reversibility of three brominated flame retardants (BFRs) known to be weak thyroid hormone disruptors on neuronal development in the hippocampal formation and apoptosis in the dentate subgranular zone. Pregnant Sprague-Dawley rats were exposed to 10, 100, or 1,000 ppm decabromodiphenyl ether (DBDE); 100, 1,000 or 10,000 ppm tetrabromobisphenol A (TBBPA) or 1,2,5,6,9,10-hexabromocyclododecane (HBCD) in the diet from gestational day 10 through to day 20 after delivery (weaning). On postnatal day (PND) 20, interneurons in the dentate hilus-expressing reelin increased with all chemicals, suggestive of aberration of neuronal migration. However, this increase had disappeared by PND 77. NeuN-positive mature neurons increased in the hilus on PND 77 with all chemicals. In the subgranular zone on PND 20, an increase in apoptotic bodies suggestive of impaired neurogenesis was observed after exposure to TBBPA or HBCD. The effects on neuronal development were detected at doses of ≥100 ppm DBDE; ≥1,000 ppm TBBPA; and at least at 10,000 ppm HBCD. On PND 20, the highest dose of DBDE and HBCD revealed mild fluctuations in the serum concentrations of thyroid-related hormones suggestive of weak developmental hypothyroidism, while TBBPA did not. Thus, DBDE and TBBPA may exert direct effect on neuronal development in the brain, but hypothyroidism may be operated for DBDE and HBCD at high doses. An excess of mature neurons in the hilus at later stages may be the signature of the developmental effects of BFRs. However, the effect itself was reversible.

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.

Preventive effects of calcitriol on the development of capsular invasive carcinomas in a rat two-stage thyroid carcinogenesis model.

We have shown phosphoinositide 3-kinase (PI3K)/Akt signaling activation in thyroid capsular invasive carcinomas (CICs), which are highly induced by promotion with sulfadimethoxine (SDM) in a rat 2-stage thyroid carcinogenesis model. To examine the potency of calcitriol, a synthetic vitamin D3 analog, on the development or progression of CICs, male F344 rats were injected with calcitriol (0.1 µg/kg body weight) three times a week intraperitoneally, during an entire period of SDM-promotion for 13 weeks (Experiment 1) or during the last 2 weeks of a 15-week SDM-promotion (Experiment 2). Initiation with N-bis(2-hydroxypropyl)nitrosamine preceded all treatments. In Experiment 1, long-term calcitriol treatment reduced the multiplicity of CICs, while cell proliferation activity, estimated by Ki-67 cell index in the induced CICs, was unchanged with SDM-promotion alone. Considering the strong dependency of promotion with SDM during the early stages on thyroid-stimulating hormone, the reduced multiplicity in Experiment 1 may be due to the effect on an early stage of neoplastic proliferation. Although the magnitude was mild, cell proliferation activity was decreased in existing CICs after short-term calcitriol treatment in Experiment 2, which was associated with a mild decrease in cyclin-dependent kinase-2-positive cells, cytoplasmic immunolocalization of phosphorylated, inactive, Rb protein and a mild increase in nucleocytoplasmic expression of p27(kip1). Although the effect was mild at the late stage of SDM-promotion in this hypothyroidism-related thyroid carcinogenesis model, our results suggest that calcitriol targets cell proliferation via inhibition of a molecular cascade downstream of PI3K/Akt signaling, controlling G1/S transition.

Ectopic Splenic Adenocarcinoma in a Dog.

A 10-year-old spayed female Border Collie developed a ductal adenocarcinoma in the spleen. Clinically, the spleen was enlarged and a small liver nodule was present but there were no other abnormalities. Most of the splenic parenchyma was diffusely infiltrated by variably shaped atypical neoplastic cells that formed small clusters or larger nests, arranged as duct or duct-like structures within a fibrous matrix. There was acinar differentiation in a few portions of the tumour with a sheet-like solid growth pattern and occasional squamous metaplasia or exocrine acinus-like structures. Mitotic figures were frequent. Neoplastic cells with ductal differentiation were diffusely immunoreactive for AE1/AE3, CAM5.2 and CK7 cytokeratins but negative for CK20, while cells with acinar differentiation were immunolabelled only for AE1/AE3 cytokeratins and were also immunopositive for mucin-1 and trypsin. A few regions of tumour with ductal or acinar differentiation were immunopositive for pancreatic lipase. All neoplastic cells were negative for mucin-2, vimentin, smooth muscle actin, chromogranin A, CD31, hepatocyte paraffin 1 and thyroglobulin antigens. Because of the formation of exocrine acinus-like structures and an immunolabelling pattern consistent with exocrine pancreas tissue, an adenocarcinoma of ectopic exocrine pancreas within the spleen was diagnosed.

Aberrant neurogenesis and late onset suppression of synaptic plasticity as well as sustained neuroinflammation in the hippocampal dentate gyrus after developmental exposure to ethanol in rats.

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. The present study investigated the effects of maternal oral ethanol (EtOH) exposure (0, 10, or 12.5 % in drinking water) from gestational day 6 until day 21 post-delivery (weaning) on postnatal hippocampal neurogenesis at weaning and in adulthood on postnatal day 77 in rat offspring. At weaning, type-3 neural progenitor cells (NPCs) were decreased in the subgranular zone (SGZ), accompanied by Chrnb2 downregulation and Grin2b upregulation in the dentate gyrus (DG). These results suggested suppression of CHRNB2-mediated cholinergic signaling in γ-aminobutyric acid (GABA)ergic interneurons in the DG hilus and increased glutamatergic signaling through the NR2B subtype of N-methyl-d-aspartate (NMDA) receptors, resulting in NPC reduction. In contrast, upregulation of Chrna7 may increase CHRNA7-mediated cholinergic signaling in immature granule cells, and upregulation of Ntrk2 may cause an increase in somatostatin-immunoreactive (+) GABAergic interneurons, suggesting a compensatory response against NPC reduction. Promotion of SGZ cell proliferation increased type-2a NPCs. Moreover, an increase in calbindin-d-29 K+ interneurons and upregulation of Reln, Drd2, Tgfb2, Il18, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunit genes might participate in this compensatory response. In adulthood, reduction of FOS+ cells and downregulation of Fos and Arc suggested suppression of granule cell synaptic plasticity, reflecting upregulation of Tnf and downregulation of Cntf, Ntrk2, and AMPA-type glutamate receptor genes. In the DG hilus, gliosis and hyper-ramified microglia, accompanying upregulation of C3, appeared at weaning, suggesting contribution to suppressed synaptic plasticity in adulthood. M1 microglia increased throughout adulthood, suggesting sustained neuroinflammation. These results indicate that maternal EtOH exposure temporarily disrupts hippocampal neurogenesis and later suppresses synaptic plasticity. Induction of neuroinflammation might initially ameliorate neurogenesis (as evident by upregulation of Tgfb2 and Il18) but later suppress synaptic plasticity (as evident by upregulation of C3 at weaning and Tnf in adulthood).