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Environmental toxicants such as mercury, manganese, pesticides and others; contaminants in designer drugs of abuse such as MPTP; and a growing inventory of industrial chemicals have been linked to neurological damage and a significant number of progressive neurological diseases such as Parkinson’s and other CNS degenerative syndromes. In addition, attrition due to neurotoxicity represents a significant issue in all stages of drug development. Traditionally, neurotoxicity testing has relied on composite data sets of functional assessments (e.g., behavior, activity, seizures) and conventional neuropathological evaluations (e.g., organ weights, gross observations, histopathology). Current histopathologic analyses often suffer from constrained spatial sampling and limited translational capability, and microscopic findings often do not correlate with the functional or neurochemical evidence typically collected. In addition, most neurotoxicants produce very specific cellular changes, restricted to either cellular compartments (e.g., somatic, axonal, or dendritic) or cell populations in different regions of the brain with distinct temporal profiles. ?These toxicities may also be species specific, further confounding the challenge of translation to humans. Thus far, few non-invasive biomarkers for neuropathologies have been qualified. In late-2012, ILSI-HESI approved a proposal for identifying and assessing new biomarkers of neurotoxicity and a consortium was formed. This poster will review recommendations from a recent ILSI-HESI workshop and highlight new approaches for identifying translational biomarkers in neurotoxicity. These innovations include fluid-based biomarkers, non-invasive imaging and functional measures. Two neurotoxicants, trimethyl tin and MPTP, were used as initial prototypic compounds to help focus a discussion around current best practices, assessment gaps and potential new biomarkers.

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The present toxicological assessment systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Human embryonic stem cells (hESC) have unique attributes that can be applied in drug discovery, from initial target ideas to clinical trials. This study tried i) to generate human pluripotent stem cell lines carrying tyrosine hydroxylase (TH) gene-driven EGFP as a reporter, by which live-cell imaging can be employed to monitor the specific cell type numbers and morphology, as well as ii) to establish an effective alternative developmental toxicity test system. We have successfully selected stable hESC lines by transfection using 10-kb rat TH promoter connected with EGFP into KhES1. The expression of EGFP only appeared during the neuronal cell differentiation culture process of hESC. The intensity of the EGFP increased day by day during the dendrite formation. Using this hESC cell, we tried to investigate the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in neuronal cell developmental process in vitro, because it has been reported that, fetal brain functions are affected by the TCDD exposure in rodent studies. The expression of EGFP might be able to monitor the real time dopaminergic neuronal differentiation during neuronal developmental process. These cells will provide a qualitative and quantitative advantage to investigators in human neuro-developmental research.

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Signal transducer and activator of transcription-1 (STAT1) has an important role in inflammation and the innate immune response, but its role in the central nervous system is less well understood. Here, we examined the role of STAT1 in spatial learning and memory, and assessed the involvement of STAT1 in mediating the memory-impairing effect of amyloid-beta (Aβ). We found that water maze training downregulated STAT1 expression in the rat hippocampal CA1 area, and spatial learning and memory function was enhanced in Stat1-knockout mice. Conversely, overexpression of STAT1 impaired water maze performance. In addition, we have found that STAT1 also mediates the toxicity of Aβ on hippocampal neuronal loss. STAT1 strongly upregulated the expression of the extracellular matrix protein laminin β1 (LB1), which also impaired water maze performance in rats. Furthermore, Aβ impaired spatial learning and memory in association with a dose-dependent increase in STAT1 and LB1 expression, but knockdown of STAT1 and LB1 both reversed this effect of Aβ. This Aβ-induced increase in STAT1 and LB1 expression was also associated with a decrease in the expression of the N-methyl-D-aspartate receptor (NMDAR) subunits, NR1, and NR2B. Our results demonstrate that STAT1 negatively regulates spatial learning and memory through transcriptional regulation of LB1 expression. We also identified a novel mechanism for Aβ pathogenesis through STAT1 induction. Notably, impairment of spatial learning and memory by this STAT1-mediated mechanism is independent of cAMP responsive element-binding protein signaling.

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It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytomery. Recently, the Pig-a mutation assay has been developed to evaluate in vivo genotoxicity in laboratory animals and humans. In this study, we used three-color staining with fluorescently labeled anti-CD24, anti-TER-119, and anti-CD71 to detect the Pig-a mutant frequency in total red blood cells (RBCs) from X-irradiated mice. Single exposures to X-irradiation resulted in dose-dependent increases in Pig-a mutant frequency. We compared Pig-a mutant frequencies between irradiated C57BL/6J (p53^+/+) mice and p53^-/- mice. After the peak in radiation-induced Pig-a mutant frequencies, a gradual decrease in mutant frequencies in irradiated p53^-/- mice was observed, while irradiated p53^+/+ mice had a rapid decrease, which suggests that Pig-a mutant cells are eliminated normally in irradiated p53^+/+ mice but not in irradiated p53^-/- mice due to lack of p53 function. In addition, we also found that the p53 function affected the regulation of Pig-a mutagenesis in aging mice. Our results suggest that p53 function, distinct types of mutation, and the life span of RBCs play key roles in the persistence of Pig-a mutation in the hematopoietic system of RBCs after irradiation.

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Evodia officinalis (EO) is natural product which has been used by oriental medicine and it has been known to have some pharmacologic effects such as testosterone secretion, catecholamine secretion, analgesic, anti-inflammatory, anti-obesity, vasodilatory, thermoregulatory and uterotonic effects. However, its toxicity has not been fully evaluated. In the present studies, we carried out a 90-day repeated dose toxicity study (orally five times per week at doses of 25, 74, 222, 667 and 2,000 mg/kg) using in F344 rats and genotoxicity studies (bacterial reverse mutation test in Escherichia coli WP2uvrA, Salmonella typhimurium TA98, TA100, TA1535 and TA1537, chromosome aberration test using Chinese Hamster Lung cell and micronucleus test using ICR mice) of EO. Increased liver weight in both sexes at 2,000 mg/kg (P<0.01), decreased alanine aminotransferase in males at 222, 667 and 2,000 mg/kg (P<0.01), in females at 667 and 2,000 mg/kg (P<0.01 and P<0.05, respectively), decreased total cholesterol in males at 667 and 2,000 mg/kg (P<0.01), in females at 222, 667 and 2,000 mg/kg (P<0.05, P<0.01 and P<0.01, respectively), and decreased glucose in females at 222, 667 and 2,000 mg/kg (P<0.05, P<0.01 and P<0.05, respectively) were relevant. The changes were not associated with histopathological alterations. Thus, the no-obsevered-adverse-effect level (NOAEL) of EO in F344 rats is considered to be greater than 2,000 mg/kg. For genotoxicity study, EO did not show the mutagenic potential, chromosome aberration and micronucleus formation.
Key word: Evodia officinalis, F344 rat, genotoxicity, repeated oral dose toxicity

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Spontaneously occurring proliferative lesions of the accessory sex gland of the male rat are infrequent, but atypical hyperplasia of the prostate which has a various incidence in many rat strains. In rodents, the ampullary glands were embedded to locate in the prostatic complex. Although spontaneous atypical hyperplastic lesions of the ampullary gland were observed, the adenoma and adenocarcinoma of this gland have not been described. The present study described atypical hyperplasias, adenomas and adenocarcinomas in bilateral ampullary glands in 52-week-old intact male Sprague-Dawley rat housed as part of a control group in toxicological experiments. At necropsy, the prostate was normal-seeming and showed similar body weight to same group rats. A gross recognized prostate was measured weight 2.75 g. Histopathologically, the most frequent changes were observed in epithelial sites as prostatic intraepithelial neoplasias, adenomas, and microinvasive adenocarciomas, but vascular invasion was not observed. Other part of prostatic complex including sminal visicle and coagulating gland did not revealed proliferative lesions.

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As the urethra is the main place of entry for sexually transmitted pathogens, histopathologic evaluation of the urethral prostate complex is an important component of drug safety assessment and evaluation environmental toxicants. In rodent, the prostate complex is designated as prostate: ventral, lateral, dorsal lobes; and coagulating gland, ampullary glands, and similar vesicle. Aumuller et al., (2012) recently described the prostatic urogenital duct system of male rat, but there are still some controversial. The ejaculatory sinus had been described to compose binding the ampullary duct and the seminal vesicle duct, and many ducts of prostatic lobs opened at this sinus. While the present study revealed that the seminal vesicle duct drained into the ampullary duct at quite nearly position at the seminal colliculus with the slit like opening into urethra, and no prostatic lobes duct drain at these structures. While the seminal vesicle duct drained independently into the ampullary duct, and the coagulating glands duct independently opened into the urethra. As histology and distribution of the ampullary gland was similar to that of prostate lobes, it should be carefully investigated the ampullary gland when the prostate complex evaluation.

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The purpose of this study is to collect and summarize historical control data for Embryo-fetal development studies using Sprague-Dawley rats (Crl:CD(SD)), laboratory animals having a uniform quality from Charles River Inc. The background data were obtained from pregnant Sprague-Dawley rats in the control groups of our previous toxicity studies. The data includes reproductive and developmental parameters associated with fetus after caesarean section and fetal external, skeletal and visceral alterations, etc. These background control data will contribute to evaluation of reproductive and developmental toxicity studies especially embryo-fetal studies for rodents.

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[in Japanese]

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This study presents comparison of the immunosuppressive effects of Azathioprine (AZA) and Cyclophosphamide (CPM) in CD-1^® mice and to generate positive control data. The mice were treated with the AZAat the dose level of 20 and 50 mg/kg b. wt. and CPM at the dose level of 20mg/kg b. wt.for 4 consecutive days. Control group animals were treated with RO water. All the animals showed no clinical signs throughout observation period of experiment. Blood samples were collected at termination and analysed for Lymphocytes’ subpopulations and NK cells by FACSVerse^TM. Iimmunosuppressive effects on T cells (CD3⁺, CD4 and CD8), B cells (CD19, CD45) and natural killer cells (CD3^-, CD16) were significant in male and female mice treated with CPM at the dose level of 20 mg/kg b. wt and AZA at 50 mg/kg b. wt. The effects in both the groups were identical. Based on these results, it could be concluded that CPM at 20 and AZA at 50 mg/kg b. wt. found to have similar immunosuppressive effects in CD-1^®mice. Female mice could be considered more sensitive than male as both helper and cytotoxic T cells (CD4^- and CD8) suppressed by CPM at 20 and AZA at 50 mg/kg b. wt. However, further experimental evaluations should confirm the findings in respect of sex sensitivity. Results of this study will be helpful for comparative assessment of lymphocytic enumeration in the future studies.
Keywords: FACSVerse^TM (Fluorescence Activated Cell Sorting), Cluster of Differentiation (CD) cells

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In vitro percutaneous absorption studies offer a valid alternative for in vivo studies. Therefore present study was conducted to evaluate dermal absorption of benzoic acid and caffeine through human and rat skin. Benzoic acid and caffeine representing different physico-chemical properties were evaluated in these experiments. Two replicates each of human and rat skin was exposed to benzoic acid and caffeine (4 mg/mL). Split-thickness skin discs were placed in flow-through diffusion cells with 0.64-cm² exposure area and exposed at 32±1ºC, at ambient humidity. The exposure time was 8h with post-exposure sampling for 16h. Mass balance analysis was conducted for samples of receptor fluid, the residues remaining in/on the skin and in the stratum corneum (at 24h) by using Liquid Scintillation Counter. The mean total recovery of benzoic acid was 95.63±4.74% in human skin, and 96.18±2.99% in rat skin. The mean cumulative absorption of benzoic acid into the receptor fluid after 24 h was 12.23 µg/cm², i.e.32.70% of the dose applied in human skin and 19.30 µg/cm², i.e.51.60% in rat skin. The mean maximal flux was 1.288 µg/cm²/h in human skin and 3.019 µg/cm²/h in rat skin. The mean total recovery of caffeine was 94.02±0.94% in human skin, and 87.49±8.16% in rat skin. The mean cumulative absorption of caffeine into the receptor fluid after 24 h was 3.48 µg/cm², i.e.9.58% of the dose applied in human skin and 6.60 µg/cm², i.e.18.16% in rat skin. The mean maximal flux was 0.948 µg/cm²/h in human skin and 0.894 µg/cm²/h in rat skin.
Keywords: Benzoic acid, Caffeine, dermal absorption, in vitro percutaneous penetration

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