Symposia
Novel knowledge and analysis for developmental neurotoxicity／遅発性神経毒性研究の新展開 |
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| 1S3-1
Development of developmental neurotoxicity testing using human iPS cells
Yasunari Kanda
Div Pharmacol, NIHS
Environmental chemicals have potential effects on children's developmental neurotoxicity (DNT), leading to neurobehavioral outcomes such as attention deficit hyperactivity disorder and autism. Current DNT guideline (OECD TG 426) requires a lot of animals and costs and large numbers of chemicals have been evaluated with no risk-based criteria. Thus, it is necessary to establish an in vitro DNT method with more accurate and better predictability. Human iPS cells (iPSCs) are expected to provide a novel platform as human cell-based assay systems to develop alternative in vitro testing approaches that are rapid and efficient. Here we report a role of mitochondrial dynamics in developmental neural toxicity (DNT) of chemicals using iPSCs. We have focused on neural differentiation process in human iPS cells, using several positive compounds and negative compounds obtained from literature. Exposure to compounds inhibited neural differentiation and ATP levels in iPSCs. We next assessed the effects of compounds on mitochondrial dynamics. Staining with MitoTracker revealed that compounds induced mitochondrial fragmentation. Immunoblot analysis revealed that levels of Mfn were significantly reduced by compounds. In addition, knockdown of Mfn inhibited neural differentiation in iPSCs. In this symposium, I would like to discuss a role of mitochondrial dynamics in iPSCs and future perspectives for iPSC-based DNT testing. | | 1S3-2
Structural and functional alteration of developing rat cerebellum by administration of autism-inducing developing neurotoxins
Sachiko Yoshida
Dept Environ Life Sci, Toyohashi Univ Tech
Background Autism, a severe neurodevelopmental disorder, becomes increased in young age. Several chemicals are known to play some roles in the onset of autism. Especially, reduction in size and number of Purkinje cells in the cerebellum is revealed in both the human postmortem studies and some drug- administrated adult animals. In this study, we investigated cerebellar cytological changes in developing rat with some drug administrations. Methods Valproate (VPA), an antiepileptic drug, and Chlorpyrifos (CPF), an organophosphorus agent, are known as the candidates of inducer of autism. Because VPA is also known as a HDAC inhibitor, the effects of other HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA) and MS-275, were also investigated. Each drug was administrated to embryonic day 14 or 16 in a single dose p.o. with VPA; 600mg/kg, MS-275; 4mg/kg, or CPF; 4.3mg/kg of mother weight, respectively, or i.p. with SAHA; 50mg/kg of mother weight. Results In VPA-administrated rat, we have observed the elongation of Purkinje cell dendrites started earlier than control and reached all over the molecular layer even in P12. It was observed also in SAHA, or CPF administrated rat, while in MS-275 administrated rats, it was not. After the facilitation of Purkinje cell development in these drugs in the first week, excess folding in cerebellar lobules, especially between lobele V and VII (primary fissure), was formed in the second week. This excess folding was maintained for long period with elimination of some Purkinje cells.Conclusions Some autism-inducing drugs changed developing cerebellar structures and functions. We suggest that drug-induced autistic model rat would become useful to evaluate the developmental neurotoxicity of a drug. | | 1S3-3
Developmental neurotoxicity evaluation: Studies on neural network activity at synaptogenic period.
Susumu Ueno1，Yukiko Fueta2
1Dept Occup Toxicol, Inst Ind Ecol Sci, Univ Occup Environ Hlth, JPN，2Dept Environ Manag Cont, Sch Hlth Sci, Univ Occup Environ Hlth, JPN
To evaluate developmental neurotoxicity (DNT) of chemicals before the appearance of neurobehavioral changes in adolescent period, we applied electrophysiological approaches, using hippocampal slices obtained from rat pups prenatally exposed to chemicals of interest, at synaptogenic period (from PND13 to PND18). We studied stimulation/response (S/R) relationships of the slope of field excitatory postsynaptic potential (fEPSP) and the amplitude of population spike (PS) evoked in the CA1 area, and found that S/R relationship was suddenly enhanced between PND15 and PND16 (the day of eye-opening). Involvement of ambient GABA, which is known to tonically inhibit intrinsic neural excitability, was additionally examined by analyzing effects of bicuculline methiodide (BMI), a GABAA receptor antagonist. We found that effects of BMI on PS amplitudes developmentally altered, suggesting a possible change in ambient GABA involvement in neural excitability around the eye-opening day.Taking these results, we evaluated valproic acid (VPA), known as a developmental neurotoxicant, and 1-bromopropane (1BP), an industrial solvent whose DNT remains unclear. In prenatally VPA- and 1BP-exposed pups, S/R relationships of fEPSP and PS gradually enhanced at PNDs14-15 and PND14, respectively, which was before the day of eye-opening. Moreover, developmental change in the effect of BMI on PS amplitudes was abolished in VPA-exposed pups. These results indicate that VPA and 1BP may hasten the developmental change in hippocampal excitability at synaptogenic period. Our electrophysiological approaches using hippocampal slices obtained from prenatally chemical-exposed rats at synaptogenic period may be useful for earlier prediction of DNT of chemicals. | | 1S3-4
DOHaD theory in human cases: Inheritable epigenetic changes caused by environmental factors
Kenichiro Hata
Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo
Inappropriate conditions in utero could result in communicable diseases in adulthood. These phenomena are recognized as developmental origin of health and disease (DOHaD), which is defined as undesirable environmental factors that act during fertilization as well as fetal and early childhood stages that could affect epigenetic profiles. Some of these changes could result in predispositions to diseases that interact with environmental factors after birth, resulting in the development of diseases.It is thought that the epigenome is a key player of molecular mechanisms underlying the DOHaD theory. Epigenetic information such as DNA methylation and histone modifications regulate gene functions, and it is especially important during early development and aberrant epigenome causes diseases. These chemical modifications are stable, and since daughter cells inherit the modification patterns from the parent cells, they are necessary for homeostasis of life. Once aberrant epigenetic information is acquired, they are stably propagated and will continue to affect gene functions even in the absence of genetic mutations. It has been concerned that epigenetic changes via environmental factors occur during early stages of human development. Detecting epigenetic changes in human cases is not trivial because of various etiological factors and physiological epigenetic variations. Here, we focused on outliers in DNA methylation that were observed for each case. For example, even though babies are diagnosed to be normal, if their mothers are too thin or fat, the DNA methylation profiles of their placentas could be potentially disturbed. In this symposium, I will summarize topics in the DOHaD field and present our data for discussion. | | 1S3-5
ROLE OF PRIOR TRAUMA & IN-UTERO SABNYSTRESS ON EPIGENETIC REGULATION & AUTONOMIC FUNCTIONING AS EARLY PRECURSOR FOR DEVELOPMENTAL PSYCHOPATHOLOGY
Yoko Nomura
Queens College and Graduate Center, the City University of New York
Background: The prenatal environment prepares the developing fetus for conditions in the postnatal world. While animal models show that prenatal stress was associated with HPA-axis reactivity and anxiety behavior, less is known about how these findings translate to humans. Capitalizing on Superstorm Sandy disaster in 2012, the study examined the role of fetal programming on the placental gene expression, temperament, and autonomic functioning (ANF) in early childhood. Methods: A subsample of 310 mother-child dyads was drawn from the Stress in Pregnancy Study (SIP Study, PI Nomura). Stored placenta tissues were used to quantify the mRNA levels of the candidate genes. Timing of Sandy exposure in the index pregnancy constitutes a measure of objective stress, and perceived level of stress due to Sandy constitutes subjective stress. Children’ ANF was measured by the skin conductance response amplitude to the startle stimuli. Result: Both an objective and subjective measures of Sandy stress show independent effects on epigenetic dysregulations in affected genes related to stress modulation and autonomic nervous system. Furthermore, there is evidence for synergistic dysregulation in ANF and stress modulation. Specifically, children exposed to both objective and subjective Sandy stress had lowest gene expression of genes that regulate stress response and brain development of offspring in the intrauterine and postnatal environment, including DYRK1A, HSD11B2, MAOA, MAOB, SEMA3B, ABCB1, and ATP1A1. Conclusions: Results suggested that offspring of mothers exposed to Sandy in-utero who perceived the event to be highly stressful, had dysregulated placental gene expression and increased physiological reactivity to stress at an early age, which in turn influenced their temperament. | | | |
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