To evaluate the molecular mechanisms underlying reproductive and developmental neurotoxicity, the regulation of genes, proteins, and miRNAs at global scale in vivo and in vitro was analyzed. The studies consist of three parts for reproductive and developmental neurotoxicity as follows; I) reproductive toxicity induced by reference chemical, 1,3-dinitrobenzene, II) reproductive toxicity induced by endocrine disrupting chemical, nonlyphenol, and III) developmental neurotoxicity induced by silver nanoparticles.
Part I. Genomic and proteomic analyses of 1,3-dinitrobenzene-induced testicular toxicity in Sprague-Dawley rats
1,3-dinitrobenzene (DNB) is an industrial intermediate and testicular toxicant that has been shown to target Sertoli cells. The mechanism of action of DNB in the testis, however, is un-clear. To investigate global alterations in gene or protein expression during testicular toxicity, testes from rats treated orally with DNB were subjected to microarray and two-dimensional gel electrophoresis (2-DE) analyses. Histopathological abnormalities were detected in the tes-tes of the DNB-treated rats. Microarray analysis revealed that, during early testicular toxicity, several genes involved in apoptosis, germ cell/Sertoli cell junction, and tight junction signaling pathways were differentially expressed. Based on 2-DE analysis, 36 protein spots showing significantly different expression during early testicular toxicity were selected and identified. Network analysis of the identified proteins revealed that these proteins are associated with cellular development or reproductive system diseases. Collectively, these data will help clarify the molecular mechanism underlying testicular toxicity in DNB-exposed rats.
Part II. miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol
It is known that some environmental chemicals affect the human endocrine system and the harmful effects of endocrine disrupting chemical (EDC) including nonylphenol (NP) have been studied. In the present study, the expression of miRNAs and their target genes in mouse Sertoli TM4 cells using microarray experiments was analyzed to better understand NP’s ac-tions on the reproductive system. Genes and miRNAs that were differentially expressed in NP-treated cells were identified and their functions were analyzed through Gene Ontology analysis. Network analysis of deregulated miRNAs suggested that Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNAs in NP-treated TM4 cells. Levels of expressions of the miRNAs $miR-135a^{\ast}$ and miR-199a-5p were validated by qRT-PCR. Finally, $miR-135a^{\ast}$ target gene analysis showed that the generation of reactive oxygen species following exposure to NP exposure may be mediated by $miR-135a^{\ast}$ through regulation of the Wnt/ $\beta$ -catenin signaling pathway. Collectively, these data help to determine NP’s actions on mouse TM4 Sertoli cells and increase the understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system.
Part III. Developmental neurotoxic effects of silver nanoparticles in human embryonic stem cells-derived neural stem/progenitor cells
Nanoparticles are being used increasingly in many fields including diagnosis, imaging and drug delivery. Together with these increases, there is increasing concern about adverse effects of nanoparticles on biological system. The recent investigations have reported that the silver nanoparticles (Ag NPs) were founded to be distributed in the brain or fetus by crossing brain-blood barrier and placenta. However, little is known about potential development neurotoxic effects of Ag NPs. Human embryonic stem cells (hESCs) provide new prospects to investigate the developmental neurotoxic effects in human. The successful derivations of neural stem/progenitor cells (NPCs) from hESCs allow testing the developmental neurotoxic effects of neurotoxicants. Here hESCs-derived NPCs which exhibit neurogenesis was derived and then the cytotoxic effects of Ag NPs were examined in hESCs-derived NPCs. The results showed that Ag NPs evoke the significant toxicity in hESCs-derived NPCs. The toxicity of Ag NPs was further evaluated using cell cycles, apoptosis, and oxidative stress and cell cycle analysis. To evaluate the molecular regulations at transcriptional and post-transcriptional level, genes and miRNAs profiling were analyzed in hESCs-derived NPCs after Ag NPs exposure. The results showed that Ag NPs induce the oxidative stress and following cytotoxic effects on hESCs-derived NPCs. Comprehensive gene and miRNA profiling suggests the molecular mechanisms underlying nanotoxicity induced by Ag NPs.
본 연구는 최근 사회적 문제로 야기되고 있는 환경 유해물질에 대한 남성계 생식독성 및 나노물질에 대한 발생 신경 독성에 대한 작용 기전에 관한 것으로, 독성유전체기술을 이용하여 독성 유발에 의해 변화하는 유전자, 단백질 및 miRNA를 비교 분석함으로써 독성물질의 작용 기전을 제시하고자 한다. 최근 독성학 분야에서는 독성유발에 대한 현상학적인 판정뿐만 아니라, 독성이 유발되는 작용기전에 대한 제시가 꾸준히 요구되고 있다. 독성 유발시 주요 표적장기인 간, 신장, 폐 및 심장독성에 대한 연구는 최근까지 활발히 진행되어 왔으나. 생식 및 발생신경독성에 대한 이해는 여전히 미비한 상황이다. 지금까지 독성연구는 동물모델을 이용하여 평가되어 왔으나 종간 상이성으로 인해 인체의 독성 예측에는 많은 한계점이 보고되고 있다. 독성 유발 작용 기전을 이해하기 위해, 본 연구에서는 동물 모델, 동물 유래 세포주 및 인간배아줄기세포 유래 신경전구세포를 이용하여 독성을 평가하고 이에 작용기전을 분석하였다. 본 연구는 아래와 같이 3 개의 주제로 구성되어 있다. 첫째, 남성계 생식독성 평가를 위해 대표적인 양성대조물질로 알려져 있는 1,3-dinitrobenzene을 랫드 모델에 노출 시킨 후 생식계 조직에서의 독성 유발 유무를 평가하고 microarray 및 2D 분석을 통해 생식독성에 관여하는 분자기전을 분석하였다. 이를 통해, 초기 생식독성 유발시 apoptotic cell death 및 Sertoli/germ cell junction이 주요 분자기전으로 선별되었으며, 생식독성이 진행됨에 따라 protein ubiquitination의 기능이 저하됨을 확인하고 이에 대한 주요 분자지표를 제시하였다. 둘째, 내분비장애물질로 알려져 있는 nonylphenol의 생식계에 미치는 영향을 평가하기 위해서, 마우스 Sertoli 세포주를 이용하여 세포 독성을 평가하고 유전자의 발현변화 및 이에 상응하는 miRNA 발현을 비교 분석하였다. 이를 통해, Wnt/ $\beta$ -catenin signaling의 조절에 관여하는 $miR-135a^{\ast}$ 의 발현이 유의적으로 변화함을 확인하였으며, Sertoli 세포의 독성 유발에 관여하는 주요 분자기전 및 이에 대한 주요 분자지표를 제시하였다. 셋째, 나노물질의 인체내 신경계 발생 단계의 이상을 평가하기 위해 인간배아줄기세포 유래 신경 전구세포를 분화시키고 이를 이용하여 은나노물질에 의한 세포독성 및 세포 주기, 자가 세포 사멸 (apoptotic cell death) 및 산화스트레스 유발과 같은 주요 독성 기전을 분석하였다. 또한, microarray 분석을 통해 인간배아줄기세포 유래 신경전구세포에서 은나노물질 노출에 의해 변화하는 유전자 및 miRNA의 발현을 비교 분석하였다. 이를 통해, 은나노물질의 노출에 의해 산화스트레스 관련 유전자 및 miRNA가 유의적으로 변화함을 확인하였으며, 이에 대한 주요 분자지표를 제시하였다. 최종적으로, 본 연구를 통하여 생식독성물질 및 은나노물질에 대한 생체내 분자의 발현 양상을 통합 분석함으로써 환경 유해물질 및 나노물질에 의해 생체내 생식독성 및 발생신경독성에 관여하는 독성 기전을 제시하였으며, 이러한 분자기전 정보는 향후 인체내 독성 평가 및 예측 기술에 활용할 수 있을 것으로 기대된다.