(A) study on toxicological evaluation and molecular mechanism underlying reproductive and developmental neurotoxicity using toxicogenomic analysis

Abstract

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.