| Objects:Using the model of zerbafish embryos, assess whether acrylamide (ACR) can causeteratogenesis and study the molecular mechanisms.Methods:(1) The zebrafish embyos were exposed in various concentrations of ACR. Retinoid acidand 0.1%DMSO was treated as positive and negative controls, respectively. Teratogenicitywas assessed through microscopy obsevation and statistics analysis of teratogenic index(TI). The following developmental endpoints were observed and scored at 48 and 96hpf:heart rats, circulation, numbers of red blood cell, heart edema, trunk edema, hemorrhage,ventricle swelling, brain necrosis, jaw malformation, abnormal caudal region andmotility. Motor neurons and proliferating of neurons were assessed by anti-acetylatedtubulin (a-AT) and proliferating cell nuclear antigen (PCNA) through immunostaining,respectively.(2) Different expression profiles induced by ACR were analyzed by Affymetrix microarray;Using annotation and statistics analysis, searched the key genes in the process ofteratogenesis; K-means cluster analyzed the expression pattern of different gene inducedby ACR; In order to predict the molecular mechanisms of teratogenesis induced by ACR,Gene Ontology enrichment was performed to evaluate gene function, and pathway analysiswas performaed to study the interactions of ACR induced genes.(3) In situ hybridization was performed to predict the candidate miRNAs in theteratogenesis induced by ACR. The significant role of the interaction between miRNAsand their targets in the the teratogenesis was predicted by Bioinformatics analysis.(4) To identify the primary function of miR-124, approaches include over expression and knock down were used in the develop embryos. Developmental malformation, the status ofneuron proliferation and the function of motor neurons were evaluated for the effectscaused by miR-124. The significant targets genes of miR-124 were screened out throughbioinformatics analysis to investigate the molecular mechanism in abnormal development.Results:(1) Zebrafish embryos were exposed in ACR with various concentration, the embryos weredied immediately in 25-50mmol/L ACR, teratogenic in multiple system including: shorterlength, abnormal somite, no heart or smaller heart in15mmol/L ACR, and the embryoswith reduced skull, delayed pectoral fin, twist tail and decreased somite were in 5-10mmol/LACR. There were significant teratogenicity in ACR treated embryos observed at48hpf and 96hpf but not 24hpf. At 48hpf, the TIs of ACR and RA was 4.4 and 11.3,respectively. While at 96hpf, the TIs were 3.6 and 8.5, respectively. Immunostainingshowed that PCNA in ACR treated embryos were more expressed than untreated embryos,while there were not significant alteration observed in RA treated embryos. Theexpression of a-AT in ACR treated embryos were not significant different compared withuntreated embryos. While the expression level and position of a-AT in RA treated embryoswere reduced and altered.(2)There were 77 up-regulated genes and 80 down-regulated genes in ACR treatedembryos compared with untreated embryos through Affymetrix expression chip. By GeneOntology enrichment analysis, we found that the primary function of up regulated geneswere cell differential and cell organization, etc. While the primary function of down-regulated genes were organ development and cellular physiological process, etc. By GCOSstatistics analysis and annotation a lot of candidate genes in teratogenesis were screen outincluding: mbtps, nkx2.5, tbx16, zar1, ttn, dcl, hsp70, ache, itgb4, glra4a, bcor and nol5a.Analyzed by K-means and GO enrichment, the genes with same time series pattern hadsimilar function, and the function of the significant changed genes were organdevelopment and cell commutation, etc. Through pathway analysis, it was found that genesinduced by ACR were taken in Wnt, Notch and Tgf-βpathway.(3) In situ hybridization used LNA modified probes showed that the expression of miRNAs in nerves system, cartilage tissues and bony tissue including: miR-124, miR-125b, miR-9, miR-21, miR-140, miR-206 were increased in different degree compare withthe untreated embryos, and the miR-1 and miR-133a were decreased in the cardiovasculartissues. While in the embryos treated with RA, miRNAs including: miR-1, miR-133a,miR-21, miR-124 were increased, miR-140 and miR-206 were decreased. Throughbioinformatics analysis, we found that partial targets of miR-140 including lmnb2,LOC554905, nr2f11, rnf11, siah1, zgc:92014, zgc:92331, mbtps2 and fgf20 were downregulated in the ACR treated embryos. But the expressions of miR-124 targets genes werevariance.(4) Through over expression of mature miR-124 in developed zebrafish embryos, showedshorten length, twist tail and abnormal brain. While knock down of miR-124 were not anysignificant abnormal observed. By bioinformatics analysis, between development stageZygote to Pharyngula the targets of miR-124 were atplb3a, fn1, atp6v1ba, jag2, zgc:85956,zic1and zic2a expressed in spinal cord and were: atp1b3a, atp6v1ba, jag2, mid1ip1 andotx2 in hindbrainConclusion:(1) ACR is a kind of teratogenic chemicals to zebrafish embryos, and the effect is dosesdependent. In high concentration exposure, the embryos were mostly appeared as death.While in lower concentration the embryos were teratogenic. Furthermore, the lowerconcentration of ACR primarily affected the morphogenetic processes of nerves system inlate development stage. It can be suggested that metabolic residue of ACR affects theneuron proliferation and differentiation through repress cell cycle.(2) The molecular mechanisms of ACR induced teratogenicity were include disrupted theWnt, Notch and Tgf-βpathway, and altered the key genes' expression such as mbtps,nkx2.5and tbx16 etc. in develop process.(3) The altered expression of miRNAs in nerves and cardiovascular system is an importantmolecular affair in teratogenesis induced by ACR. And the interaction of miRNAs andtheir targets probably play a role in the mechanisms of teratogenicity.(4) Over expression of miR-124 can cause ventral spinal cord and hindbrain with abnormal differentiated in zebrafish embryos. The mechanism was probably related to disrupt Notchand Wnt pathway. |
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