| Silver nanoparticles(AgNPs) have been widely used in human industrial and commercial applications due to its specific antibacterial properties,but the research of silver nanoparticles’ safety assessment is far from enough.Recently,AgNPs have been reported to inhibit specification of erythroid cells and to induce spinal cord deformities and cardiac arrhythmia in vertebrates,but have not been implicated in development of neural crest(NC)and pigment cells in an in vivo model yet.In this work,zebrafish(Danio rerio)was chosen as study subject.First,microarray and bioinformatics were used to screen out the down-regulated genes related to chromogenesis in zebrafish embryos.Next,the results were verified by quantitative real time polymerase chain reaction(qRT-PCR)and whole-mount in situ hybridization(WISH).Then,in order to find the molecular mechanisms underlying AgNPs induced hypopigmentation in zebrafish embryos,melanogenesis agonists palmitic acid and dibutyryl cyclic AMP(dbcAMP)were used to rescue the down-regulated expressions of chromatogenesis genes in AgNPs-exposed embryos,and Ag+ chelating and AgNPs coating compound L-cysteine was used to distinguish the effect of Ag+ and AgNPs.The main results are as follows:1.AgNPs induced hypopigmentation in zebrafish embryosReduced pigmentation was observed in AgNPs-exposed embryos compared to embryos exposed to supernatant and to wild-type embryos,and obviously reduced melanophores and slightly reduced xanthophores were observed in AgNPs-exposed embryos.2.AgNPs suppressed transcription of melanophore and xanthophore genesThere are sixteen down-regulated genes related to chromogenesis have been selected by microarray and bioinformatics analysis.Then,melanophore genes mitfa,tyrp1 b,oca2 and dct,xanthophore genes gch2 and aox5,and iridophore gene csf1 b were tested further in AgNPs-treated embryos by qRT-PCR detections.Compared to the controls,expressions of melanophore genes mitfa,tyrp1 b,oca2,gch2,aox5,and dct were all down-regulated significantly both in AgNPs-exposed embryos and in Ag+-exposedembryos.However,AgNPs and Ag+ elevated expression of gene csf1 b significantly compared to its expression in WT control.Moreover,WISH was used to test the transcription levels and distributions of the above mentioned genes in AgNPs-exposed embryos.Melanophore genes dct and tyrp1 b exhibited obviously reduced expressions in embryos from AgNPs-exposed groups and from Ag+-exposed groups.3.AgNPs impacted zebrafish NC development and pigmentation mostly by their releasing Ag+Effects of different concentrated palmitic acid or dbcAMP on expressions of chromotogenesis genes in embryos were firstly tested,and 10 μM dbcAMP and 1 μM palmitic acid were used in the following co-exposure experiments in this study.Expressions of genes crestin,mitfa,dct,and gch2 were suppressed significantly in AgNPs-treated embryos,however,their expressions were recovered to normal level or even higher than normal level after being co-exposed to dbcAMP or to palmitic acid.In order to distinguish the effect of Ag+ and AgNPs,we next detected expressions of chromatophore genes in embryos co-exposed to AgNPs and L-cysteine which was used in chelating Ag+and coating AgNPs for times.Obviously reduced melanophores and xanthophores were observed in AgNPs-exposed embryos,however,xanthophores and melanophores came back again in embryos which were co-treated with AgNPs and L-cysteine.In addition,recovered expressions of dct and gch2 were also observed in embryos co-treated with AgNPs and L-cysteine compared to their expressions in AgNPs-exposed embryos by WISH detections.According to all of the above observations,we speculated that AgNPs suppressed chromotogenesis from NC formation stage mostly by their releasing Ag+,and specifically suppressed specification and differentiation of NC derived melanophores and xanthophores.In addition,AgNPs might act up-stream of mitfa and pax7 in genetic cascade to regulate melanogenesis and xanthogenesis respectively. |
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