The Teratogenic Mechanisms Of Triphenyltin And RXR Antagonist UVI3003 In Xenopus Embryos

Retinoid X receptor(RXR)is one of the most important targets of pollutants and drugs.Previous studies suggest that the agonists of RXR lead to severe teratogenicity in embryos of amphibians.Unexpectedly,the phenotypes induced by triphenyltin(TPT,RXR agonist)are most different from those induced by 9-cis retinoic acid(9c-RA)and fluorobexarotene(FBA),while are very similar to those induced by UVI300(RXR antagonist).Therefore,we hypothesized that UV13003 was exerting its teratogenic effects through a mechanism similar to TPT(e.g.,through RXR-PPAR?),but perhaps not through RXR to induce malformations in Xenopus tropicalis embryos.In this study,X.tropicalis and X.laevis embryos were used as model species for the study of the teratogenic mechanisms of TPT and UVI3003.We exposed X.tropicalis embryos to UVI3003 in seven specific developmental windows and identified changes in gene expression.Exposure to UVI3003 during different developmental windows induced obviously developmental delay and multiple malformations.The most common phenotypes were reduced forehead,turbid eye lens and narrow fin in UVI3003 treatment groups.The teratogenic ability of UVI3003 was weak in NF 10-25 stages,while it was significantly increased in NF 25-39 and then decreased in NF 39-43.The expression of mRNAs encoding RXRs and their heterodimeric partners RARs;PPARs and TRs were evaluated after 7 different UVI3003-exposure windows.We found RAR? was down-regulated in early exposure periods,whereas RXRs,TRa and TR? were affected after late embryogenesis treatment.Those various changing of gene expression was correlated well with the multiple and stage specific phenotypes induced by UVlI3003.Unexpectedly,the expression of PPARy was clearly decreased during all the treatment periods.PPARy was also down-regulated after TPT treatment.Mmp1(matrix metallopeptidase 1),which is known to be inhibited by PPARy agonists,was significantly up-regulated by UVI3003 and TPT.The results indicate that PPARy is likely to play a critical role in inducing malformations inX.tropicalis embryos after exposure to UVI3003.Microarray analysis showed that seven genes(rps15,serp2,fmr1,cyp2e1,lrrc9,ugtla6 and LOC100490188)were differentially regulated in all three treatment groups after exposure of UVI3003 duing moderate window.The most significantly affected pathway was galactose metabolism,insulin signaling pathway and steroid biosynthesis.In sensitive window exposure groups,18 genes were differentially regulated,mainly comprising components of the PPAR signaling pathway,glutathione metabolism and nitrogen metabolism.In summary,the results suggest that UVI3003 is a teratogen to X.tropicalis embryos and that the teratogenicity is stage-specific.Gene expression profiles indicate that PPAR,rather than RA,pathways may be the underlying cause of the teratogenic effects observed.We further measured the ability of UVI3003 to activate Xenopus RXR?(xRXR?)and PPAR?(xPPAR?)in vitro and in vivo.We found that UVI3003 activated xPPAR?either in Cos7 cells(in vitro)or Xenopus embryos(in vivo).UVI3003 did not significantly activate human or mouse PPAR? in vitro;therefore,the activation of Xenopus PPAR? by UVI3003 is novel.The ability of UVI3003 to activate xPPAR?explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos.We documented for the first time that a well-known RXRs antagonist(UVI3003)could also activate xPPAR?.We explored the role of PPAR? in teratogenicity of TPT in X.tropicalis embryos.PPAR? overexpression resulted small eyes,which could be rescued by TPT treatment.PPAR? MO showed strong teratogenicity and the mortality rate was obviously increased after 20 ?g Sn/L TPT treatment.Wholemount in situ hybridization(WISH)revealed that the spatial expression of PPAR? was restricted in the neral tube and neural crest during neurula stage.The results indicate that the strong teratogenicity of PPAR? MO likely plays a critical role in the teratogenic process of TPT.We comparatively investigated the phenotypic and global gene expression changes in X.tropicalis embryos induced by three different agonists,including a RAR selective ligand(all-trans retinoic acid,at-RA),a RXR selective ligand(fluorobexarotene,FBA)and their common ligand(9-cis retinoic acid,9c-RA).All three agonists induced striking and similar malformations in X.tropicalis embryos.We further used a phenotypic based system to distinguish the differences in the malformations of embryos induced by the three agonists.The hierarchical clustering analysis suggested that malformations induced by 9c-RA separated from those by at-RA and FBA.Microarray analysis showed that RAR selective agonist at-RA primarily affected the retinol,glycolysis,starch and sucrose metabolisms while RXR selective agonist FBA led to disturbances in more wide-ranging pathways such as the adipocytokine,insulin,PPAR,FoxO signaling pathways,alanine,aspartate and glutamate metabolism,in addition to pathways that involved in energy homeostasis.The gene profile specificity of ligands for RAR and RXR was established though the corresponding phenotypic changes were very similar to each other.Our data indicate that not only the well-known RXR-RAR heterodimer but also PPAR signaling pathways play important roles in the control of metabolism with retinoid treatment in X.tropicalis embryos.In conclusion,we documented for the first time that a well-known RXR antagonist(UVI3003)could also activate xPPAR?.The ability of UVI3003 to activate xPPAR? explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos.We established a link between the response of PPAR? gene to TPT exposure and the phenotypic characteristics of ocular malformations.We screened out a large number of differentially expressed genes using microarray analysis,which provide a large amount of potential biomarkers for evaluation of RXR and RAR interfering activity.