Studies Of Establishing Rabbit Post-implantation Whole Embryo Culture Model And Mouse Developmental Toxicity Of Three Nanomateirals In Vitro

The research of rodent whole embryo culture and its application for developmentaltoxicity had been for more than30years.Now it had been improved by ECVAM for itsconsistency with the results in vivo,and recommended to alternative method.Moreover,thedevelopment and applicantion of rabbit whole embryo culture delayed than the rodentgreatly, recently its method was brokenthrough.It was reported that the rabbit wholeembryo culture was more excellence in developmental toxicity research than rodent insome studies.It was required in the guide of developmental toxicitiy test for two kinds ofanimals,including rodent and non-rodent,and the rabbit was choosed for non-rodentfirstly.Thus,no matter developmental toxicity research or alternative method,the rabbitwhole embryo culture model had important perspective in application.It wasn’t reportedthe culture research in dostmetic,therefore the aim of the study was introducing andestablishing the model,and trying to explore the developmental toxicity of nanomaterials.Nanomaterials had special physical and chemical characters,wide applicationvalues,but its difference from the non-nano materials became problems for reason thatunknown health harm to producers and users.The developmental toxicity was oneimportant content in chemical safety evaluation or risk assessment.,and wheather the recentevaluation methods suited to nanomaterials was one problem.Thus the study choosed threerepresentatives of three kinds of nanomaterials（metal oxide,carbon nanotubes,organicChemicals）,and studied their developmental toxicity by mouse whole embryo culture,andcompared it of Nano-TiO₂with rabbit.The aim was to explore the toxicity characters ofnanomaterials, and provide scientific proof to decision if the recent method suited fornanomaterials.Our study had three parts.1. To establish the rabbit post-implantation whole embryo culture and validate itTo use NZW pregnant rabbit,get its GD9embryos.The method based on Carney andNaya’s and modificated a little, and the different place was cutting the uterus from middlewall to both side.To separate the embryos,culture one embryo in one bottle.Themorphology scoring system was Carney’s.Afer48h, compared the results in vitro to whichin reports or literatures.Then5.0mmol/L MAA was used for positive control to furthervalidate the results.The results showed that23GD9embryos100%survived in vitro,the average headlength was2.15times more than beginning,it was similar to literatures and reports in VYS closure reducing,flexion,somites,nature malformation rate, morphology score,and soon.The positive contol MAA can induce evident developmental toxicity,including reducingVYS closure,swelling head region,lacking face organ,disordering somites,unclosing caudalnerve tube,et al,the morphology scoring was much lower than the solvent control（P<0.05）.The malformation ratio was88.24%,them were same as literatures’.So wethought it was successful in establishing rabbit post-implantation whole embryo culture.2. The influence of three nanomaterials on development of mouse embryos in vitroTo separate GD8.5ICR embryos,and culture in64.0,160.0,400.0μg/mL Nano-TiO₂,Nano-PS, SWNCT for48h,observe the end point in dissecting microscope according toVan’s mouse morphology scoring system,study the influence of the three chemicals onmouse embryos.2.1Nano-TiO₂There was no evident influence observed on mouse VYS diameter, CRL,head length,and somites number in each dosage,but the malformation ratio had statistic meaning（P<0.05）, the more the concentration,the higher the malformation ratio,and it had cleardosage-reaction raltionship.Beginning at64.0μg/mL,the embryos showed developmentaltoxicity,referring to VYS blood circling,somites,heart,cadual nerve tube, midbrain,hindlimbs（P<0.05）,in middle dosage hindbrain was also effected, the morphology scoreswas lower than the solvent control（P<0.05）,in high dosage there were more effected organsincluding Auditory system and Smell system,and the total score was the lowest among thegroups（P<0.05）.It can be concluded that the higher the dosage,the more effected organs,andthe lower the morphology scores.2.2Nano-PSThere was no evident influence observed in mouse growth in64.0,160.0,400.0μg/mL60nm PS,the malformation ratio was higher than the control,but no statistic difference,each dosage group’s score was lower than solvent control’s,no statistic difference inmorphology scoring between groups,and the main effected organs were heart, brain,auditory system and hindlimb.2.3SWCNTsThere were evident effect in middle dosage, the somites were influenced and themorphology scoring was lowest, malformation ratio was clearly higher than other groups.The middle dosage induced many organs’ abnormality.The developmental toxicity mainlyshowed bad VYS blood circling, unfull flexion, disordering somites, heart filled with liquid or bad developmental otic, optic, branchial bars, lacking hindlimbs. The low andhigh dosages were no statistic difference compared to solvent control in growth and organdifferentiation.2.4Comparing with three nanomaterials’ developmental toxicity in mouseThe embryo developmental toxicity can be seen from the lowest dosage in Nano-TiO₂.and in the middle dosage of SWCNTs, and there were a few organs effected in Nano-PS inthe three dosage.The abnormality ratio was statistic different from the lowest dosage inNano-TiO₂, and showed dosage-reaction trend, SWCNTs had different ratio in middledosage, and no dosage-reaction trend, but worst abnormality.As the type of abnormality,Nano-TiO₂mainly effected VYS circulation,midbrain, heart,visual system and hind neuraltube,Nano-PS mainly effected heart, brain, auditory system and hindlimb, SWCNTs effected almostall the organs including brain, heart, facial organ, and hind neuraltube and so on.3. Comparing developmental toxicity of Nano-TiO₂in mouse and rabbitThere was no evident influence observed on rabbit length growth and somits number in160.0,400.0,1000.0μg/mL, the malformation ratio was higher than solvent control in160.0,400.0μg/mL, and had statistic difference, but no dosage-reaction trend. Themorphology score at all concentrations was lower than solvent control, no statisticdiiference. But the malformation ratio of mouse was statistic different comparing tosolvent control in all groups. As to the malformation and effected organ, they were silimarbetween the rabbit and the mouse, but more organs were effected in mouse.Through the experiment, we can finally get the conclusions below:1. We firstly introduced and successfully established the rabbit post-implantationwhole embryo culture model in domestic.2. Among the three nanomaterials, there were no influence to embryos’ survival, butNano-TiO₂can induce mouse developmental toxicity from64.0μg/mL,and showeddosage-reaction trend.SWCNTs showed toxicity in160.0μg/mL, no dosage-reactiontrend,but the malformation ratio and severe degree was more higher than Nano-TiO₂.Nano-PS only showed retarding effect at the three concentrations,its developmentaltoxicity should be validated further.3. Nano-TiO₂can induce developmental toxicity in rabbit and mouse,the effectcharacters were silimar,but the malformation ratio and the reactive dosage showed that themouse seemed more sensitivity than rabbit.