Optimized Matching Of Positive Control Drug In Embryo-foetus Developmental Toxicity Tests

1.ObjectiveTo search a better positive control drug by comparing the malformation sensitivity of N,N'-methylene-bis-(α-amino-1,3,4-thiadiazole)(Bis-A-TDA) and cyclophosphamide(CP) on SD rats for Embryo-fetal development toxicity test.(Ⅱsection reproduction toxicity),of which we investigate the mechanism.2.MethodsHealthy adult female rats were choosed by 1:1 mated with male rat at 17:00 every afternoon,and the vagina embolism and vagina smear checked in next morning.Once the vagina embolism or sperm in vagina smear were detected,we determined they were mated successfully.60 female SD rats were divided into 6 groups at random which included two negative control group administrated with 0.5 %CMC intragastric or physiological saline by injected in the caudal vein respectively;two Bis-A-TDA experimental groups were administered with Bis-A-TDA 11.0 mg/kg or 13.8mg/kg;two CP experimental groups were injected with CP 7.2 mg/kg or 9.0mg/kg in the morning at 10^th of gestation.Pregnant rats were weighed per triduum from the first day of gestation,checked up corpus luteum, foetus weight(including uterus),placental weight,living foetus,dead foetus, absorbed foetus,foetus weight,crown rump length,appearance and skeletal malformation of foetus,and incidence of malformation on the day 20 of gestation. Collected pregnant rats' blood serum were used for mensuration of SOD,MDA,NO. 1/3 foetus were dealed with alizarin red stain for skeletal malformation examination, and 2/3 foetus were put into 4%Formaldehyde Solution after removing internal organs to make internal organs pathological section for myeleterosis examination. We detected the protein expression of PCNA of spinal cord of Bis-A-TDA experimental groups compared with control group by immunohistochemistry SABC methods.We detected the apoptotic cells and the protein expression of Bcl-2 and Bax in spinal cord of CP group compared with control group with immunohistochemistry SABC methods.3.ResultsThere was no statistics difference in skeletal deformity incidence between low-dose Bis-A-TDA group,high-dose Bis-A-TDA group(92.59%and 100%) and low-dose CP group,high-dose CP group(78.5%and 100%)(P＞0.05).The appearance deformation incidence of two Bis-A-TDA groups(84.62%and 86.84%) were much higher than which of two CP groups(2.6%and 0.0%)(P＜0.01).A variety of malformation was induced by Bis-A-TDA,like limbs microsoma,heart revealed,exomphalocele,abdominal cleft,spina bifida,tail malformation,anal atresia,cranial anostosis,rib deformity,vertebra absence,breast hypoevolutism,et al,but less malformation was appeared in CP experimental groups,which were mainly skeletal hypoevolutism.Under the electron microscope,82.35%(14/17) thoracic cord and 75%(12/16) isthmus cord pathological section of low-dose Bis-A-TDA group appeared changed, including central canal disappearance,offset,basal plate and alarpalate fuzziness, nerve cells obviously reduction,spinal cord seriously emarcid(P＜0.01),the same to the high-dose Bis-A-TDA group with observing 66.67%(4/6)thoracic cord and 100%(7/7)isthmus cord pathological section.There were no pathological changes in spinal cord of CP group.Apoptotic cells appeared typical morphological changes,spinal cord nerve cell caryosome minished,chromatin condensation,nuclear fragmentation,apoptotic body and apoptotic cells dissociated from peripheral cell.There was no difference among negative control CMC group,low-dose Bis-A-TDA group and high-dose Bis-A-TDA group in apoptotic index of thoracic cord and isthmus cord anterior horn cell.low-dose CP group was higher than negative control physiologic saline group in apoptotic index of thoracic cord and isthmus cord angulus anterior (P＜0.01).Calculating masculine cell showed that there was no difference between negative control CMC group and Bis-A-TDA group in PCNA expression(P＞0.05).Bcl-2 and Bax mainly expressed in anterior horn cell endochylema.Compared with negative control physiologic saline group,Bax expression in thoracic cord and isthmus cord anterior horn cell of low-dose CP group increased significantly(P＜0.01),and Bcl-2 expression in thoracic cord and isthmus cord anterior horn cell of low-dose CP group are lower(P＜0.05).MDA density in pregnant rats' blood serum had no difference between negative control physiologic saline group and low-dose CP group,but was higher in high-dose CP group(P＜0.01).Density of NO in pregnant rats' blood serum of low-dose CP group and high-dose CP group are both higher than negative control physiologic saline group(P＜0.05),activity of blood serum SOD in low-dose CP group is higher than which of negative control physiologic saline group,while is lower in high-dose CP group(P＜0.01).4.Conclusion1.Compared with cyclophosphamide,(Bis-A-TDA) was an excellent positive control drug on SD rats which posses high malformation sensitivity.It is the best positive control design proposal that rats were intragastric administered with Bis-A-TDA 11.0 mg/kg in the morning of 10^th of gestation2.We could state that Bis-A-TDA and CP may possibly induce malformation via different mechanism in the rats from the below viewpoints:the different methods and the different doses required at the same time of drug applieation, to produce malformation and the difference on anatomy and spina cord pathology.3.There maybe an internal relation between multi-target tissue defection and deformity of spinal cord.Under the certain condition,we found that Bis-A-TDA induced NTDs(neural tube defect) and Deformity of spinal cord was not by apoptosis.4.NTDs teratogenic dose of CP maybe between 7.2mg/kg and 9.0mg/kg.Excessively apoptotic nerve cells appearing in the fetal spina cord were closely related to the occurrence of the spina bifida malformation in fetal rats.5.The stronger expression of Bax in spina cord facilitates nerve cell apoptosis of the CP group,and the stronger expression of Bcl-2 in the normal spinal cord inhibiits the programmed cell death.The correlative genes Bcl-2 and Bax participate in the apoptosis process.6.That the density of MDA and NO in blood serum of CP groups were higher than control group accounted that oxidative damage played significant roles in teratogenic effect of CP.There were cause-effect relation among oxidation, neurons apoptosis and spina cord deformity.