Study On Effects Of Gestation Exposure To Tributylyin Chloride On Developmental Toxioligy, Sex Hormone Level In Female Offspring Rats And Their Primary Mechanism

Tributyltin(TBT),for its long-term biocidal effect for a wide range of halobios, has been extensively used for the hull coating addictives to prevent the hull organisms' attachment and accretion.However,with organic tin compounds entering the ocean environment,they have also affected many non-target organisms,posing a tremendous threat to the marine ecosystem and function;therefore they have been banned by many countries.And due to the difficulty of TBT's degradation in the environment,it still exists in the seawater,especially in the sediment even after a quite long time of withdrawal,and would consequently disserve the people through marine biological functions such as its amplification and enrichment.TBT's current research has been focused on the study of the neurotoxicity,immunotoxicity, embryotoxicity,organ toxicity and the effect to male reproductive system,while little has been concerned about its adverse effects on female off-springs.This study is to explore the effects of TBTC which were exposed to offspring rats in the key duration of gender differentiation in the fetal rats in order to provide experimental information and reference for the possible impacts on the human offspring made by the TBTC prenatal exposure.Objective:To explore the endocrine disrupting effects and possible mechanisms made by the TBTC exposure on the offspring of female Wistar rats in the key duration of gender differentiation of fetal rats.Methods:In the critical duration of the fetal rats' gender differentiation(12-19 days of gestation),the oral gavage for the pregnant Wistar rats was with TBTC solution of different concentrations(0,1,2.5,5 mg/kg,ten pregnant rats in each group).At 20^thof duration of pregnancy five pregnant rats were randomly chosen in each group and killed.Abdominal to remove foetus,whose body weight,body length were measured.It was observed whether there were external deformities;by measuring the anogenital distance of fetal rats,it was the impact of TBTC on the development of fetal rats was assessed and the male and female ratio was judged.The alizarin red dye staining was applied to observe the bone development situation.After the birth-giving of other pregnant rats,kept record of the number born,and after the second day(PND2)of the offspring's birth,their body weight,body length,tail length and anogenital distance were measured.Offspring PND21 were weaned and offspring were separated into different cages according to gender.The body weights of female offspring were measured and their food consumption was recorded once a week.Ten were selected randomly within each group at PND 70.All were decapitaed after weighting to obtain blood with the purpose of measuring serum LH,FSH,T and E₂ level.Uterus,liver,kidneys and ovaries on both sides were all separated and weighed and the organ coefficient was calculated.Parts of liver,kidney,uterus and ovary were fixed in neutral formaldehyde buffer(4%formaldehyde),and would be made paraffin section for observation of histopathological changes.The remaining organizations were restored at -80℃.The RT-PCR method was applied to examine the impact of offspring rats' ovarian aromatase gene expression situation.Result:The body weight,body length of fetal rats in the 2.5,5 mg/kg group has significantly decreased compared with the control group at GD20.The male and female ratio has not changed greatly,and the TBTC exposure in the critical period of fetal rats' gender differentiation didn't lead in the external deformity and no obvious defects was shown in their bone development.2.5,5 mg/kg TBTC exposure leaded to the NAGD's significant increase of the female rat offspring compared with the corn oil control group,suggesting that the TBTC exposure might play a role of females androgenization in the fetal rats.After the fetal rats were newly born,the observation of the average litter size and the ratio between male and female in each group of pregnant rats showed no obvious difference compared with the control group.No anisotrophy was observed in newly born rat,body weights,body lengths and tail lengths of which are significantly different from control group,and a dose response relationship exists;For NAGD,significant difference only exists between 2.5mg/kg group and control group.The results mentioned above suggest that exposure of TBTC in the critical period of fetal gender differentiation bears embryo toxicity,but shows no obvious teratogenic property,and may lead to the female fetal rats' androgenization.Comparing with control group,body weight gain from PND 21 to PND 70 and utilization rate of food of female off-spring rats are significantly lower in treatment groups,which suggests the possible developmental toxicity of low dose TBTC exposure in GD 12-19.The difference of viscera coefficient between treatment groups and control group is not statistically significant.Pathologic sections of all viscera show no evident morphologic changes,which suggest that organic toxicity of TBTC exposure in GD12-19 is not enough to cause organ hyperplasy or morphologic changes.The measurement results of sex related hormones of mature female offspring rats suggest levels of follicle stimulating hormone and estradiol at 5mg/kg group are significantly higher than control group,and levels of testosterone in 2.5mg/kg and 5mg/kg groups are significantly lower than control group.These results suggest possible effects of low dose TBTC exposure in the middle and late period of duration of pregnancy on filial endocrine system.While the further results of RT-PCR for P450_aromshow no abnormal expression of related genes,so the cause of changes of sex hormone levles needs to be further studied.Conclusions:Low dose TBTC exposure(≤5mg/kg)in the critical period of fetal gender differentiation bears embryo toxicity,but shows no obvious teratogenic property,and may lead to the female fetal rats' androgenization tendency.The results show that the TBTC treatment may have endocrine disrupting effect under the circumstances of causing no hormone-sensitive organs' pathological changes. Through the exploration of its mechanism,it's found that TBTC has effect on the hypothalamus-pituitary-sex gland axis but no significant impact on the aroma of the p450stoma's gene expression.