| Tetrabromobisphenol A(TBBPA)and tetrachlorobisphenol A(TCBPA)are typical and persistent toxic environmental pollutants,which are mainly used as flame retardants and widely exist in various environment medium.TBBPA and TCBPA have been proved that both of them have potential developmental and reproductive toxicity to animals.TBBPA and TCBPA were set as target pollutants,the Rana nigromaculata tadpoles were the object,the effects of TCBPA and TBBPA exposure on external morphological parameters,development stage,thyroid hormone contents and thyroid hormone related gene expression of Rana nigromaculata tadpole were tested in this study;The male Rana nigromaculata were set as the object of study to study the effects of TCBPA and TBBPA exposure on the testis function,morphological damage,sex hormone contents,sex hormone synthesis related genes and the expression of sex hormone receptor genes in the testis of male Rana nigromaculata.Gosner 26 Rana nigromaculata tadpole were exposed to 100,250 ?g/L TBBPA and TCBPA for 7,14 and 21 days.The results of external morphological parameters and development stage of Rana nigromaculata tadpoles showed that:(1)Treatment with 250 ?g/L TBBPA or TCBPA for 21 days,tadpoles hind limb length and tail length were significantly reduced by 0.351 double and 0.346 double,0.110 double and 0.099 double,respectively.The growth of tadpoles were inhibited by the exposure of 250 ?g/ TBBPA and TCBPA for 21 days;(2)Treatment with 250 ?g/L TBBPA or TCBPA for 21 days,the average developmental stages of tadpoles reached Gosner 34,which were 2 developmental stages lower than that of the control group and delayed the development of tadpoles.Gosner 26 Rana nigromaculata tadpole were exposed to 100,250 ?g/L TBBPA and TCBPA for 7,14 and 21 days.The results of thyroid hormone contents and thyroid hormone related gene expression of Rana nigromaculata tadpole showed that:(1)Treatment with 250 ?g/L TBBPA or TCBPA for 21 days,the T4 contents of tadpole head were decreased by 0.45 double and 0.39 double,thereby TBBPA and TCBPA resulted in thyroid endocrine disruption to the Rana nigromaculata tadpole;(2)Compared with the corresponding control group,250 ?g/L TBBPA and TCBPA downregulated the expression of Dio2,TR? and TR? of HTP axis-related genes by 40.8% and 24.2%,28.6% and 20.9%,36.9% and 25.3%,respectively.TBBPA and TCBPA limited the production of TH in tadpole brain tissue through the low level expression of Dio2,reduced the activity of TR regulated by hormone and led to the decrease of TR? and TR? expression,thus delayed the metamorphosis development of tadpoles.The male Rana nigromaculata were exposed to 0.001,0.01,0.1 and 1 mg/L TBBPA and TCBPA for 14 days.The function,histomorphology and ultrastructure,oxidative stress index of the testis of Rana nigromaculata showed as follows:(1)Compared with the corresponding control group,1 mg/L TBBPA or TCBPA resulted significant decrease of the sperm number and sperm activity at 10.3% and 29.3%,51.5% and 46.9%;and significant increase of the sperm malformation rate at 39.7% and 54.9%;(2)Compared with the corresponding control group,1 mg/L TBBPA or TCBPA resulted significant decrease of ROS levels at 10.3% and 29.3% and MDA and GSH contents at 39.7% and 9.36%,44.7% and 37.7%;The activities of related antioxidant enzymes CAT,GSH-PX were decreased significantly by 33.42% and 24.29%,58.47% and 41.93% and the activities of T-SOD were increased significantly by 68.31% and 43.35%.TBBPA and TCBPA reduced ROS levels,and destructed the steady state of the anti-oxidation system in the testis of Rana nigromaculata,which showed the characteristics of anti-oxidation.(3)HE staining results showed that TBBPA and TCBPA treatment resulted in sperm neck swelling,abnormal agglutination and deficiency of the tail,increased lysosomes and a large number of vacuoles.(4)The results of transmission electron microscopy showed that abnormal vacuoles appeared in sperm and spermatogenic cells were disordered,scattered and shed.The male Rana nigromaculata were exposed to 0.001,0.01,0.1 and 1 mg/L TBBPA and TCBPA for 14 days.The result of sex hormone contents,sex hormone synthesis related genes and the expression of sex hormone receptor genes in the testis of male Rana nigromaculata showed as follows:(1)TBBPA and TCBPA significantly increased the contents of T and E2 hormones in serum and significantly reduced the contents of LH and FSH.Compared with the corresponding control group,1mg/L TBBPA or TCBPA resulted significant increase of T,E2 contents at 72.16% and 10.19%,24.29% and 8.48% and significant decrease of LH,FSH contents at 42.83% and 86.8%,26.67% and 8.02%.TBBPA and TCBPA disrupted the endocrine system and resulted in sperm formation disorders.(2)1 mg/L TBBPA or TCBPA resulted CYP11A1,CYP17A1 and CYP19A1 genes relate to sex hormone synthesis upregulation by 33.06% and 89.24%,31.77% and 8.71%,57.18% and 26.04%,while HSD17B3 downregulated by 64.6% and 50.7%,respectively.TBBPA and TCBPA increased the synthesis of T by downregulated the expression level of HSD17B3 gene,and increased the synthesis of E2 by upregulating the expression level of CYP19A1 gene;(3)Compared with the corresponding control group,1 mg/L TBBPA or TCBPA resulted the significant increase levels of AR mRNA at 84.55% and 60.10%,significant increase of the ESR1 mRNA levels at 65.2% and 61.79%.Through the abnormal expression of AR and ESR1 gene,TBBPA or TCBPA caused endocrine disorder in the testis of Rana nigromaculata,which affected spermatogenesis and caused severe reproductive toxicity.In conclusion,TBBPA and TCBPA inhibited the growth of hind limbs length and tail length,delayed the development stage of Rana nigromaculata tadpole,induced thyroid hormone secretion disorder and abnormal expression of HTP axe-related genes Dio2,TR? and TR?,disturbed the thyroid endocrine system and delayed the growth and development of the Rana nigromaculata tadpoles.TBBPA and TCBPA affected spermatogenesis,caused the histopathological damage of the testis,destructed the stable state of the anti-oxidation system of the testis of Rana nigromaculata,caused endocrine disorders and caused severe reproductive toxicity.Those results of this study provided a new scientific basis and theoretical basis for the toxic effects of both TBBPA and TCBPA on amphibian population. |
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