Reproductive Toxicity And Its Molecular Mechanism Of Environmental Endocrine Disruptor4-nonylphenol To The Invertebrate Bombyx Mori

As an environmental endocrine disruptor,4-nonylphenol （4-NP） severely affects the developmentof the reproductive system within various animals, including mammals. Right until now there is noreport on the effect of4-NP on reproductive development of terrestrial invertebrates. Bombyx mori （B.mori） was set as an invertebrate insect model, adopting the full-instar larvae diet exposed to4-NP,employed by histopathology, physiological and biochemical detection, gene expression profile analysis,IHC, HPLC analysis, tissue and cell culture and other experimental technologies, the reproductivetoxicity of4-NP to B. mori and its molecular mechanism were investigated. On the basis of thetraditional reproductive toxicity testing in vivo, a easy-handed, fast and convenient in vitro toxicitytesting model was estabished which could be used to detect the endocrine disruptor such as4-NP. Themajor results are as follows:1.4-NP through oral exposure has reproductive toxic effects on silkworm, B. moriAdded with0.05～0.4g kg^-14-NP in diet at B. mori’s larval stage, there was no significantdifference in the sex ratio （female/male） at pupal stage, while the gonad somatic index decreased by70.0%～48.7%; At the adult stage, the sex ratio （female/male） fell by29.2%in the group exposed to0.4g kg^-14-NP, the fecundity and the hatching rate decreased by80.7%and91.8%, respectively.Histopathology demonstrated that the growth of testis germ cells of B. mori was retarded and the cellstructure was injured in4-NP exposed groups.4-NP could be accumulated in the silkworm tissues, andthe content of4-NP in fat body was higher than in intestine, epidermis and gonads; the content of4-NPin females was higher than that in males. The results showed that4-NP through oral exposure hadreproductive toxic effects on B. mori. 2. Oxidation protective enzyme system in the gonads of silkworm, B. mori is sensitiveto4-NP exposureB. mori exposed to0.05～0.4g kg^-14-NP in whole larval stage, the lipid peroxide MDA content ingonads was induced, and the GSH level was exhausted, the enzyme activities of SOD, CAT and GPXwere decreased,4-NP broke the balance of cell oxidation and antioxidant and led to cell injury.Meanwhile, the cooperation of GSH and GST played a role in4-NP detoxification.The antioxidant enzymatic systems in gonads of pupae and adults were more sensitive than inlarvae, suggesting that the oxidation protective enzymatic systems in the pupae and adults when germcells developed quickly, were still reactive to the4-NP exposed in larvae.The antioxidant enzymatic activities in the gonads of male could be activated easier than in femalegonads, because the male gonads had a better mechanism in cooperation of GSH and GST, and theattenuation of antioxidant enzyme activities in female gonads aggravated the lipid peroxidation of cells.The results suggested that4-NP could produce more reproductive toxicity in female silkworm throughoxidative damage. The adult was the sensitive stage which could be selected as the best period fordetecting insect reproductive toxicity.3.4-NP has different molecular mechanisms in affecting the reproductive toxicity ofmale and female silkworm, B. mori4-NP exposure induced the expression levels of female special genes Vg and Esp, in which Vgprotein content in ovaries was reduced in0.1g kg^-14-NP exposed group analyzed by IHC. Takentogether, these results showed that4-NP impacted the expression level of the EcR gene which mightlead to reduced activity of ecdysteroids in the ovary and subsequent disturbance of the Vg productionpathway thus affecting its transfer from the fat body to the ovary in this in vivo model. Also,4-NP couldaffect the egg development by down-regulated the expression level of Jun in JNK signal.Vg gene expression in male gonad silent in normal stage was induced in larval, pupal and adultstage, while4-NP was added in diet in whole larval stage. Vg protein in testis of pupae was detected in0.05g kg^-14-NP exposure group by IHC assay. The results suggested that4-NP had estronic effect onmale silkworm. Vg gene expression could be used as a typical biomarker in evaluating the reproductivetoxicity of B. mori exposed to4-NP and other related environment endocrine disruptors. The expressionlevel of SoxE and Stat in gonad of male B. mori was significantly reduced in4-NP exposure groups,while the expression level of EcR was not affected. The effects of4-NP on the development of malegonad or germ cells might be related to the JAK/STAT signal pathway. 4. Establishing the model of B. mori’s testis and spermatotheca in vitro toxicityculture, which can be used to analyze the male reproductive toxicity of4-NP in insectsTestes of silkworm were exposed to7～28mg L^-14-NP in vitro, and the balance of antioxidantdefense system in tissue were damaged but the expression level of SoxE gene were not affected much.4-NP was restrained in the growth development of spermatocyst in vitro which could be used as amodel in analyzing male reproductive toxicity of4-NP. The concentration of4-NP used in detectionwas0.02～2μg L^-1, which was close to that reported in the actual environment in TaiHu. It meant themale reproductive cells of B. mori were sensitive to4-NP and could be used for the quick analysis of4-NP’s male reproductive toxicity.Conclusion: Silkworm, B. mori is a good invertebrate model organism in studying reproductivetoxicity of4-NP.