Research On The Toxic Effect Of Butyl Benzyl Phthalate And Its Metabolites On Sertoli Cells

Plastifier BBP is a environmental estrogen, when it,entered the body, it can simulated or interfered the natural estrogen’s synthesis, secretion, transporting, binding, excretion and other regular physiological functions, especially injure the male reproductive system. So BBP had become a new global environmental pollutant. Experiments confirmed that there were not BBP in the urine of rat, but it is metabolized into metabolites, they are PA, MBuP, HA, Monobutyl Phthalate with glucuronic acid content and Benzyl butyl phthalate with glucuronic acid content. And MBuP has high fat water distribution coefficient, it can across the testisbarrier. So many domestic and overseas scholars consider that perhaps the reproductive toxicity is caused by its metabolites, however, relevant investigation is very seldom. This experiment compare BBP and several metabolites which has the strong androgen depriation activity, and cause male reproductive system damage.This experiment select sertoli cells of rat as the study object, comparison the cytotoxicity of BBP and its metabolites (MBuP, PA, HA). Testicle is the main reproductive organ of male mammal, sertoli cell is one of the typical cells of testicle, it has vital function for testicle and even whole male reproductive system. Substances can damage sertoli cells which also can injure the male reproductive system. We use double enzyme digest method, separating sertoli cells by collagenase and trypsinase sequentially. Then the Sertoli cells are identified with Oil Red O staining, HE staining and immunocytochemical method. And using MTT assay to comparison the cytotoxicity of BBP and its metabolites, Flow cytometry detect the influence of cell cycle after added BBP and its metabolites, then using Western Blot to research the change of expression of ABP and Vimentin.Oil Red O staining, HE staining and immunocytochemical method show the ratio of Sertoli cells was more than80%. MTT show BBP(1.0×10-7,1.0×10-5,1.25×10mol/L) and low dose of HA(1.0×10-6mol/L) can promote the proliferation of sertoli cells. But MBuP, PA(1.0×10-6,10×10-5,10×10-4mol/L) and medium or high dose of HA(1.0×10-5×10-6,1.0×10-5,1.0×10-4mol/L) and medium or high dose of HA(1.0×10-5,1.0×10-4mol/L) all inhibit the growth of sertoli cells. The result of FCM show that BBP(1×10-7mol/L) and HA(1×10-6mol/L) can increase the contents of S phase cells, G2phase cells, proliferation obvious with extended poisonous time, MBuP(1×10-6mol/L)s PA(1×10-6mol/L) will reduce the contents of S phase cells, inhibit the proliferation of sertoli cell. Western Blot show MBuP(1×10-6mol/L) and PA(1×10-6mol/L) can reduce the protein expression of ABP, and BBP(1×10-7mol/L), MBuP(1×10-6mol/L) and PA(1×10-6mol/L) can reduce the protein expression of Vimentin, diversity has significance(p<0.01). So we infer MBuP and PA have the strong androgen depriation activity, and they may inhibit the proliferation of sertoli cell in vary degrees, alter the normal cell cycle distribution, influence the expression and distribution of some proteins in sertoli cells, destroy the normal function of sertoli cells and the cytoskeleton system, damage the testicle of mice, these changes cause developmental toxicity of male reproductive finally.