Study On The Disrupting Effects Of Low Doses Of Di(N-butyl) Phthalate (DBP) On Male Rat Reproduction

Phthalates are industrial chemicals used primarily as plasticizers to impart flexibility to polyvinylchloride plastics. They are not covalently bound to the plastic matrix and can easily leach out to contaminate the external environment. Generally, PAEs enter human body through ingestion of contaminated food , inhalation, dermal contact and is hazardous to human health. DBP is one of PAEs which is has been most commonly identified in air, water and soil because of its large production and diverse uses. Although DBP shows very low-acute toxicity, experimental data indicate that the reproductive system is susceptible to high dose of DBP (eg. 500 mg/kg/d). Our Previous studies showed that low doses of DBP metabolites influenced androgen synthesis in vitro. The present study is to determine whether low doses of DBP will affect the reproduction of pubertal male SD rats Part one: Disrupting effects of low doses of di(n-butyl) phthalate (DBP) on the testis function of pubertal male SD ratObjective To investigate the effect of DBP on the structure and function of rats testes in the pubertal male SD rats. Method Sprague-Dawley (SD) rats at age of 5 weeks received corn oil (vehicle control) or DBP orally at 0.1, 1.0, 10, 100, 500 mg/kg/d for 30 days. Different reproductive endpoints were evaluated comprising testis weight, testis histopathology, serum hormones, genes expression (StAR, SR, PCNA et al.) Results DBP affected the development of testis and epidydimis, induced severe atrophy of the seminiferous tubules and loss of spermatogenesis histologically, interfered with the serum homones balance, depress selectively the expression of StAR, PCNA at 500 mg/kg/d dose. Low doses of DBP (0.1, 1.0, 10 mg/kg/d) was found to exert a stimulative effect on the serum homones: Serum E2 level was increased in the 0.1mg/kg/d treatment group; Serum LH level was elevated in the 0.1 and 10 mg/kg/d treatment groups; Serum FSH level was increased in the 1 and 10 mg/kg/d treatment groups. Conclution 500 mg/kg/d DBP results in testis injuries characteristic of testis atrophy, atrophy of the seminiferous tubules and loss of spermatogenesis in pubertal male SD rats. In addition, Low doses of DBP (0.1, 1.0, 10 mg/kg/d) exhibite a stimulative effect on the serum homones, but the mechanism responsible for this remain unclear.Part two: The protein expression profile change induced by low doses of di(n-butyl) phthalate (DBP) in pubertal male SD ratsObjective to identify the differentially expression protein induced by DBP at low dose (0.1, 1.0, 10 mg/kg/d); to explore the mechanism by which DBP exerts effects on rat testes. Method Rats aging 5 weeks dailly receive DBP orally at 0, 0.1, 1.0, 10 mg/kg/d for 30 days. Three testes from each group were processed for 2-DE after exposure. Polyacrylamide gel electrophoresis was analyzed by ImageMaster 2D Platinum software and differentially expressed proteins were identified through MALDI-TOF/TOFTM. PathwayStudio software was used for the analysis of cellular processes influenced by differentially expressed proteins. Among these differentially expressed proteins, four proteins (HnRNP A2/B1, Vimentin, and SOD1) were selected for further validation using Western Blot. Immunohistochemistry was performed to study the localization of the three above-mentioned proteins in rat testes. Results Consequently, 45 differentially expressed proteins were identified. Most of these proteins were localized in Cytoplasm, Nucleus, Cytoskeleton, Mitochondria. Many cellular processes were influenced included: cell differentiation, proliferation, apoptosis, mitosis, meiosis, oxidative stress, etc. Expression tendency of three proteins (HnRNPA2/B1, Vimentin, SOD1 ) in Western Blot was consistent with the 2-DE. Immunohistochemistry demonstrated that these proteins were located in germ cell, Sertoli cells or Leydig cells respectively. Conclution DBP can change the proteins expression profile without morphological change at low doses. Differentially expressed proteins were widely distributed in cells and involved in diverse cellular processes, suggesting that the impact of DBP on the testis is complex and multifaceted. Especially, DBP can exert its effects on spermatogenesis, Sertoli cell structure and function and oxidative stress in rats testes and in-depth study of these aspects can help us to reveal the mechanism underlying the reproductive and developmental toxicity of DBP in male rats.