Combined Effects Of Two Environmental Disruptor Chemicals On Male Rat Testis Development And Tight Junctions Between Sertoli Cells

Research on toxic effects of environmental endaocrine disruptors(EEDS)on the reproductive system is an important topic of reproductive toxicology.At present,most of the researches focus on the toxicological effects of an endocrine disruptor.The combined effects of mixtures those belonging to the different types of compounds have not been extensively studied.Taking into account that humans and other animals in the environment usually expose to a variety of EEDs,the study on the combined effects of EEDs on the male reproductive is of great significance to male health risk assessment.We chose two EEDs widely distributed in the water environment,nonylphenol and dibutyl phthalate,to investigate the combined effects by performing in vivo and in vitro experiments.We used the two mathematical models to analyze the type of the combined effects of the compounds.Part I The exploration of mathematical models applied to analysis the combined effects of two environmental disruptor chemicalsObjectiveTwo mathematical models applied to analyze the combined effects of environment endocrine disruptors(EEDs)including NP and MBP are discussed.Methods1.Sertoli cells derived from the testes of rat were cultured in vitro and administrated by NP(range 0.01-50?M)and MBP(range 10-20000?M)in single and combined forms.2.Employing cell viability and lactate dehydrogenase(LDH)leakage rate of SCs as endpoints after exposure for 24h.3.A nonlinear regression based on Hill function is adopted to analyze the concentration-response relationship of single chemical.Two mathematical models were applied according to the concentration addition(CA)theory and the response addition(RA)theory to predict the combined effect of mixture based on the data of indicidual mixture components,and the difference between the predicted values and observed values were compared.Results1.Using the two models,a significant potential antagonist effect was observed both in the viability assay and in the LDH assay(at concentrations provoked LDH leakage rate>20%).2.At the lower concentrations which induced LDH leakage rate<15%the combined effect was possible synergism.Considering the environmental contaminants are frequentlyConclutionWhen several chemicals are used together in terms of different dose combination the relevant interaction type is also different.The synergism of two chemicals at low concentration suggested that the environmental risk assessment procedures about single chemical are insufficient to evaluate the combined effects of mixture.The two mathematical models are suitable for analyzing the combined effects of EEDs.Part II Combined effects of two environmental disruptor chemicals on male rat testis development and Tight junctions between Sertoli cellsObjectiveNP and DBP are regarded as widespread EEDs which at certain doses in wildlife animals and human have adverse effects on male reproduction and development.Given the ubiquitous coexistence of various classes of EEDs in the environment,their combined effects warrant investigation.In this study,we attempted to clarify the interactions of NP and DBP in testes and tight junctions(TJs)of male rats.Methods1.In the in vitro experiment,monobutyl phthalate(MBP),the active metabolite of DBP,was used instead of DBP.Sertoli cells were isolated from Sprague-Dawley rats,and treated with NP and MBP,singly or combined.The viability,morphology,apoptosis rate and functionality of TJs were measured.2.The dual-chamber culture system was established to culture SCs monolayer and detect transepithelial resistance.LDH leakage rates of cells were measured.TJs Western blotting was applied to detect TJs-associated proteins such as occludin,claudin-11 and ZO-1.Using Q-PCR to detect the gene expression levels of occludin and claudin-11.3.In the in vivo experiment,rats were gavaged on postnatal day 23-35 with a single or combined NP and DBP treatment.Testicular weight,serum hormone levels and morphology of TJs were recorded.4.The structure and function of TJs were detect using TEM and PCR.Results1.High concentrations of NP and MBP alone and combined exposure,lead to support the reduction of cell viability,morphological changes,increased apoptosis rate;2.Lower concentration of NP and MBP caused LDH leakage rate of Sertoli cell increased,transepithelial electrical resistance decreased and disordedr gene and protein expression of TJs-protein.The MAPK and PI3K/Akt pathway were activated in a short period of time;3.High concentrations of DBP can upregulate the rat testis coefficient significantly.NP and DBP can reduce testosterone levels and elevate FSH and LH levels;4.Visible damage of TJs is found in high concentrations of NP group.NP and DBP can disturb the gene expressionof TJs-proteins;5.The combined effects of NP and DBP(MBP)are mainly additive effects.Antagonistic effects exist between the mixture when they increased apoptosis rate and lowered testosterone levels at high concentration.ConclutionNP and MBP induced a range of toxicological effects on Sertoli cells.NP and DBP can interfere with the normal levels of the reproductive hormone in vivo.By affecting the tight junction protein expression,NP and DBP(MBP)induced the structural damage of tight junctions and increased membrane permeability.The response additive model more accurately predicts the combined effect between the two compounds.Innovative outcome obtained in this study:1.Due to alkyl phenols and phthalates widespread in the environment simultaneously,the human can contact with the mixture through a variety of ways.Therefore,the subject is first evaluating the combined effects of the compound NP and DBP on male reproductive system,and then exploring the mechanism,which will be of great significance to male health risk assessment.2.The focus of his study is located in the tight junctions between Sertoli cells in the spermatogenic epithelium s.We found that exposure with NP and DBP(MBP)in a short time activated the MAPK and PI3K/Akt signaling pathway,leading to disorder of gene and protein expression of tight-junction protein.Then membrane integrity was impaired,LDH leakage rate was increased,and apoptosis rate of cell was decreased.Through in vivo studies,we found that prepubertal exposure to NP and DBP interfered with the level of reproductive endocrinology,and the development of ti^it junctions between cells.3.In this study we used two mathematical models to analyze the combined effects of the mixture,explored the scientific and rational analyze methods to clarify the combined effects of environmental endocrine disruptors.