Combined Effects Of DEHP And Cadmium On Reproductive Function In Puberty Male Rats

ObjectiveIn this study,Di-2-ethylhexyl phthalate（DEHP）and cadmium（Cd）were used as test substances to study the combined effects of DEHP and Cd on reproductive function in puberty male rats.Methods1.Using a 3×3 factorial design,54 healthy 6-week-old clean male Sprague-Dawley rats were randomly divided into 9 groups according to their body weights:solvent control（corn oil+distilled water）group,250mg/kg DEHP,1 000mg/kg DEHP,1mg/kg cadmium chloride（CdCl₂）,3mg/kg CdCl₂,250mg/kg DEHP+1mg/kg CdCl₂,250mg/kg DEHP+3mg/kg CdCl₂,1 000mg/kg DEHP+1mg/kg CdCl₂,1 000mg/kg DEHP+3mg/kg CdCl₂ exposure group,6 in each group.The drug was administered by intragastric administration once a day for 4 weeks,and the exposure capacity was 10ml/kg.2.After the last exposure for 24 hours,the rats were weighed and the eyeballs were removed to collect blood samples.The serum was separated by centrifugation and the serum testosterone content was measured by ELISA kit.Specimens were collected after the rats were sacrificed.The sperm motility and density were recorded by CASA sperm analysis system.Analysis of the number and morphology of testicular tissue cells by HE staining.Testicular cells apoptosis was determined via TUNEL assay.The testis tissue concentrations of MDA,activity of T-SOD and GSH-Px were detected by colorimetry method.Total RNA was extracted from testis tissue,and the relative mRNA expression levels of Akt,bcl-2,caspase-3 and caspase-9 were detected by Real-Time PCR.Results1)Weight change and organ coefficientThe weight gain of rats exposed to 250mg/kg DEHP+1mg/kg CdCl₂ and 1000mg/kg DEHP+3mg/kg CdCl₂ was smaller than that of the control group（P<0.05）;combined exposure increased the liver coefficient of rats（hepatomegaly）,but the effect of DEHP and Cd on testicular coefficient was not statistically significant（P>0.05）.2)Sperm vitality and densityCompared with the control group,the sperm density and sperm motility of the rats exposed to 1 000mg/kg DEHP+1mg/kg CdCl₂ and 1 000mg/kg DEHP+3mg/kg CdCl₂were lower（P<0.05）;The results of factorial analysis showed that there was no interaction of DEHP and Cd on sperm toxicity and density in rats（P>0.05）.3)Testicular tissue pathologyDEHP and Cd exposure could lead to the atrophy of the testicular tubules,and the outer wall edge was not smooth;spermatogenic cells were reduced and arranged disorderly;part of the tissue were dissolved and lost to form a cavity,and the mature sperms in the lumen were significantly reduced.4)Testicular tissue apoptosisDEHP and Cd exposure led to abnormal apoptosis of testicular cells,which are mainly spermatogonia and spermatocytes.The results of factorial analysis showed that the interaction of DEHP and Cd on testicular cell apoptosis rate was antagonistic（P<0.05）.5)Serum testosterone contentCompared with the control group,the serum testosterone content of the rats in other groups were lower（P<0.05）.The results of factorial analysis showed that there was no interaction of DEHP and Cd on serum testosterone content in rats（P>0.05）.6)Oxidative stress in testicular tissueDEHP and Cd alone and in combination can lead to decreased T-SOD,GSH-Px activity and increased MDA content in testis（P<0.05）.The interaction of DEHP and Cd on T-SOD activity in rat testis showed antagonistic effect（P<0.05）,and there was no interaction on GSH-Px activity and MDA content（P>0.05）.7)Relative expression levels of Akt,bcl-2,caspase-3,caspase-9 mRNA in testisCompared with the control group,there was no significant difference in the expression levels of bcl-2,caspase-3 and caspase-9 mRNA in the exposed group（P>0.05）,while the expression level of Akt mRNA in testis tissue of rats increased in the 3 mg/kg Cd exposure group and all of the combined exposure groups（P<0.05）.The interaction of DEHP and Cd on caspase-9 mRNA expression was antagonistic（P<0.05）,and there was no interaction on the expression levels of Akt,bcl-2 and caspase-3 mRNA（P>0.05）.Conclusion1)DEHP and Cd exposure to puberty can lead to weight loss,hepatomegaly,destruction of testicular tissue morphology,reduction of support cells and spermatogenic cells,decline of sperm motility and density,thereby affecting male reproductive function.2)Possible mechanisms of DEHP and cadmium-induced reproductive toxicity are endocrine dysfunction and apoptosis induced by testicular oxidative stress.3)The combination of DEHP and Cd on the testicular tissue T-SOD activity,apoptosis rate and caspase-9 mRNA expression levels showed antagonism.