Effects Of Nano Titanium Dioxide On Blood-testis Barrier And MAPK Signaling Pathway In Male Mice

Objective This study aims to investigate the adverse effects of nano titanium dioxide（Nano-TiO₂）on male reproductive,blood testis barrier（BTB）and MAPK signaling pathway.The study can provide reference for reproductive hazard assessment and prevention of Nano-TiO₂.Methods Sixty healthy male ICR mice were randomly divided into control group and Nano-TiO₂ groups with the dosage of 10,50 and 100 mg/kg BW intragastric administration for 30 days.The ultrastructural changes of the mouse testicular tissue were observed by Transmission electron microscopy（TEM）.The micronucleus rate of testicular germ cells was measured.Furthermore,HE staining was used to observe the changes of testicular histopathology,spermatogenesis was evaluated using the Johnsen score.Sperm concentrations,sperm motility and sperm deformity rate were also measured in this research.The contents of BTB-associated proteins（Occludin,ZO-1,Claudin-11,Connexin-43 and F-actin）,testosterone and estradiol were assessed by ELISA methods.The mRNA expression of ERK,JNK and p38 in the testis were detected by qRT-PCR,whereas the protein expression levels of ERK,p-ERK,JNK,p-JNK,p38 MAPK and p-p38 MAPK were detected by Western blot.Results1.After Nano-TiO₂ exposure,the ultrastructure of the testicular tissue showed that the basement membrane and extra-basement membrane collagen tissue in the mice of Nano-TiO₂ exposed groups were loose and thick,and the thickness of the collagen tissue was uneven.The vacuolation of sertoli cells,abnormal membrane and chromatin of germ cells were observed in the testicles of 50 and 100 mg/kg BW dosage groups.The micronucleus rate of germ cells in the testicles of the 50 and 100 mg/kg BW dosage groups increased by 5.50‰、8.33‰compared with that in the control group（P<0.05,P<0.01）.Testicular histopathology showed that the distributions of spermatogenic cells were irregular and the vacuolization of epithelial cells of the seminiferous tubules in the testicles of the 50 and 100 mg/kg BW dosage groups.The spermatogenesis score in the testicles of 50 and 100 mg/kg BW dosage groups decreased compared with that in the control group（P<0.001）.The sperm count showed that there was no significant difference in all groups（P>0.05）.The sperm progressive motility decreased by 13.62%、19.12%and sperm motility decreased by 17.93%、24.87%while the sperm malformation rate increased by 2.50%、5.48%in the mice of 50 and 100 mg/kg BW dosage groups compared with that in the control group（P<0.05）.The contents of testosterone and estradiol in the mice serum of 50 and 100mg/kg BW dosage groups decreased compared with that in the control group（P<0.05）.2.The structure and the associated proteins of BTB changed after Nano-TiO₂ exposure.The ultrastructure of BTB in 50mg/kg BW dosage group was disordered and broken.In the BTB structure of 100mg/kg BW dosage group,the tight junction voids were enlarged,and the expansion of endoplasmic reticulum were expanded.The content of ZO-1 in the testicles of 100 mg/kg BW dosage group significantly increased compared with that of the control group（P<0.01）.The content of Claudin-11 in the testicles of 50 and100mg/kg BW dosage groups increased compared with that of the control group（P<0.01）.The content of F-actin in the testicles of 10,50 and 100 mg/kg BW dosage groups increased compared with that of the control group（P<0.01）.There was no significant differences in the contents of Connexin-43 and Occludin of testis in the control and treatment groups（P>0.05）.3.The gene and protein expression levels of MAPK signaling pathway changed after Nano-TiO₂ exposure.The expression levels of ERK and JNK1 mRNA in the testicles of 100mg/kg BW dosage group were significantly higher than that of the control group（P<0.05）.There was no significant differences in the expression levels of p38 mRNA of testis in the control and treatment groups（P>0.05）.The expression levels of ERK and JNK protein in the testicles of 50 and 100mg/kg BW dosage groups significantly increased compared with that of the control group（P<0.01）.The expression levels of p-ERK,p-JNK protein in the testicles of 50 and 100mg/kg BW dosage group increased compared with that of the control group（P<0.01;P<0.05）.The expression levels of p-p38 MAPK protein in the testicles of 100mg/kg BW dosage group increased compared with that of the control group（P<0.05）.The ratio of p-ERK/ERK and p-JNK/JNK were not significant different between the control group and treatment groups（P>0.05）.The ratio of p-p38 MAPK/p38 MAPK in the testicles of 10 and 100mg/kg BW dosage group was significantly higher compared with that of the control group（P<0.05）.Conclusion Nano-TiO₂ can up-regulate the mRNA and protein expression levels of MAPK signaling pathway,resulting in the imbalance of estrogen and androgen levels.Causing disturbance of the structure and function were altered through the overexpression of ZO-1,Claudin-11 and F-actin of the BTB.The spermatogenesis and sperm quality also decreased under the coaction of these toxic effects.All of the damages may eventually lead to male reproductive toxicity.