Effects Of Melamine Alone Or In Combination With Cyanuric Acid On Blood-testis Barrier In Male Mice

In2008in China, the occurrence of an outbreak of urinary stones in infants draw critical attention to the toxicity of melamine (MA) or the mixture of MA and cyanuric acid (CA) in human and animals.Until now, a large number of researches focused on the toxic effects of MA and its analoges on the urinary organs. In this study, based on our prelimitary results, we focused on the blood-testis barrier and Sertoli cells to investigate the toxic effects of MA and its analogues on male reproductive system on different levels with methods of histochemical and ultrastructural pathology,immunohistochemical staining, Western blot and flow cytometry. The research contents are as follows:1. In vivo studyEight-week-old male Kunming mice were administered either melamine (MA,30,140, or700mg/kg/day), a melamine and cyanuric acid mixture (MC, each at15,70, or350mg/kg/day), or vehicle (control) for3consecutive days. Testicular toxicity was evaluated on days1(Post Id) and5(Post5d) after the final exposure. Furthermore, the structure of blood-testis barrier and possible cellular target for MA and MC was investigated.With the methods of clinical observation, serum chemistry analysis、autopsy、histopathological examination and sperm detection, on Post1d, we found that the body weight gain of the MC-treated mice significantly decreased (P<0.05or P<0.01), with obvious clinical sighs in the higher groups. In the highest MC group, the serum T level significantly decreased(P<0.01), and the organ indexes of testis and epididymis decreased (P<0.05or P<0.01). In both MA and MC groups, the testes and epididymis tissue showed dose-related histopathological lesions, and the characterize changes were germ cell exfoliation and tubular vacuolation. Additionally, the sperm abnormality were significantly increased in all treated groups (P<0.05or P<0.01). On Post5d, the sperm abnormality were still significantly increased in all treated groups (P<0.05or P<0.01).The other indexes indicated that the degree of testicular injury became slighter at this time point. These results suggested that MA and MC had direct toxicity on the testicular epithelium and caused decrease of sperm quality.We detected the apoptosis and the protein expression of Fas/Fas-L in the testes with TUNEL staining, immunohistochemistry staining and Western Blot. On post Id, the results showed that the germ cells exhibited apoptosis in the testes among all treated groups, and apoptotic index was significantly increased in low MA group and all MC groups(P<0.05or P<0.01). The Fas and Fas-L distributed in the germ cells and Sertoli cells, respectively. On post5d, apoptotic index in the highest MA group and the highest two MC groups(P<0.05or P<0.01). In the highest MC group, the Fas and Fas-L protein showed strong expression in germ cells. These results suggested that MA and MC could induce germ cell apoptosis, and the Fas/Fas-L system may involved the apoptotic process.The structure of blood-testis barrier and related protein was evaluated with TEM, immunohistochemistry staining and Western Blot. On Post Id, the results showed that structure of the blood-testis barrier was damaged dose dependently. The vimentin filament in Sertoli cell collapsed in all treated groups. The expression of vimentin and β-actin protein decreased with difference degree among the treated groups (P<0.05or P<0.01). On Post5d, the lesions of the blood-testis barrier structure was still serious in the treated groups, with decreased expression of vimentin protein(P<0.05or P<0.01). These results suggested that MA and MC could disrupt the blood-testis barrier and decreased cytoskeleton protein expression. Sertoli cells appear to be a cellular target for MA and MC.2. In vitro studyWith the methods of MTT, TEM, Flow cytometry and Western Blot, we evaluated the cytotoxicity of MA (0-1000ug/mL) on TM4Sertoli cells. When cells were treated with MA at50ug/mL or above for24h, the cell viability was significantly decreased(P<0.05or P<0.01). When cells were treated with MA at range of30to500ug/mL for24h, the ultrastructure was obviously damaged, and high concentration of MA induced significant apoptosis(P<0.05or P<0.01). When cells were treated with MA at50ug/mL MA for0,1,3,6or24h, the expression of vimentin protein decreased with time. It can be seen that MA even at low concentration could induce obvious injury in TM4cells, suggesting that Sertoli cells maybe sensitive to MA toxicity.Taken together, the in vivo and in vitro studies showed that oral exposure to MA and MC in male Kumming mice could produce obvious testicular toxicity including germ cell sloughing and apoptosis and high rate of sperm abnormality. MC exhibited much more toxicity than MA. The damage of blood testis barrier and Sertoli cell play a critical role in the MA-and MC-induced male reproductive toxicity.