Study On The Mechanism Of The Carbendazim-induced Reproductive Toxicity In Male Rats

Carbendazim also known as delsene,benzimidazole 44,BCM,bavistin (Taiwan) etc.,is a kind of benzimidazole fungicide commonly used for industrial and agricultural, and also is the major metabolite of benomyl in mammalian species and degradation products in the environment. China is the most production and use of carbendazim with an annual production of more than 10,000 tons, accounting for a quarter of Chinese total fungicide. Although carbendazim toxicity test results show low toxicity, but the degradation of carbendazim is slow, and it also easily residues. Some reports have showed that carbendazim can cause reproductive damage in male rats, including testicular atrophy, vas deferens obstruction, and sperm abnormalities. Therefore, it is necessary to carry out a deep study on the pathway and mechanism of the carbendazim-induced reproductive toxicity in male rats.[Objective]To observe the anti-androgen/androgenic effects of carbendazim by establishing Hershberger test model, study on the pathway and features of the carbendazim-induced reproductive toxicity in male rats. Then by studying Sertoli cells in vitro model to clarify if Sertoli cells are targets of the effect of carbendazim. Through the above two part study to preliminarily explore the toxic mechanism, provide data for the male reproductive toxicity of benzimidazoles chemicals in-depth research, awareness benzimidazole chemicals harm the ecological balance of the environment and human reproductive health threats and to take effective preventive measures. [Methods]56 healthy clean degree Wistar male rats,5 weeks old, after 1 week for the rats to adapt to the environment, according to Hershberger test methods, the implementation of bilateral orchiectomy,7 days later, were randomly divided into 7 groups,containing solvent control group (corn oil), negative control group (testosterone propionate + corn oil), positive control group (testosterone propionate + flutamide), carbendazim group (testosterone propionate + carbendazim 20/100/200mg/kg.bw), solvent + carbendazim group (corn oil + carbendazim 200mg/kg.bw), and each group with 8 rats, exposed for 10 days, the corn oil as the solvent of testosterone propionate and flutamide. After the last exposure 24 hours, each group rats were weighed, and then killed, separated the androgen dependent tissues, and finally separated the liver and kidney. Weighed them immediately and calculated the organ coefficient.Primarily, Sertoli cells were harvested from rat testicles aged 18～20 days by the modified method of two-step digestion by enzymes, and then handled by Tris-Hcl low permeability of cell purification, the Sertoli cells were identified with HE staining, Wright-Giemsa staining. Sertoli cells proliferations were tested with MTT method. Detected the effects of expression of the Sertoli cells vimentin, tubulin-a and tubulin-P by Carbendazim with immunocytochemistry.[Results]1. Non- endogenous androgens animal modelAfter castration, the rats recovery well, no wound infection and mortality occurred. There were no obvious symptoms of poisoning.All the animals'body weights compared with the negative control group, there were no significant differences (P> 0.05). Each dose group animals, liver, kidney organ coefficient with the negative control group, no statistically significant difference (P> 0.05). Negative control coefficient of androgen relevant sex accessory glands and tissue was significantly higher than the solvent control group, there were significant differences (P<0.05;P<0.01); positive control coefficient of androgen relevant sex accessory glands and tissue was significantly lower than the Negative control group, the difference was statistically significant (P<0.05; P<0.01).2. The study of the androgen and anti-androgenic effects of CarbendazimUsing the established non-endogenous androgens animal model found that 3 Carbendazim experimental groups coefficient of androgen relevant sex accessory glands and tissue was compared with the negative control group, there were no significant differences (P>0.05); solvent and Carbendazim group coefficient of androgen relevant sex accessory glands and tissue was compared with the solvent control group, there were no significant differences (P>0.05), compared with the negative control group were significantly lower and was statistically significant (P< 0.05;P<0.01).3. The culture and identification of Sertoli cellsEach Wistar rat testis can be obtained about 10'Sertoli cells by the modified method of two-step digestion by enzymes, the cell survival rate was 95%.Sertoli cells in vitro close to the bottom growth, cell body was elongated or irregular, there are multiple processes, the nucleus was oval or irregular, the nucleus shows a typical satellite nucleosomes.4. The effects of proliferation of sertoli cells by carbendazimMTT results showed that at 24 h,48 h and 72 h, the cell growth inhibition rate of 0,0.1,and 1μg/ml dose of carbendazim was under 5%, but 10,100μg/ml dose of carbendazim significantly inhibited the growth the cells, most obvious when 72 h, respectively,31% and 34.87%.5. The effects of expression of the vimentin,α-tubulin andβ-tubulin of the Sertoli cells by CarbendazimImmunocytochemistry showed that 10,100μg/ml dose of carbendazim can inhibit vimentin,α-tubulin andβ-tubulin of Sertoli cell function and cytoskeleton. The sertoli cells may be the target of the male rats reproductive toxicity.[Conclusions]1. According the Hershberger test, the stable animal model of Non- endogenous androgens can be established, and the successful model can be used in the follow-up study. 2. In this dosage, the androgen dependent tissues in male rats haven't been affected by Carbendazim. The anti-androgen/androgenic effects of carbendazim didn't be detected. The carbendazim-induced reproductive in male rats may not be interfered by hormone pathway.3. The Sertoli cells were harvested by the modified method of two-step digestion by enzymes, and then handled by Tris-Hcl low permeability of cell purification, and identified with HE staining, Wright-Giemsa staining. The cells that we obtained were relatively high purity Sertoli cells.4. MTT results showed that 10,100μg/ml dose of carbendazim can inhibit the proliferation of the Sertoli cells, longer time, more seriously.5. The results showed that 10,100μg/ml dose of Carbendazim could inhibit Sertoli cells proliferation, and interfere the expression of vimentin, tubulin-a, tubulin-β.Carbendazim affect the Sertoli cells cytoskeleton. The Sertoli cells may be the targets of the carbendazim-induced reproductive toxicity in male rats.