The Relationship Between Anti-androgenic Activity Of Environmental Endocrine Disruptors And The Pathogenesis Of Gonadal Dysgenesis, And The Mechanism Of Antagonistic Effect Treated With Chinese Herbal Medicine

Recent years, the potential of environmental endocrine disruptors (EEDs) to cause adverse effects on reproduction and development in children have been received great concerns. Most of EEDs have both estrogenic effects and anti-androgenic effects, which can cause not only precocious puberty in girl but also gonadal dysgenesis in boy. Currently, research predominantly focused on the relationship between the estrogenic effects of EEDs and pathogenesis of precocious puberty in girl. While, potential adverse effects of EEDs on reproduction and development in boy got gradual attention. However, the molecular mechanism(s) of action of the environmental toxicants on the testis have yet to be elucidated, and need to be expanded. More importantly, there still have not effective preventions and treatments to antagonize against the anti-androgenic effects of EEDs. Traditional Chinese medicine suggests that gonadal dysgenesis is renal essences deficiency, and tonifying kidney drugs are believed to be an appropriate and beneficial treatment and have been achieved significant clinical efficacy. Hence, the present study was designed to investigate the relationship between anti-androgenic activity of environmental endocrine disruptors and the pathogenesis of gonadal dysgenesis, and the mechanism of antagonistic effect treated with Chinese herbal medicine for tonifying kidney to replenish essence (TKRE).Di (2-ethylhexyl) phthalate (DEHP) and Cypermethrin (CYP) are common environmental endocrine disruptors (EEDs) in China which are well-known testicular toxicant inducing adverse effects in androgen responsive tissues. The prepubertal period (age from postnatal day 21 to postnatal day 48 is a transitional period for weaning to sexual maturity in rodents, with gonads being particularly sensitive to EEDs-induced injury. Hence, the prepubertal male rats were used in our experiment. A total of 40 healthy 3-week-old SPF male Sprague-Dawley rats were randomly and evenly divided into four groups, namely, the control group (corn oil), the DEHP group (500 mg/kg, dissolved in corn oil), the CYP group (80mg/kg, dissolved in corn oil) and the combined group exposed to both DEHP and CYP (500mg/kg DEHP+CYP 80mg/kg, dissolved in corn oil). Rats were exposed to EEDs via gavage administration once a day for 28 days. After the exposure, the animals were decapitated for the blood collection. The testicular descent, preputial separation, wet weight of the testis, testis coefficient and the serum testosterone level as well as testis histopathological parameters and ultrastructural lesions were calculated to verify the anti-androgen effects of DEHP and CYP. The relative gene and protein expressions of follicle stimulating hormone receptor (FSHR), androgen binding protein (ABP), inhibin beta-B (INHB) and vimentin (VIM) which were analyzed by real time quantitative RT-PCR and Western Blot were detected to explore the probable mechanism of the EEDs inducing gonadal dysgenesis. Statistical analysis was done using the 2×2 variance of factorial design.Based on the anmimal model of gonadal dysgenesis, we further investigate the antagonistic effect of Chinese herbal medicine on environmental endocrine disruptors inducing gonadal dysgenesis in prepubertal male rats. Seventy male Sprague-Dawley rats (21 days old) were randomly divided into seven groups (N=10/group). The exposure groups were gavaged for 28 days with 500mg/kg of DEHP,80mg/kg of CYP and mixture of DEHP and CYP, respectively. The three corresponding treatment groups were simultaneous supplemented with 40mg/kg of Chinese herb medicine. Testicular descent, preputial separation, testes weight, testis coefficient, serum testosterone level and morphology paremeters were analyzed to test the antagonistic effects of Chinese herb medicine for tonifying kidney to replenish essence (TKRE) on DEHP and CYP inducing gonadal dysgenesis in prepubertal male rats. The paremeters including oxidative stress, and relative gene and protein expressions of FSHR, ABP, INH, VIM and PPARy were further detected to investigate the mechanism of the antagonistic effects of Chinese medicine on prepubertal male rats of gonadal dysgenesis induced by EEDs. Data were analyzed by one-way ANOVA and the Fisher’s least significant difference (LSD) method to determine treatment differences.It is found that compared with the control group, testicular descent and preputial separation of the other three exposure groups were obvilously delayed. Testes weights, testes coefficients and serum level of testosterone in exposure groups were significantly decreased. And all the exposure groups showed damages in different stages in histology and ultrastructure of testes. Rats exposed to DEHP and CYP showed severe alterations of testicular morphology and Sertoli cell function. However, those changes were significantly reversed by simultaneous Chinese herb medicine supplementation to EEDs-exposed rats. Testicular descent, preputial separation, testis weight, serum hormone of testosterone, androgen-dependent process and reliable marker of progression of puberty, were improved significantly in simultaneous supplemented with Chinese medicine. Oxidative stress was significantly decreased. Relative gene and protein expressions of Sertoli cellular receptors (FSHR), prepubertal testicular marker (INH) and Sertoli cell functional protein (ABP) were increased significantly in TKRE supplementation compared to the EEDs-treated group. PPARy expression which is related to Sertoli cell apoptosis and proliferation presented with a significant down-regulation in the Chinese herb medicine supplementation.Taken together, these results validated the antiandrogenic effects of DEHP and CYP exposure on prepubertal male rats. DEHP showed a significant toxicity on Sertoli cell which could markedly interfere with the gene and protein expressions related to Sertoli cell function. CYP showed slight toxicity on Sertoli cell. The Chinese herb medicine for tonifying kidney to replenish essence (TKRE) could significantly antagonize against the anti-androgenic effects and reproductive toxicity of EEDs. TKRE could significantly suppress the oxidative stress induced by EEDs, boost the secretion of testosterone and ameliorate the Sertoli cell function, which might be one of main mechanism of TKRE antagonist agaist the antiandrogenic effects of EEDs.