Androgen Receptor Plays a Vital Role in Benomyl- or Carbendazim-Induced Reproductive and Developmental Toxicity and Endocrine-Disrupting Activity in Rats

Abstract

Benomyl and its metabolite carbendazim were reported to induce reproductive and developmental toxicity and endocrine-disrupting activity in rats. The exactly underlying mechanism of reproductive and developmental toxicity and endocrine-disrupting activ- ity still remain unclear. Based on our unpublished data it showed that the antiandrogen flutamide can completely recover the reproductive and developmental toxicity including embryolethality induced by benomyl and carbendazim in rats. This manuscript aimed to review and generalize the results based on our previous reports. Androgen receptor might play an important role in benomyl- and carbendazim-induced reproductive and developmental toxicity and endocrine-disrupting activity. The evidences were (1) andro- gen- and androgen receptor-dependent mechanisms are possibly involved in carben-dazim-induced toxicity; (2) carbendazim exposure in utero displays a transient and weak androgenic effect and reduces flutamide antiandrogenicity in male rats; (3) antagonistic effect of flutamide on the carbendazim-androgenic effect on mRNA and protein levels; (4) benomyl and carbendazim exhibit an androgenic effect, leading to increase weight of ventral prostate and seminal vesicles and uterine fluid retention in young adult rats. The molecular underlying mechanism of reproductive and developmental toxicity and endocrine-disrupting activity induced by benomyl and carbendazim through androgen receptor need to be further investigated. HAP for using of didymis by from of of on of in This study aimed to investigate the endocrine-disrupting activity of carbendazim-induced reproductive and developmental toxicity in rats. The male rats were co-treatment with 675 mg/kg carbendazim and 50 or 100 mg/kg flutamide, an androgen receptor antagonist, once daily for 28 days decreased testis weight induced by treatment with carbendazim alone. Co-treatment of carbendazim and flutamide blocked losses of spermatozoa and cell morphology and decrease of sperm concentration induced by carbendazim. An important evidence for endocrine disrupting activity induced by carbendazim and benomyl was that premating treatment of male and female rats with 200 mg/kg carbendazim for 28 days resulted in androgenic effects including incomplete development of uterine horn, enlargement of urethra, absence of vagina, and induction of semi nal vesicles in female offspring, without significant effects in male offspring. Also, premating treatment with 100 mg/kg benomyl, the parent compound of carbendazim, produced incom plete Both carbendazim (methyl 2-benzimidazolecarbamate) and benomyl are reported to exhibit reproductive and developmental toxicity in male rats. This study was mainly to detect the ability of carbendazim exposure in utero to alter androgen-dependent development indicators in rat offspring and measure the effects of antiandrogen flutamide on the carbendazim-induced repro ductive and developmental alterations. All pregnant female rats were administered with 6.25, 12.5 or 25 mg/kg carbendazim, 25, 50 or 100 mg/kg benomyl, and 0.6, 2.5 or 10 mg/kg flutamide by gavage once daily from gestational day 0 to 20. Also, group of female rats was co-treated with 25 mg/kg carbendazim or 100 mg/kg benomyl and 0.6, 2.5, and 10 mg/kg flutamide. The results showed that the various treatments decreased the survival rates of pups on PND 1 and 21. For male offspring, 12.5 and 25 mg/kg carbendazim increased AGD, an androgen-dependent indica tor, on PND 2. Also, benomyl increased AGD of offspring. Co-treatment with 25 mg/kg carben dazim with 0.6, 2.5, and 10 mg/kg flutamide relieved the androgenic effect on AGD induced by carbendazim. The androgenic effects of AGD induced by carbendazim and benomyl on AGD were reversible on PND 22 and later. Carbendazim had no effects on other androgen-dependent indicators such as testis and epididymis malformations, hypospadias, nipple retention, and organ weights of seminal vesicle and levator ani bulbocavernosus muscle on PND 56. Quite surprisingly, carbendazim antagonized the antiandrogenic effects on these indicators induced by flutamide cotreatment. For female offspring, carbendazim exhibited synergistic effects on the flutamide cotreatment-mediated increases of organs weights in liver and kidney on PND 56. No significant effects on female reproductive organs were induced by carbendazim. These findings suggested that carbendazim exposure in utero exhibited a transient and weak androgenic effect and reduces flutamide antiandrogenicity in male rats. These two fungicides enhance flutamide-mediated increases of liver and kidney weight in female rats. The antagonistic and synergistic interactions between carbendazim and flutamide in utero need to be further investigated. 7.5% polyacrylamide. The proteins were transferred to nitrocellulose membranes. were then blocked for non-specific binding and incubated with polyclonal primary The both benomyl and carbendazim are widely used systemic fungicides. It has been shown that benomyl and carbendazim induce endocrine-disrupting activity, resulting in reproductive and developmental toxicity, as well as androgen receptor (AR) gene expression in rats. The aim of this study was to link AR induction by benomyl and carbendazim, observed in our previous reports, with the results of Hershberger and uterotrophic assays. In an uterotrophic assay, nei ther benomyl nor carbendazim, except at 800 mg/kg/day, affected weight of uterus and vagina when compared to the ovariectomized control rats. Co-treatment with 17β-estradiol (E 2 ) and 200 mg/kg/day benomyl or co-treatment with E 2 and 200, 800 mg/kg/day carbendazim signifi cantly increased uterine weight when compared to treatment with E 2 alone in an uterotrophic assay. This uterotrophic activity might be mediated through AR. Treatment with flutamide alone or in combination with E 2 had no effect on uterine weight. In the Hershberger assay, treat ment with 50 and 100 mg/kg/day benomyl increased weight of ventral prostate plus seminal vesicles. Carbendazim or flutamide alone exhibited no effect on reproductive accessory gland weight. Co-treatment with testosterone propionate (TP) and 50 or 100 mg/kg/day carbendazim, but not benomyl, significantly increased the weight of ventral prostate plus seminal vesicles. Co-treatment with TP and 50 or 100 mg/kg/day flutamide significantly decreased these repro ductive accessory gland weights when compared with TP alone. Based on our previous report, carbendazim increases mRNA and protein expression of AR in testis, epididymis and prostate and antagonizes the reduced tissue weights of