農藥貝芬替與其前驅物免賴得誘發大鼠生殖與發育毒性作用機制之研究

Abstract

貝芬替與免賴得屬benzimidazole類化合物之殺菌劑農藥，可廣泛用於各種農作物抗菌與殺蟲用。本研究目的在探討農藥貝芬替與其前驅物免賴得誘發大鼠生殖與發育毒性可能作用機制。貝芬替在低、中劑量時隨劑量增加而降低睪丸絶對與相對重，而在高劑量時因睪丸腫脹而增加組織重。貝芬替也隨劑量增加而降低副睪絶對與相對重。貝芬替隨劑量顯著增加睪丸與副睪組織傷害指數，也隨劑量降增加而低副睪尾精子活動力與濃度。免賴得也有相似作用。生體外，貝芬替顯著隨濃度增加而增加取代放射性標幟二氫睪固酮與睪丸中雄性素受體結合率；生體內，貝芬替隨劑量增加而增加睪丸與副睪中雄性素受體結合活性。因此，隨貝芬替劑量增加，睪丸與副睪在組織重降低、精子品質下降及組織病理傷害程度等均與雄性素受體活性呈正相關。同時處理貝芬替與雄性素受體拮抗劑Flutamide，隨Flutamide劑量增加而顯著緩解貝芬替降低雄大鼠睪丸絶對與相對重，也改善副睪尾精子活動力與濃度，但未達顯著水準，及顯著改善睪丸與副睪組織病理傷害指數。聚合酶鏈鎖反應、組織免疫染色及西方墨點法分别顯示，貝芬替隨劑量增加睪丸中雄性素受體mRNA及蛋白表現，Flutamide則有相反作用，同時處理貝芬替與Flutamide，隨Flutamide劑量可抑制睪丸中雄性素受體mRNA及蛋白表現。 雌、雄大鼠分別處理貝芬替或免賴得28天後配種，兩藥劑均誘發8週齡仔代雌大鼠子宮角單側萎縮、尿道口雄性化及陰道口缺失，貝芬替更在兩側子宮角誘發一對貯精囊，免賴得誘發8週齡仔代雄大鼠左右睪丸與左右副睪萎縮。雌大鼠於懷孕第0至20天時口服投予6.25、12.5及25 mg/kg貝芬替，0.6、2.5及10 mg/kg flutamide，或25與50 mg/kg免賴得。對仔代雄大鼠而言，處理12.5與25 mg/kg貝芬替可誘發增加出生後第2天肛門-陰莖距，它是一種雄性素作用指標。投予免賴得同樣可增加仔代雄大鼠肛門-陰莖距。當同時處理貝芬替與flutamide時可阻斷貝芬替所增加之肛門-陰莖距。貝芬替與免賴得對仔代雄大鼠誘增之肛門-陰莖距，在仔代出生後第22天及其後即漸漸消失。貝芬替對出生後56天仔代雄大鼠其它與雄性素有關之指標如睪丸與副睪異常、陰莖尿道下裂及貯精囊與球海棉體肌等組織重無明顯作用，貝芬替可拮抗flutamide對上述與雄性素有關之組織重。對仔代雌大鼠而言，貝芬替可與flutamide產生協同作用，較單處理flutamide更增加出生後56天雌大鼠之肝與腎重。貝芬替對仔代雌大鼠生殖器官無明顯作用。這些結果顯示，懷孕期間暴露貝芬替對仔代雄大鼠誘發暫時性且微弱的雄性素激性作用，另外，貝芬替對仔代雌大鼠促進flutamide所誘發增加肝與腎重。 研究結果發現雄性素受體在雄大鼠生殖毒性與雌、雄大鼠發育毒性扮演重要角色，雄性素受體可能參與雄大鼠睪丸毒性及仔代雌、雄大鼠生殖器官發育毒性作用。

Both carbendazim and benomyl are antifungal benzimidazole compounds used to control fungal proliferation on a large variety of crops. The study was conducted to study the possible mechanism of reproductive and developmental toxicity induced by carbendazim and its precursor benomyl in rats. Treatment with low and middle doses of carbendazim decreased the absolute and relative testis weights, while high doses increased the testis weight more than that of middle dose due to inflammation. Carbendazim also decreased the absolute and relative weights of the epididymis in a dose-dependent manner. In a addition, carbendazim increased the lesion index of testis and epididymis, and decreased sperm motility and concentration of cauda epididymis in a dose-dependent manner. Benomyl exerted the same effect on the male rat as previously described for carbendazim. Carbendazim replaced binding of [3H]-5α-dihydrotestosterone to androgen receptors (AR) of testis extract from untreated rats in a concentration-dependent manner in vitro. Treatment of male rats with carbendazim for 56 d produced dose-dependent increases of androgen receptor concentrations in testis and epididymis. Therefore, there was a high correlation between reduction of testis and epididymis weights, decrease in sperm quality, and increase in the lesion index and androgen receptor concentration in male rats. Co-treatment of male rats with carbendazim and flutamide once daily for 28d prevented the decrease of testis weight induced by treatment with carbendazim alone. In addition, the co-treatment prevented losses of spermatozoa and in cell morphology and the decrease of sperm concentration induced by carbendazim. Treatment with carbendazim increased the expressions of AR mRNA and protein of rat testis while flutamide decreased the same expressions. Co-treatment with carbendazim and flutamide decreased the expressions of AR mRNA and protein of rat testis in a dose-dependent manner. Premating treatment of male and female rats with carbendazim for 28 d produced androgenic effects including incomplete development of the uterine horn, enlargement of the uretha, absence of the vagina, and induction of seminal vesicles in female offspring, without marked effects in male offspring. Premating treatment with benomyl resulted in incomplete development of the uterine horn and absence of the vagina in female offspring and produced testis and epididymis atrophy in male offspring. Pregnant female rats were treated with 6.25, 12.5, or 25 mg/kg carbendazim and 0.6, 2.5, or 10 mg/kg flutamide by gavage once daily from day 0 to 20. Another group of rats were treated with 25 and 50 mg/kg benomyl, the parent compound of carbendazim for comparison purposes. In male offspring, 12.5 and 25 mg/kg carbendazim increased anogenital distance (AGD), an androgen-dependent marker, on PND 2. Treatment with benomyl also increased AGD. Cotreatment with 0.6, 2.5, and 10 mg/kg flutamide blocked the androgenic effect on AGD induced by 25 mg/kg carbendazim. The androgenic effects of carbendazim and benomyl on AGD were reversible on PND 22 and later. Carbendazim had no effects on other androgen-dependent markers including testis and epididymis malformations, hypospadias, and organ weights of the seminal vesicle and levator ani bulbocavernosus muscle on PND 56. Surprisingly, carbendazim antagonized the antiandrogenic effects on these markers induced by flutamide cotreatment. In female offspring, carbendazim produced synergistic effects on the flutamide cotreatment-mediated increases of organs weights in liver and kidney on PND 56. Carbenazim had no marked effects on female reproductive organs. These findings show that carbendazim exposure in utero displays a transient and weak androgenic effect and reduces flutamide antiandrogenicity in male rats. The fungicide enhances flutamide-mediated liver and kidney weight increases in female rats. We concluded that AR plays an important role in the reproductive and developmental toxicity in rats.