康唑類殺菌劑和人造雌激素混合暴露干擾青鱂魚內分泌作用相關基因表達之研究

Abstract

康唑類［conazoles，含咪康唑 (imidazole) 類和三康唑 (triazole) 類］藥劑為廣泛應用於農業及醫療中的殺真菌劑。研究指出此類殺菌劑會干擾試驗生物的生殖及發育，但其毒理作用機制、及與環境中雌性激素物質的交互作用對水生生物內分泌系統的影響至今尚未清楚。本研究目的為利用青鱂魚 (Oryzia latipes, medaka) 作為模式生物，探討康唑類殺菌劑與雌激素混合暴露下對魚體內分泌作用相關基因表達之影響。本實驗我們選擇ketoconazole (KET)及letrozole (LET)分別作為咪康唑類和三康唑類的代表化合物，並且利用不同性別之成熟的青鱂魚進行長達14天的暴露試驗，試驗共分成3組，第一組為控制組（除氯自來水）和溶劑控制組（含有0.01% acetone），第二組為200 ng/L EE2 單一化合物暴露組，第三組為KET（10 μg/L、 50 μg/L 及250 μg/L） 或LET（10 μg/L、50 μg/L 及250 μg/L） 與200 ng/L EE2 的混合物暴露組。暴露結束後以即時定量聚合酶連鎖反應 (RT-qPCR) 分析肝臟中生殖相關基因（VTGI、VTGII和 ERα）、雌激素代謝相關基因（CYP1A 和3A）以及性腺中雌激素生成相關基因（CYP17A、11B和19A）的表達量變化。與控制組相較，雄魚或雌魚受到200 ng/L EE2單獨暴露後之VTGI、VTGII和 ERα基因表達量都顯著提高。與200 ng/L EE2單一暴露組相較，KET (10-250 μg/L) 與EE2 (200 ng/L) 的混合暴露造成雄魚體中VTGI、VTGII和 ERα基因表達量增加；在雌魚體中則觀察到相反的狀況：KET (10-250 μg/L) 或LET (10-250 μg/L) 與EE2 (200 ng/L) 混合暴露是造成VTGI和VTGII基因表達量降低。而KET或LET對雌激素代謝或生成相關基因的表達並無造成顯著的影響。此研究結果表示，當水中有康唑類殺真菌劑與EE2共同存在時，不同性別的青鱂魚受到EE2所誘導的雌激素活性（VTGs和ERα的mRNA表達）完全相反。然而我們未來需要更多關於相關基因之蛋白表達或活性等資訊，才能由機制面詳細解釋康唑類殺真菌劑與EE2共同存在時對魚體之雌激素代謝、生合成及生殖等方面的影響。

Conazoles are a class of imidazole- or triazole-containing pesticides widely used in agricultural or medical field. Conazoles are environmentally-important fungicides that have received significant attention from regulatory agencies and scientists due to frequent occurrence in the aquatic environment. While all conazoles have anti-antifungal activity, some members of the conazole family have potential adverse effects in humans as they induce liver or thyroid tumors in rodents and cause endocrine disrupting effects in experimental animals. The objective of the study is to evaluate ecotoxicological effects of two prototypic conazoles, ketoconazole (KET, an immidazole) and letrozole (LET, a triazole) and assess their modes of toxic action via assessing combined endocrine relative gene expression disrupting potency of mixtures of conazoles and an environmental estrogen (17α-ethinyl estradiol, EE2) in medaka fish (Oryzias latipes). We have treated adult medaka of males and females respectively with three sets of experimental solutions for a 14 days’ aqueous exposure including a single EE2 (200 ng/L), a binary mixture of EE2 (200 ng/L) with KET (10, 50 or 250 μg/L) or LET (10, 50 or 250 μg/L) and blank controls. Gene expression of reproduction relative gene (VTGI, VTGII & ERα), and gene encoded cytochrome P450 (CYP) enzymes responsible for metabolism of endogenous or exogenous hormones in liver or gene encoded CYP enzymes responsible for synthesizing endogenous hormones in gonad of fish from control and treated groups was assessed with real time quantitative polymerase chain reaction (RT-qPCR). Results show that EE2 (200 ng/L) significantly increased hepatic VTGI, VTGII and ERα gene expression in both male and female medaka compared to the blank control. Compared with the single EE2 (200 ng/L) exposure, mRNA expression of both VTG genes was elevated in male fish from mixtures of EE2 (200 ng/L) with KET (10-250 μg/L); however, mixtures of EE2 (200 ng/L) with either KET (50-250 μg/L) or LET (10-250 μg/L) significantly decreased expression of VTGI or VTGII in livers from female fish. The correlation between VTG and ERα and the expression of the estrogen-metabolizing enzymes (e.g. CYP1A & 3A in liver) and estrogen- synthesizing enzymes (CYP19A in gonad) is not clear in this study. Our results show gender-dependent response of medaka fish to EE2-induced estrogenicity in the presence of conazole fungicides in water. More information about protein and activity of estrogen-metabolizing and estrogen-synthesizing enzyme after mixture of EE2 and conazole are needed to better understand the mechanism about endocrine disrupting action of KET or LET in medaka fish.