長期暴露環境相關濃度的三泰隆透過DAF-16加速秀麗隱桿線蟲的老化過程

Abstract

Triadimenol (三泰隆) 是農業上廣為使用的廣效型抗真菌劑，因為性質穩定不易分解，容易殘留於環境與食物中造成長期暴露風險。Triadimenol可造成基因體不穩定及干擾細胞許多轉錄因子的功能，然而，鮮少研究探討長期暴露triadimenol是否加速生物老化。本研究以C. elegans探討環境相關濃度的triadimenol的老化相關毒性及機制。研究結果發現0.1 % ETOH (酒精) 的溶劑控制組並不會顯著影響C. elegans的移動行為 (locomotive behaviors) ，而且也不會對C. elegans的繁殖與生長造成不良影響，因此所有實驗皆以0.1 % ETOH作為控制組。 Triadimenol在環境相關濃度 (3、30、300 μg/L) 的暴露顯著影響C. elegans在第0天成蟲的體長，在30 與300 μg/L暴露下顯著減少C. elegans的繁殖能力；並且在3、30、300 μg/L顯著抑制C. elegans的身體彎曲次數 (body bends) 與頭部擺動頻率 (head thrashes)，而且毒性呈劑量-反應關係。結果顯示暴露於最有效應濃度300 μg/L triadimenol的C. elegans的平均壽命、壽命中位數與最大壽命由控制組的17.3、18、29天分別縮短為15、15、25天。綜合觀察C. elegans的老化相關行為如咽喉部收縮 (pumping rate) 與排泄週期 (defecation cycle)，發現長期暴露triadimenol (300 μg/L) 顯著加速了以上兩種行為的退化，證明triadimenol加速了C. elegans的生理老化。此外，triadimenol (300 μg/L) 顯著增加第4、8天成蟲的脂褐素累積量，但是在第0、4天成蟲沒有顯著改變脂質過氧化物的累積量，只有第8天成蟲才顯著增加脂質過氧化程度。進一步量測生物體內氧化壓力如H2O2與O2•‒指標，發現在第0、4天成蟲皆無顯著差異，而在第8天成蟲triadimenol顯著增加氧化活性物質的累積量，與脂質過氧化物累積的變化趨勢相似。本研究更進一步利用轉基因C. elegans發現長期的triadimenol (300 μg/L) 暴露造成C. elegans體內DAF-16 (DAuer Formation) 的去磷酸化轉移入核，顯示triadimenol的暴露對C. elegans造成了氧化壓力；但是SKN-1 (SKiNhead) 並未對triadimenol暴露有所反應，顯示SKN-1與triadimenol造成之毒性效應無直接關係。綜合結果，本研究發現長期暴露於環境相關濃度的triadimenol可對C. elegans造成氧化壓力，並透過DAF-16調控機制加速C. elegans的老化。

Triadimenol is a widely used agricultural antifungal which is commonly detected in the environment due to its stable property. Triadimenol is able to disrupt genomic stability and modulate function of several transcription factors, yet the prolonged exposure of triadimenol and its age-related toxicity effects remain elucidated. This study utilized Caenorhabditis elegans (C. elegans) to study the toxicity of triadimenol on aging and the underlying mechanisms. The results showed that 0.1 % ETOH (ethanol) as solvent control did not affect the locomotive behaviors, reproduction, and growth in C. elegans. Therefore, 0.1 % ETOH was used as control throughout this study. Triadimenol at environmentally relevant concentrations (3, 30, 300 μg/L) significantly affected worm’s growth, and reduced the total brood size at 30 and 300 μg/L. Additionally, prolonged exposure to 3, 30, 300 μg/L triadimenol significantly inhibited locomotive behaviors of C. elegans in a dose-dependent manner. Moreover, under the most effective concentration, 300 μg/L triadimenol reduced C. elegans mean (17.3 days), median (18 days), and maximum (29 days) lifespan at 15, 15, 25 days, respectively. Further evidence showed that triadimenol (300 μg/L) accelerated the decline in pharyngeal pumping rate and the increase in defecation cycles, implying physiological aging. In addition, triadimenol (300 μg/L) significantly increased lipofuscin accumulation in day- 4, 8 adulthood, but did not significantly increase lipid peroxidation until day 8 adulthood. Similarly, triadimenol (300 μg/L) did not significantly elevate internal ROS (reactive oxidative species) levels such as H2O2 and O2•‒ until day 8 adulthood. By using the transgenic GFP strains, we found that triadimenol (300 μg/L) triggered DAF-16 translocation from cytosol into nucleus in C. elegans, suggesting that triadimenol exerted oxidative stress on C. elegans. In contrast, triadimenol did not affect SKN-1 translocation, suggesting that triadimenol toxicity in C. elegans is not SKN-1 mediated. In conclusion, this study demonstrated that prolonged exposure to triadimenol at environmentally relevant concentration resulted in oxidative stress and accelerated aging process in C. elegans via DAF-16.