普克利誘導青鱂魚氧化壓力及相關毒性效應之研究

Abstract

普克利 (propiconazole) 為農業生產中常用之三唑類 (triazoles) 系統型殺真菌劑，廣泛用於預防或控制蔬菜、水果、穀物、種子和木材等農產品的真菌感染。而環境水體如河流、表面水、城市和工業廢水等已廣泛檢測出普克利之殘留 (0.012 -13 μg/L)。文獻研究指出普克利會誘發小鼠肝腫瘤的產生，因而被歸為人類可能致癌物；此外，普克利也會誘導虹鱒體內活性氧物種 (reactive oxygen species, ROS)，干擾魚體內抗氧化系統的衡定，進而導致氧化壓力 (oxidative stress) 或傷害之發生。然而普克利對於水生生物引起之氧化壓力與其致癌性間之關聯性研究仍有限。因此，本研究以日本青鱂魚 (Oryzias latipes, Japanese Medaka)野生品系及其p53突變品系之魚苗為模式生物進行連續28天之普克利 (2.5 – 250 μg/L) 暴露試驗，以量測日本青鱂魚苗體內總ROS強度與抗氧化酵素包含超氧歧化酶 (superoxide dismutase, SOD)、過氧化氫酶 (catalase, CAT) 和穀胱甘肽硫轉移酶 (glutathione s transferase, GST) 活性，並分析脂質與蛋白質過氧化等氧化傷害相關生物標記的變化。實驗結果顯示，日本青鱂魚苗連續暴露於普克利14 - 28天時，魚體ROS強度隨濃度上升而增加；普克利 (250 μg/L) 誘導魚體內GST之活性，並抑制SOD及CAT活性，進而導致魚體內脂質過氧化物丙二醛 (malonaldehyde) 及蛋白質氧化物蛋白質羰基 (protein carbonyl group) 含量顯著提高。此結果顯示普克利會誘導魚體產生氧化壓力，進而造成氧化傷害。普克利引起之氧化壓力或傷害，於停止暴露後會些微恢復。為探討普克利引起之氧化傷害是否會誘發肝臟進入癌化過程，將暴露後之魚苗飼養至成魚階段觀察組織病理改變，以瞭解普克利誘導之氧化壓力與肝臟癌化間之關係。結果顯示野生品系成魚之肝臟並無顯著病變發生，但在p53 突變種則可觀察到普克利促進肝細胞空泡化 (hepatocellular vaculoation)、海綿狀肝臟組織 (spongiosis hepatis)、肝囊腫(hepatic cyst) 和嗜酸性細胞 (eosinophilic foci)增多等組織形態之變化。此外，p53 肝臟當中有較多巨噬細胞 (macrophage) 或發炎殘體累積，顯示肝臟可能已呈現慢性發炎狀態。綜合以上結果，長時間暴露於非致死劑量的普克利會誘發青鱂魚魚苗體內ROS強度增加，並破壞抗氧化酵素系統的平衡，進而造成體內脂質與蛋白質氧化等氧化傷害效應，使肝臟組織病變及慢性發炎之產生，因而可能促使肝臟進入癌化過程。

Propiconazole is an environmentally important fungicide widely used in agriculture to prevent fungal growth on grasses, fruits, vegetables, cereals or seeds. It is frequently detected in wastewater, rivers and surface water at the μg/L level. Studies have demonstrated propiconazole is hepatotoxic and hepatotumorigenic in mice and induces oxidative stress (OS) in aquatic animals. The objective of this study is to understand modes of propiconazole-induced OS associated with hepatocarcinogenesis using early life stages of Japanese medaka (Oryzias latipes) and p53 mutant as model organisms. We have treated larvae of wildtype or p53 mutant medaka (14 day post hatching, dph) with propiconazole solutions at sub-lethal concentrations (2.5, 25 and 250 μg/L) for a 28-day continuous aqueous exposure and then reared fish in embryo rearing medium until adults. At each time point, wildtype larvae were harvested for analyses of intracellular reactive oxygen species (ROS) levels and biomarker assays of OS and oxidative damages. Both strains of matured fish were sacrificed for histopathology analyses. Our results show that propiconazole induced intracellular ROS levels during 14-35 day’s exposure especially at higher concentration in wildtype larvae. A dose-dependent increase in glutathione transferase (GST) activity was revealed, while catalase (CAT) and superoxide dismutase (SOD) activities were inhibited with the dosage during the exposure period. Also, CAT and SOD activities were recovered to the control level without the propiconazole exposure. Also propiconazole were induced malondialdehyde and protein carbonyl groups contents in 28 and 14 day’s exposure respectively at 250 μg/L treatment. These results indicate that propiconazole can induce ROS and cause OS in fish, and result in oxidative injury. However, the propiconazole induced-OS or injury could be recovered if stressor was removed, but some oxidative damage may be irreversible and may result in carcinogenesis in fish. The adults of wildtype medaka fish didn’t have significant pathological changes in liver, but propiconazole induced hepatocellular vaculoation, spongiosis hepatis, hepatic cyst and eosinophilic foci in livers of p53 adults. We also found livers of p53 mutant had macrophage accumulation that may lead to chornic inflammation. Overall, propiconazole induced oxidative stress and oxidative injury and these promoted hepatocarcinogenesis in medaka fish.