Environmental Toxicology, Department of Organismal Biology, Uppsala University, Norbyvägen 18A, SE-75236 Uppsala, Sweden.
Environmental Toxicology, Department of Organismal Biology, Uppsala University, Norbyvägen 18A, SE-75236 Uppsala, Sweden.
Aquat Toxicol. 2018 May;198:73-81. doi: 10.1016/j.aquatox.2018.02.016. Epub 2018 Feb 21.
Antifungal azoles are widely used in medicine, agriculture, and material protection and several antifungal azoles have been found in environmental samples. Although these compounds were designed to inhibit fungal enzymes such as lanosterol-14-demethylase (cytochrome P450 (CYP) 51), it is well established that the inhibitory actions of azoles are not specific for fungal CYP isozymes. We refined a gill filament assay to determine the inhibition of CYP1, measured as reduced 7-ethoxyresorufin-O-deethylase (EROD) activity, in rainbow trout (Oncorhynchus mykiss) gill tissue ex vivo. The advantage of this method is that both induction and inhibition of EROD are performed ex vivo. Among thirteen azoles studied, the five that caused the strongest inhibition of gill EROD activity at a concentration of 5 μM were selected for concentration-response assessment. These compounds (bifonazole, clotrimazole, imazalil, miconazole, and prochloraz) showed IC values ranging from 0.1 to 1.5 μM. CYP19 (aromatase) inhibition was measured using microsomes from rainbow trout brains. Concentration-response curves for CYP19 inhibition were determined for letrozole, bifonazole, clotrimazole, imazalil, miconazole and prochloraz, which gave IC values ranging from 0.02 to 3.3 μM. It was further found that mixtures of the five most potent azoles reduced both CYP1 and 19 catalytic activity in an additive fashion (IC = 0.7 μM and 0.6 μM, in the respective assay). Bifonazole (IC = 0.1 μM) is not previously known to inhibit CYP1 activity. The additive inhibition of CYP1 and CYP19 catalytic activity is an important finding of the present study. We conclude that this additive action of azoles could mediate adverse impacts on CYP regulated physiological functions in environmentally exposed fish.
抗真菌唑类药物在医学、农业和材料保护中被广泛应用，并且已经在环境样本中发现了几种抗真菌唑类药物。虽然这些化合物旨在抑制真菌酶，如羊毛甾醇 14-脱甲基酶（细胞色素 P450（CYP）51），但已经确定唑类化合物的抑制作用并非针对真菌 CYP 同工酶具有特异性。我们改进了一种鳃丝试验来确定 CYP1 的抑制作用，该抑制作用通过在虹鳟鱼（Oncorhynchus mykiss）鳃组织离体中减少 7-乙氧基resorufin-O-脱乙基酶（EROD）活性来测量。这种方法的优点在于，EROD 的诱导和抑制都是在离体中进行的。在所研究的 13 种唑类化合物中，选择了在 5µM 浓度下对鳃 EROD 活性抑制最强的五种唑类化合物进行浓度反应评估。这些化合物（双氯芬酸、克霉唑、咪康唑、咪康唑和丙环唑）的 IC 值范围为 0.1 至 1.5µM。使用来自虹鳟鱼大脑的微粒体测量 CYP19（芳香酶）抑制。确定了用于来曲唑、双氯芬酸、克霉唑、咪康唑、咪康唑和丙环唑的 CYP19 抑制的浓度反应曲线，这些化合物的 IC 值范围为 0.02 至 3.3µM。进一步发现，五种最有效的唑类混合物以相加的方式降低了 CYP1 和 19 的催化活性（在各自的测定中，IC 值分别为 0.7µM 和 0.6µM）。双氯芬酸（IC 值为 0.1µM）以前未知具有抑制 CYP1 活性的作用。CYP1 和 CYP19 催化活性的相加抑制是本研究的一个重要发现。我们得出的结论是，唑类化合物的这种相加作用可能会介导环境暴露的鱼类中 CYP 调节的生理功能的不良影响。
