EPOC-LPTC, UMR CNRS 5805, Université de Bordeaux 1, Talence 33405, France.
Mar Environ Res. 2013 Mar;84:17-23. doi: 10.1016/j.marenvres.2012.11.003. Epub 2012 Nov 20.
Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3',4,4',5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.
二恶英类化合物（DLC）通过激活芳烃受体（AhR）诱导鱼类早期生命阶段产生毒性反应，而 AhR 通常通过乙氧基 RESO 脱乙基酶（EROD）活性来评估。本研究开发了一种新的荧光分光光度法来定量评估海洋模式鱼类美洲蟾鱼个体胚胎和幼鱼的 EROD 活性。该方法基于在 5 小时孵育乙氧基 RESO 后，通过单个活胚胎或幼鱼产生的 RESO 进行测量。从 2.5 到 50 pg·卵 ⁻¹ 用 3,3',4,4',5-五氯联苯（PCB126）处理刚受精的卵，PCB126 是一种典型的 DLC。在胚胎（受精后 7 天）和幼鱼（受精后 16 天）中评估 EROD 活性。RESORUFIN 分别在培养基（25‰海水）和整个鱼匀浆中进行测量，以评估体内保留的百分比。体内产生的 RESO 中约有 95%和 17%分别保留在胚胎和幼鱼中。胚胎匀浆和幼鱼培养基中的 EROD 活性随剂量呈线性增加。体内法测量的 EROD 活性与使用 S9 部分的传统体外技术高度相关，无论是胚胎还是幼鱼均如此。用 PCB126 预处理的胚胎和幼鱼中，体内和体外 EROD 活性均高于对照组。体内和体外方法均能测量到幼鱼的 EROD 诱导，而胚胎的诱导则是体内法高于体外法。体内法比体外法更敏感、更可靠，所需的鱼数量更少（4 条鱼比 3 个 5 条鱼的池少）。该体内法可用于将个体胚胎或幼鱼的 EROD 诱导与其他机体水平的反应联系起来。应进行其他类别的外源性化学物质的进一步研究，以评估对 RESO 吸收/排泄过程的潜在毒性影响，这可能会影响体内测量。
