Longo V, Marini S, Salvetti A, Angelucci S, Bucci S, Gervasi P G
Laboratory of Pharmacogenetic and Drug Metabolism, Istituto di Fisiologia Clinica, CNR, via Moruzzi, 1-56100 Pisa, Italy.
Aquat Toxicol. 2004 Aug 25;69(3):259-70. doi: 10.1016/j.aquatox.2004.06.001.
In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with beta-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with beta-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin-O-demethylase and ethoxyresorufin-O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin-O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.
在本研究中，我们通过使用这些酶的典型底物以及主要针对哺乳动物酶产生的多克隆抗体，检测了对照及经β-萘黄酮、苯巴比妥和二氯苯腈预处理的意大利水蛙肝脏和鼻黏膜中I相和II相药物代谢酶的存在情况及诱导性。当进行报道时，发现这种青蛙肝脏中的细胞色素P450（CYP）含量以及各种单加氧酶和II相酶活性与大部分水生和半水生青蛙的相似。用β-萘黄酮处理导致肝脏中CYP1A的诱导，这种诱导通过以下方式表现出来：（a）使用抗大鼠CYP1A1进行免疫印迹分析；（b）CYP1A介导的甲氧基试卤灵-O-脱甲基酶和乙氧基试卤灵-O-脱乙基酶活性增加。用苯巴比妥和二氯苯腈处理在肝脏中对所检测的酶均未产生任何影响。当分析水蛙的鼻黏膜时，测定了各种单加氧酶和II相酶活性，这些活性通常与肝脏中的相当。然而，通过使用抗三种不同类别的谷胱甘肽S-转移酶（GST）抗体，我们发现在这两种组织的胞质溶胶中有不同的反应性，表明这两种组织对外源化合物毒性作用的敏感性存在差异。在鼻黏膜中，还发现了一种与CYP2A相关的蛋白质以及与该同工型在哺乳动物中相关的单加氧酶活性（即乙氧基香豆素-O-脱乙基酶和香豆素-7-羟化酶）。用水蛙除草剂二氯苯腈处理会降低上述活性以及免疫反应性CYP2A载脂蛋白。用细胞色素P450抑制剂甲吡酮预处理可保护CYP2A载脂蛋白及其相关活性免受二氯苯腈的毒性作用，表明该酶在这种除草剂的生物活化中起关键作用。本研究结果表明，肝脏中CYP1A的诱导和鼻黏膜中类似CYP2A的抑制情况可能为意大利水蛙接触环境和水生污染物提供两种潜在的生物标志物。
