Siebert Marília Nardelli, Mattos Jacó Joaquim, Toledo-Silva Guilherme, Razzera Guilherme, Bainy Afonso Celso Dias
Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry - LABCAI, Federal University of Santa Catarina, UFSC, Florianópolis, Santa Catarina, Brazil.
Aquaculture Pathology Research Center - NEPAQ, Federal University of Santa Catarina, UFSC, Florianópolis, Santa Catarina, Brazil.
Aquat Toxicol. 2017 Aug;189:142-149. doi: 10.1016/j.aquatox.2017.06.004. Epub 2017 Jun 9.
Vertebrate cytochrome P450 1 (CYP1) enzymes metabolize endogenous and xenobiotic compounds and usually demonstrate a substrate-inducible response. Ethoxyresorufin O-deethylase activity (EROD) is a common method to quantify CYP1 enzymes activity in these organisms. Despite the absence of this gene family in protostomes, CYP1-like genes were identified in several species, even though no evolutionary relationship has been established with the vertebrate CYP1 family. In the present study, EROD activity was evaluated in microsomal fraction of gills, digestive gland and mantle of Crassostrea gigas. Enzyme activity was quantified in gills, although no activity was detected in digestive gland and mantle. EROD kinetic characterization in gills using typical Michaelis-Menten equation demonstrated an apparent K of 1.15μM and V of 229.2 fmol.minmg.protein . EROD activity was analyzed in the presence of CYP1 inhibitors, ellipticine (ELP), furafylline (FRF), clotrimazole (CTZ), α-naphthoflavone (ANF), and the non-ionic surfactant Triton X-100. CTZ inhibited EROD activity in all tested concentrations while Triton X-100 (0.5mM) caused 16% inhibition. Transcript levels of four CYP1-like genes were determined in gills, digestive gland and mantle. In general, CYP1-like genes showed higher transcript levels in gills compared to other tissues. The transcript levels of CYP1-like 1 and 2, analyzed together, positively correlated with EROD activity observed in gills, suggesting the possible involvement of these two gene products in EROD activity in this tissue. Homology models of translated CYP1-like 1 and 2 were generated based on human CYP1A1 structure and were similar to the general canonical cytochrome P450 fold. Molecular docking analysis showed that the two putative oyster CYP1-like structures have the potential to metabolize 7-ethoxyresorufin (7-ER), although the contribution of other CYP1-like genes needs to be investigated. Proteins encoded by CYP1-like 1 and 2 genes are plausible candidates for EROD activity observed in gills of C. gigas.
脊椎动物细胞色素P450 1（CYP1）酶可代谢内源性和外源性化合物，通常表现出底物诱导反应。乙氧基异吩恶唑酮-O-脱乙基酶活性（EROD）是定量这些生物体中CYP1酶活性的常用方法。尽管原口动物中不存在这个基因家族，但在几个物种中发现了CYP1样基因，尽管尚未与脊椎动物CYP1家族建立进化关系。在本研究中，对太平洋牡蛎鳃、消化腺和外套膜的微粒体部分进行了EROD活性评估。在鳃中对酶活性进行了定量，但在消化腺和外套膜中未检测到活性。使用典型的米氏方程对鳃中的EROD进行动力学表征，结果显示表观K为1.15μM，V为229.2 fmol·min·mg蛋白。在存在CYP1抑制剂椭圆玫瑰树碱（ELP）、呋拉茶碱（FRF）、克霉唑（CTZ）、α-萘黄酮（ANF）以及非离子表面活性剂曲拉通X-100的情况下，对EROD活性进行了分析。CTZ在所有测试浓度下均抑制EROD活性，而曲拉通X-100（0.5 mM）导致16%的抑制。测定了鳃、消化腺和外套膜中四个CYP1样基因的转录水平。总体而言，与其他组织相比，CYP1样基因在鳃中的转录水平更高。共同分析的CYP1样1和2的转录水平与在鳃中观察到的EROD活性呈正相关，表明这两种基因产物可能参与了该组织中的EROD活性。基于人CYP1A1结构生成了翻译后的CYP1样1和2的同源模型，其与细胞色素P450的一般典型折叠相似。分子对接分析表明，两种假定的牡蛎CYP1样结构具有代谢7-乙氧基异吩恶唑酮（7-ER）的潜力，尽管其他CYP1样基因的作用仍需研究。CYP1样1和2基因编码的蛋白质可能是在太平洋牡蛎鳃中观察到的EROD活性的候选者。
