Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis.
Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis.
复制标题
DOI：
10.1016/j.aquatox.2012.02.028
复制
发表时间：
2012-06-15
期刊：
影响因子：
--
通讯作者：
Powell WH
中科院分区：
文献类型：
--
作者：
Iwamoto DV;Kurylo CM;Schorling KM;Powell WH
细胞色素P450家族1（CYP 1）包括四个酶亚家族：CYP 1A、CYP 1B、CYP 1C和CYP 1D。在许多脊椎动物中，CYP 1A、1B和1C的表达由芳香烃受体的激动剂诱导，包括有毒污染物，如氯化二恶英、共面氯化联苯和多核芳烃。在mRNA、蛋白质或酶活性水平上进行评估，CYP 1 s（尤其是CYP 1A）是水生脊椎动物污染物暴露的有效和流行的生物标志物。烷基化试卤灵是用于检测、量化和描述细胞色素P450催化活性的合成底物。氧化特定试卤灵底物的能力可以区分单个CYP 1的催化活性。非洲爪蟾（Xenopus laevis）是水生毒理学中应用最广泛的两栖动物模型，但其CYP 1的数量、诱导和活性尚未得到系统的研究。在这里，我们报告的cDNA编码的两个新的CYP 1家族成员，X。沿着对4种已知X. laevis CYP 1B和CYP 1C的诱导作用进行了综合评价。奶牛CYP 1 s：CYP 1A 6、CYP 1A 7、CYP 1B和CYP 1C。使用XLK-WG，X.通过对小鼠肾上皮细胞系的EROD（乙氧基试卤灵底物）和MROD（甲氧基试卤灵）的诱导，我们确定了在2，3，7，8-四氯二苯并-p-二恶英（TCDD）暴露至250 nM后，EROD（乙氧基试卤灵底物）和MROD（甲氧基试卤灵）均被诱导3000- 5000倍，而BROD（苄氧基试卤灵）和PROD（戊氧基试卤灵）活性无论TCDD处理与否都检测不到。TCDD诱导CYP 1A 6和CYP 1A 7 mRNA增加2-3个数量级，而CYP 1B和CYP 1C无变化。更有效的AHR激动剂FICZ（6-甲酰吲哚并[3，2-B]咔唑）在0.1和250 nM之间的浓度下诱导CYP 1 B高达10倍，而CYP 1C诱导小于3倍。CYP 1B mRNA表现出最高的组成型mRNA表达，比其他CYP 1转录本高5- 75倍。总之，这些结果表明，CYP 1A 6和CYP 1A 7执行我们在这些细胞中观察到的大部分EROD和MROD活性。每个X的能力。laevis CYP 1催化各个试卤灵底物的氧化仍有待确定。将CYP 1 mRNA和诱导的AROD活性联系起来是澄清这些生物标志物的生化意义和CYP 1酶在X中的作用的重要一步。光滑。细胞培养的方法是一个重要的补充，长期使用的青蛙胚胎和蝌蚪的毒理学研究，提供了一个非常合适的模型系统，用于确定这些重要的生物标志物的污染物暴露的调节的分子机制。
Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250 nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2–3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250 nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75-fold greater than the other CYP1 transcripts. Taken together, these results suggest that CYP1A6 and CYP1A7 perform the bulk of EROD and MROD activities we observed in these cells. The ability of each X. laevis CYP1 to catalyze oxidation of individual resorufin substrates remains to be determined. Correlating CYP1 mRNA and induced AROD activity is a significant step toward clarifying the biochemical meaning of these biomarkers and the roles of CYP1 enzymes in X. laevis. The cell culture approach represents an important complement to the long standing use of frog embryos and tadpoles in toxicological studies, providing a well suited model system for determining the molecular mechanisms underlying the regulation of these important biomarkers of contaminant exposure.