Brzuzan P, Jurczyk L, Luczyński M K, Góra M
Department of Environmental Biotechnology, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury in Olsztyn, Słoneczna 45G, 10-723 Olsztyn, Poland.
Pol J Vet Sci. 2006;9(2):139-43.
We developed a real-time PCR assay for measuring relative quantities (RQ) of p53 tumor suppressor mRNA in the whitefish (Coregonus lavaretus, Salmonidae, Teleostei). Real-time PCR primers for the p53 gene were designed from a region that was found to be conserved among salmonid p53 genes. To test for the usefulness of the assay we performed a treatment study, using benzo[a]pyrene (B[a]P) a putative p53-inducer. Two groups of hatchery raised whitefish, with an average body mass of 15 g and total length of 12 cm were either given an intraperitoneal injection (10 mg x kg(-1)) of B[a]P in corn oil (2 mg B[a]P ml(-1) corn oil) or corn oil alone (Control). After treatment (48 h, 7 degrees C), two random fish from each group were anesthetized and the liver, head kidney and brain were collected for mRNA isolation and analysis. In the control fish, relative quantification analysis based on the p53 mRNA levels in liver (RQ=1.00) showed higher basal levels of p53 mRNA in the head kidney (RQ= 1.69), and lower in the brain (RQ=0.41). In all three tissues sampled, p53 mRNA was affected by treatment with B[a]P. Liver tissue showed the greatest induction (RQ=1.53) from base levels (RQ=1.00), followed by brain (RQ=1.36), and head kidney (RQ=1.23). These results confirm that p53 mRNA is generally present at lower levels in differentiated tissues (liver and brain) than in those tissues with cell lines (head kidney), and demonstrate that p53 is moderately inducible by B[a]P in the whitefish. The approach presented here has the advantage of providing rapid and accurate measures of p53 induction in various tissues of fish responding to PAH contaminant exposure.
我们开发了一种实时聚合酶链反应（PCR）检测方法，用于测定白鲑（Coregonus lavaretus，鲑科，硬骨鱼纲）中p53肿瘤抑制基因mRNA的相对含量（RQ）。p53基因的实时PCR引物是根据在鲑科p53基因中发现的一个保守区域设计的。为了测试该检测方法的实用性，我们进行了一项处理研究，使用苯并[a]芘（B[a]P），一种假定的p53诱导剂。两组孵化场养殖的白鲑，平均体重15克，全长12厘米，一组腹腔注射（10毫克/千克）溶于玉米油（2毫克B[a]P/毫升玉米油）中的B[a]P，另一组仅注射玉米油（对照组）。处理后（48小时，7摄氏度），每组随机选取两条鱼麻醉，采集肝脏、头肾和脑用于mRNA分离和分析。在对照鱼中，基于肝脏中p53 mRNA水平的相对定量分析（RQ = 1.00）显示，头肾中p53 mRNA的基础水平较高（RQ = 1.69），而脑中较低（RQ = 0.41）。在所有三个采样组织中，p53 mRNA都受到B[a]P处理的影响。肝脏组织从基础水平（RQ = 1.00）诱导程度最大（RQ = 1.53），其次是脑（RQ = 1.36），头肾（RQ = 1.23）。这些结果证实，p53 mRNA在分化组织（肝脏和脑）中的水平通常低于具有细胞系的组织（头肾），并表明白鲑中p53可被B[a]P适度诱导。本文介绍的方法具有能够快速、准确地测量鱼类各种组织中p53对多环芳烃污染物暴露反应诱导情况的优点。
