B Rees Christopher, McCormick Stephen D, Vanden Heuvel John P, Li Weiming
Department of Fisheries and Wildlife, 13 Natural Resources Building, Michigan State University, East Lansing, MI 48824, USA.
Aquat Toxicol. 2003 Jan 10;62(1):67-78. doi: 10.1016/s0166-445x(02)00062-0.
Environmental pollutants are hypothesized to be one of the causes of recent declines in wild populations of Atlantic salmon (Salmo salar); across Eastern Canada and the United States. Some of these pollutants, such as polychlorinated biphenyls and dioxins, are known to induce expression of the CYP1A subfamily of genes. We applied a highly sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), for measuring the levels of CYP1A induction in Atlantic salmon. This assay was used to detect patterns of CYP1A mRNA levels, a direct measure of CYP1A expression, in Atlantic salmon exposed to pollutants under both laboratory and field conditions. Two groups of salmon were acclimated to 11 and 17 degrees C, respectively. Each subject then received an intraperitoneal injection (50 mg kg(-1)) of either beta-naphthoflavone (BNF) in corn oil (10 mg BNF ml(-1) corn oil) or corn oil alone. After 48 h, salmon gill, kidney, liver, and brain were collected for RNA isolation and analysis. All tissues showed induction of CYP1A by BNF. The highest base level of CYP1A expression (2.56 x 10(10) molecules/microg RNA) was found in gill tissue. Kidney had the highest mean induction at five orders of magnitude while gill tissue showed the lowest mean induction at two orders of magnitude. The quantitative RT-PCR was also applied to salmon sampled from two streams in Massachusetts, USA. Salmon liver and gill tissue sampled from Millers River (South Royalston, Worcester County), known to contain polychlorinated biphenyls (PCBs), showed on average a two orders of magnitude induction over those collected from a stream with no known contamination (Fourmile Brook, Northfield, Franklin County). Overall, the data show CYP1A exists and is inducible in Atlantic salmon gill, brain, kidney, and liver tissue. In addition, the results obtained demonstrate that quantitative PCR analysis of CYP1A expression is useful in studying ecotoxicity in populations of Atlantic salmon in the wild.
环境污染物被认为是导致加拿大东部和美国野生大西洋鲑（Salmo salar）种群数量近期下降的原因之一。其中一些污染物，如多氯联苯和二恶英，已知会诱导CYP1A基因亚家族的表达。我们应用了一种高度灵敏的技术，即定量逆转录-聚合酶链反应（RT-PCR），来测量大西洋鲑中CYP1A的诱导水平。该检测方法用于检测在实验室和野外条件下暴露于污染物的大西洋鲑中CYP1A mRNA水平的模式，CYP1A mRNA水平是CYP1A表达的直接测量指标。两组鲑鱼分别适应11摄氏度和17摄氏度。然后，每组实验对象腹腔注射（50毫克/千克）玉米油（10毫克β-萘黄酮/毫升玉米油）中的β-萘黄酮（BNF）或仅注射玉米油。48小时后，采集鲑鱼的鳃、肾、肝和脑用于RNA分离和分析。所有组织均显示BNF诱导了CYP1A的表达。在鳃组织中发现CYP1A表达的最高基础水平（2.56×10¹⁰分子/微克RNA）。肾脏的平均诱导倍数最高，达五个数量级，而鳃组织的平均诱导倍数最低，为两个数量级。定量RT-PCR还应用于从美国马萨诸塞州两条溪流采集的鲑鱼样本。从已知含有多氯联苯（PCBs）的米勒斯河（南罗亚尔斯顿，伍斯特县）采集的鲑鱼肝和鳃组织，与从一条无已知污染的溪流（四英里溪，北菲尔德，富兰克林县）采集的样本相比，平均诱导倍数高出两个数量级。总体而言，数据表明CYP1A存在于大西洋鲑的鳃、脑、肾和肝组织中且可被诱导。此外，所获得的结果表明，对CYP1A表达进行定量PCR分析有助于研究野生大西洋鲑种群的生态毒性。
