Marine Biotech. Fellowship: Molecular Characterization of Cytochrome P450(s) in a Marine Fish: Applications to Environmental and Ecological Questions

本课题的目的是克隆和鉴定诱导细胞色素P450cDNA(S)，并建立细胞色素P450mRNA和酶水平的定量检测方法。细胞色素P450基因表达的诱导将使用两种模型底物来表征：1)β-萘黄酮(β-NF)，一种3-甲基胆蒽类型的异源化合物和2)CurcuH.Quinone，以及来自柳珊瑚Pseudopterogorgia rigida的具有鱼类威慑作用的化感物质。利用第一种底物进行表征将解决卡氏拟线虫在珊瑚礁环境中作为环境生物监测器的潜在用途。利用第二底物的鉴定将解决鱼类威慑化感物质对P450诱导的影响。综上所述，这两类外来化合物的解毒作用之间的进化关系可以得到解决。此外，还将调查饮食中的化感物质对外源诱导的P450的潜在混杂效应。有或没有最近接触多环芳烃(PAH)的现场采样点将测试使用卡氏弧菌细胞色素诱导作为环境污染生物监测的可行性。珊瑚礁是生态和经济上重要的环境，正日益受到有害的人为影响，但缺乏有效的监测机制。由多环芳烃和合成有机化学品污染造成的慢性亚致命影响，虽然不是很明显，但可能是致命的。检测低水平污染物的能力可能对防止长期损害至关重要。目前还没有分子研究表征多环芳烃在热带海洋生物中诱导的P450。Capstratus P450的特性不仅将增加我们对这类具有生物重要性的酶的多样性的了解，更重要的是将提供一个分子探针，用于高度灵敏地评估生态和环境意义的问题。特别是，细胞色素P450负责卡氏梭子虫以柳珊瑚为食的能力，否则这些珊瑚会被鱼类威慑化感物质防御。这一发现对于理解海洋环境中的捕食和草食模式具有潜在的广泛意义。因此，Capstratus P450的分子特征和分子探针的开发以测试化学物质在亚致死水平上的影响，因此对重要的环境和生态问题具有适用性。

The objectives of this project are to clone and characterize induced cytochrome P450 cDNA(s) from hepatic tissue of the fish, Chaetodon capistratus, and develop quantitative assays for cytochrome P450 mRNA and enzyme levels. Induction of cytochrome P450 gene expression will be characterized in C. capistratus using two model substrates: 1) beta-naphthoflavone (beta-NF), a 3- methylcholanthrene type xenobiotic and 2) curcuhydroquinone, and ichthyodeterrent allelochemical from the gorgonian coral, Pseudopterogorgia rigida. Characterization utilizing the first substrate will address the potential use of C. capistratus as an environmental biomonitor in coral reef environments. Characterization utilizing the second substrate will address the effect of an ichthyodeterrent allelochemical on P450 induction. Taken together, the evolutionary relationship between detoxification of these two classes of foreign compounds can be addressed. Furthermore, the potentially confounding effect of dietary allelochemicals on xenobiotically induced P450 will be investigated. Field sampling sites with and without recent polycyclic aromatic hydrocarbon (PAH) exposure will then test the feasibility of using C. capistratus cytochrome induction as a biomonitor of environmental contamination. Coral reefs are ecologically and economically important environments which are increasingly being subjected to detrimental anthropogenic influences, but effective monitoring mechanisms are lacking. Chronic, sublethal effects of pollution due to contamination by PAH and synthetic organic chemicals, though not readily apparent, can become lethal. The ability to detect low levels of contaminants may be crucial for preventing long-term damage. There are presently no molecular studies which have characterized a PAH-induced P450 in a tropical marine organism. Characterization of C. capistratus P450 will not only add to our knowledge of the diversity of this biologically important class of enzymes, but more importantly will provide a molecular probe with which to assess questions of ecological as well as environmental significance with great sensitivity. In particular, is cytochrome P450 responsible for the ability of C. capistratus to feed on gorgonian corals which are otherwise defended by ichthyodeterrent allelochemicals. This finding has potentially broad significance for understanding patterns of predation and herbivory in the marine environment. Molecular characterization of C. capistratus P450 and the development of molecular probes to test the effects of chemicals at sublethal levels thus has applicability to important environmental and ecological questions.