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

本项目的目的是克隆和鉴定毛羽鱼（Chaetodon capistratus）肝组织诱导的细胞色素P450 cDNA，并建立细胞色素P450 mRNA和酶水平的定量分析。利用两种模式底物：1)β -萘黄酮（β - nf），一种3-甲基胆蒽型外源物；2)曲库对苯二酚，以及来自柳珊瑚的鱼阻化物，来表征C. capistratus细胞色素P450基因表达的诱导。利用第一种基质进行表征将解决C. capistratus在珊瑚礁环境中作为环境生物监测仪的潜在用途。利用第二底物的表征将解决鱼威慑化感化学物质对P450诱导的影响。综上所述，这两类外来化合物解毒的进化关系可以解决。此外，还将研究膳食化感物质对外源诱导的P450的潜在混淆作用。有或没有最近暴露于多环芳烃（PAH）的现场采样点将测试使用C. capistratus细胞色素诱导作为环境污染生物监测仪的可行性。珊瑚礁是生态和经济上重要的环境，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引起的慢性亚致死效应，虽然不容易明显，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。目前尚无多环芳烃在热带海洋生物中诱发P450的分子研究。C. capistratus P450的鉴定不仅将增加我们对这类重要的生物学酶的多样性的认识，更重要的是将提供一个分子探针，以高灵敏度评估生态和环境意义问题。特别是，细胞色素P450是否负责C. capistratus以柳珊瑚为食的能力，否则柳珊瑚会受到鱼阻化化物质的保护。这一发现对于理解海洋环境中的捕食和食草模式具有潜在的广泛意义。capistratus P450的分子特征和亚致死水平化学物质影响的分子探针的开发具有重要的环境和生态问题的适用性。

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.