Measurement of DNA damage in marine organisms

Several methods have been described to quantify UV-B-induced DNA photo-products. Assays can be divided into chromatographical, biochemical and immunochemical assays. In chromatographical assays, DNA is degraded by acid hydrolysis to the bases, after which the undamaged bases and photoprodilcts are separated by paper chromatography [54,55], thin-layer chromatography [56] or high-performance liquid chromatography [57]. In biochemical assays [58,59], photoproduct specific endonucleases are used to cleave DNA at photoproduct sites. The amount of photoproducts is then calculated from the molecular weight distribution of the single-stranded DNA fragments. Immunochemical assays use antibodies directed to a DNA photoproduct. Antibody binding can be quantified using radiolabeled DNA [9,60,61] or a secondary antibody which may be coupled to a fluorescent label [10,62], a gold particle [63] or an enzyme. This enzyme converts a substrate into a fluorescent [64] or colored product [65,66], or catalyzes a reaction resulting in the emission of light [67].

Karentz et al. [9] were the first to describe the occurrence of CPDs in (Antartic) marine phytoplankton cultures. In their study, DNA was isolated from the cells before photoproducts were quantified. Later, Buma et al. [10] developed a method to monitor UV-B-induced DNA damage in individual phytoplankton cells using immunofluorescent labeling in combination with flow cytometry. This method allows for the study of DNA damage in individual cells in relation to other flow cytometric parameters (e.g., cell size, pigments or DNA content). The application of this method for field studies, however, has some major disadvantages. First of all, results may be very difficult to interpret for complex field samples because the immunofluorescence of damaged cells may be masked by background fluorescence of other (larger) species. Moreover, small cells such as bacterio- or picophytoplankton cells may not contain enough DNA to give reliable fluorescence signals. The commonly used methods to quantify CPDs in aquatic organisms are, therefore, those where DNA is extracted from biological material collected by filtration. Often samples are fractionated during filtration to roughly distinguish between functional groups, (i.e., viruses, bacteria or phytoplankton). DNA extracts are then labeled with a CPD specific antibody, after which detection of antibody binding is done either using radiolabeling or using a secondary antibody coupled to an enzyme that converts a reaction substance resulting in luminescence [50].

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