257  2002 American Society for Photobiology 0031-8655/02 $5.00+0.00 Photochemistry and Photobiology, 2002, 75(3): 257–265 Development and Application of a Novel Immunoassay for Measuring Oxidative DNA Damage in the Environment ¶ David L. Mitchell* 1 , Jarah Meador 1 , Laxmi Paniker 1 , Didier Gasparutto 2 , Wade H. Jeffrey 3 and Jean Cadet 2 1 Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park/Research Division, Smithville, TX; 2 Laboratoire des Le ´ sions des Acides Nucle ´ iques, DRFMC/SCIB and UMR 5046, CEA Grenoble, Grenoble, France and 3 University of West Florida, Center for Environmental Diagnostics and Bioremediation, Pensacola, FL Received 8 August 2001; accepted 6 December 2001 ABSTRACT We developed a facile, cost-effective competitive binding assay for the analysis of 8-oxo-7,8-dihydro-2-deoxygua- nosine (8-oxodGuo) in DNA, using a polyclonal rabbit antiserum raised against an 8-oxodGuo hapten coupled to bovine serum albumin and radiolabeled synthetic li- gand containing multiple 8-oxodGuo residues. This ra- dioimmunoassay (RIA) displays a high affinity for 8- oxodGuo in DNA, with a detection limit of 1 adduct in 10 5 bases of DNA. 8-oxodGuo standards for RIA were quantified by high-performance liquid chromatography and electrochemical detection in DNA diluted in methy- lene blue and exposed to visible light. As an initial ap- plication we quantified 8-oxodGuo in dosimeters de- ployed at increasing depths in the Southern Ocean dur- ing the austral spring of the 1998 field season or at the surface at Palmer Station, Antarctica, throughout the 1999 field season. Cyclobutane pyrimidine dimers (CPD) were quantified using an established RIA. We found that the frequency of both photoproducts decreased with depth. However, CPD induction was attenuated at a fast- er rate than 8-oxodGuo, correlating with the differential attenuation of solar ultraviolet wavelengths in the water column. CPD induction was closely related with ultravi- olet-B radiation (UVB) attenuation, whereas the lower ¶Posted on the website on January 8, 2002. *To whom correspondence should be addressed at: The University of Texas M.D. Anderson Cancer Center, Science Park/Research Division, Smithville, TX 78957, USA. Fax: 512-237-2437; e- mail: dmitchell@sprd1.mdacc.tmc.edu Abbreviations: BSA, bovine serum albumin; CMGHM, 4-(carbox- ymethyl)-2-(guanosyl-9-yl)-6-(hydroxymethyl)morpholine; CPD, cyclobutane pyrimidine dimer; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography; HPLC– ECD, high-performance liquid chromatography and electrochem- ical detection; mBSA, methylated BSA; 8-oxoGuo, 8-oxo-7,8-dih- ydroguanosine; 8-oxodGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosi- ne; PAGE, polyacrylamide gel electrophoresis; RIA, radioimmu- noassay; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; TEAA, triethylammonium acetate; UVA, ultraviolet-A ra- diation; UVB, ultraviolet-B radiation; UVC, ultraviolet-C radia- tion; UVR, ultraviolet radiation. attenuation of 8-oxodGuo suggests that oxidative damage is more closely related to ultraviolet-A radiation (UVA) irradiance. The ratio of 8-oxodGuo : CPD was also found to covary with changes in stratospheric ozone concentra- tions at Palmer Station. These data demonstrate the use- fulness of these assays for environmental photobiology and the potential for their use in studying the relative impacts of UVB versus UVA, including ozone depletion events. INTRODUCTION There is strong evidence that ultraviolet radiation (UVR) is increasing over many locations on the earth’s surface (1–4), and there is no doubt that significant ozone depletion has been observed over Antarctica and parts of the Southern Ocean and Australia (4,5). Because ozone absorbs strongly in the ultraviolet-B radiation (UVB) portion of the solar spectrum (i.e. 290–320 nm), the result of ozone depletion is an increase in the flux of UVB reaching the surface of the earth. Phytoplankton and bacterioplankton comprise the base of oceanic food webs and are sensitive to UVB irradiance. Indeed, there is growing evidence that both ultraviolet-A ra- diation (UVA) (i.e. 320–400 nm) and UVB inhibit both pri- mary and heterotrophic production (6–9). Because UVA has greater penetration into aqueous environments than UVB, it is probable that these wavelengths have greater biological impact at greater depths. In addition, because the DNA ab- sorption spectrum (10), and consequently the action spec- trum for direct damage in DNA (e.g. cyclobutane pyrimidine dimers [CPD]), is greatly reduced at wavelengths 320 nm, indirect DNA damage produced by reactive oxygen species (ROS) predominates in the UVA portion of the solar spec- trum. By virtue of their indirect formation and association with cellular biochemistry and metabolism, oxidative lesions in DNA are produced by diverse xenobiotic routes. Although a variety of oxidative reactions occur in cellular DNA, 8- hydroxylation of guanine is probably the most prevalent (11–13). A wide range of environmental agents generate H 2 O 2 and other ROS and are capable of producing 8-oxo- 7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in cellular