Photochemistry and Photobiology, 2003, 77(4): 390–396 Prompt and Delayed Nonsteroidal Anti-inflammatory Drug–photoinduced DNA Damage in Peripheral Blood Mononuclear Cells Measured with the Comet Assay { Anne L. Vinette 1 , James P. McNamee 2 , Pascale V. Bellier 2 , J. R. N. McLean 2 and J. C. Scaiano* 1 1 Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada and 2 Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, Ontario, Canada Received 19 August 2002; accepted 19 January 2003 ABSTRACT Nonsteroidal anti-inflammatory drug (NSAID)–photoinduced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid–photoinduced DNA damage was promptly induced (i.e. observed at relatively low radiation doses), Ketoprofen-photoinduced DNA damage was delayed. This prompt and delayed effect is observed with UVA (320– 400 nm), UVB (290–320 nm) and solar-simulated radiation and is attributed to the different photochemical properties of NSAID. The results from these experiments, carried out in living cells, confirm the speculations of NSAID-photoinduced DNA damage brought up by the many experiments conducted in solution. INTRODUCTION Approximately 10 000 measurable DNA modification events occur every hour in each mammalian cell because of intrinsic causes, which include depurination, depyrimidination, deamination, sin- gle-strand breaks (SSB), double-strand breaks, base modification and protein–DNA cross-links (1). These events are caused by metabolic processes that lead to the formation of free radicals such as hydroxyl radicals (HO  ), peroxides and other reactive oxygen species (ROS) (1). Other external forces such as UV radiation (200–400 nm), photosensitizers and free radical species can also cause DNA damage. DNA bases absorb in the UV region, especially from 220 to 320 nm, with a maximum at 260 nm (2). This region of the electromagnetic spectrum therefore is deemed particularly relevant to photobiology. At wavelengths from 250 to 320 nm, where DNA itself is the chromophore, pyrimidine photoadducts and [212] pyrimidine cyclobutane dimers are the principal photoproducts formed after radiation absorption by DNA, although photooxida- tion also occurs. The pyrimidine cyclobutane dimers are not alkali- labile sites (ALS) but are repaired by enzymes (3). The Dewar valence isomer of the pyrimidine(6-4)pyrimidone photoadduct (cytidyl-(39-59)-thymidine monophosphate) (4), however, is an alkali-labile lesion (5) and is formed ca 50% less often than cyclobutane dimers (6). Overall, pyrimidine cyclobutane dimers are the most common product of direct DNA damage, constituting ca 70% of the total damage (7). Although UVA radiation is only weakly absorbed by DNA bases, it is considered to be a generator of intracellular oxidative stress. UVA radiation can cause cellular DNA damage through photosensitization reactions as opposed to a direct reaction with DNA (8). The resulting photoexcited sensitizer can potentially mutate DNA or kill cells through direct interaction with cellular DNA or, more commonly, through an energy transfer to molecular oxygen, generating singlet oxygen ( 1 O 2 ), which can then react with cellular DNA (8). ROS can be generated by one of two mech- anisms, both involving initial radiation absorption by a sensitizer; the type-I mechanism generates radical oxygen species, whereas the type-II mechanism generates 1 O 2 . On the other hand, the direct irradiation of DNA with UV leads to a variety of lesions, many of which are centered on the nucleobases and are alkali labile (9); the wavelength and radiation intensity determine to some extent the type of lesions obtained. For example, UVA irradiation of DNA will generate alkali-labile 7,8-dihydro-8-oxoguanine (8-oxoG) through direct oxidation of a guanine base, from hydrogen abstraction by a photosensitizer and via 1 O 2 . UVB irradiation of DNA, on the other hand, generates 8-oxoG as well as the Dewar valence isomers of the pyrimidine(6-4)pyrimidone dimer, both of which are ALS. Nonsteroidal anti-inflammatory drugs (NSAID) are a large class of compounds, which inhibit cyclooxygenase isoenzymes 1 and 2 that are responsible for catalyzing the rate-limiting step in prostaglandin synthesis from arachidonic acid (10). In light of their pharmacological significance, NSAID have seen worldwide popu- larity as anti-inflammation therapeutics. During the past 30 years, NSAID have undergone critical scrutiny, from a pharmacological {Posted on the website on 1 February 2003. *To whom correspondence should be addressed at: Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario, Canada K1N 6N5. Fax: 613-562-5633; e-mail: tito@photo.chem.uottawa.ca Abbreviations: ALS, alkali-labile sites; ddH 2 O, 18 MX water; DMSO, dimethyl sulfoxide; EB, 3-ethylbenzophenone; EDTA, sodium tetra- ethylenediamine tetraacetate; EtBr, ethidium bromide; FBS, fetal bovine serum; FPG, formamidopyrimidine glycosylase; HBSS, Hanks’ balanced salt solution; 8-hydroxyquinoline, 8-hydroxy-1-azanaphthalene; KP, ketoprofen; LMP, low-melting point; NSAID, nonsteroidal anti-inflamma- tory drug; 1 O 2 , singlet oxygen; 8-oxoG, 7,8-dihydro-8-oxoguanine; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; ROS, reactive oxygen species; RPMI, Roswell Park Memorial Institute; SCGE, single-cell gel electrophoresis; SLS, N-lauryl sarcosine sodium salt; SSB, single-strand breaks; SSR, solar-simulated radiation; TP, tiaprofenic acid; Triton X-100, isooctylphenoxypolyethoxyethanol; WB, whole blood. Ó 2003 American Society for Photobiology 0031-8655/01 $5.0010.00 390