Comparison of DNA Damage Photoinduced by Ketoprofen, Fenofibric Acid and Benzophenone via Electron and Energy Transfer(para)
Ketoprofen (KP) and fenofibrate, respectively, anti-inflammatory and hypolipidemiant agents, promote anormal photosensitivity in patients and may induce photoallergic cross-reactions correlated to their benzophenone-like structure. Here, their ability to photosensitize the degradation of biological targets was particularly investigated in DNA. The photosensitization of DNA damage by KP and fenofibric acid (FB), the main metabolite of fenofibrate, and their parent compound, benzophenone (BZ), was examined on a 32P-end-labeled synthetic oligonucleotide in phosphatebuffered solution using gel sequencing experiments. Upon irradiation at lambda > 320 nm, piperidine-sensitive lesions were induced in single-stranded oligonucleotides by KP, FB and BZ at all G sites to the same extent. This pattern of damage, enhanced in D^sub 2^O is characteristic of a Type-II mechanism. Spin trapping experiments using 2,2,6,6-tetramethyl-4-piperidone have confirmed the production of singlet oxygen during drug photolysis. On double-stranded oligonucleotides, highly specific DNA break occurred selectively at 5'-G of a 5'-GG-3' sequence, after alkali treatment. Prolonged irradiation led to the degradation of all G residues, with efficiency decreasing in the order 5'-GG > 51-GA > 51-GC > 5'-GT, in good agreement with the calculated lowest ionization potentials of stacked nucleobase models supporting the assumption of a Type-- I mechanism involving electron transfer, also observed to a lesser extent with adenine. Cytosine sites were also affected but the action of mannitol which selectively inhibited cytosine lesions suggests, in this case, the involvement of hydroxyl radical, also detected by electronic paramagnetic resonance using 5,5-dimethyl-1-- pyrrolidine-1-oxide as spin trap. On a double-stranded ^sup 32^P-end-labeled 25-mer oligonucleotide containing TT and TTT sequences, the three compounds were found to photosensitize by triplet-triplet energy transfer the
formation of cyclobutane thymine dimers detected using T4 endonuclease V.
Abbreviations: ATP, adenosine triphosphate; BZ, benzophenone; DMPO, 5,5-dimethyl-1-pyrrolidine-1-oxide; DMSO, dimethylsulfoxide; EDTA, ethylenediamine tetraacetic acid; EPR, electronic paramagnetic resonance; FB, fenofibric acid; 8-OxodAdo, 8-oxo-7,8-dihydro-2'-deoxyadenosine; KP, ketoprofen; NSAID, nonsteroidal antiinflammatory drug; SCE, saturated calomel electrode; SOD, superoxide dismutase; TMP, 2,2,6,6-tetramethyl-4-- piperidone.
Comparison of the photosensitizing activities of KP and FB to that of BZ on two double-stranded oligonucleotides, chosen to easily characterize the participation of electron and energy transfer, clearly shows that the three compounds have the similar ability to promote DNA damage by both processes. As shown by the sequence specificity of the alkali-labile sites, KP, FB and BZ promote transfer of an electron through a Type-I mechanism involving guanines and to a lesser extent adenines. They also cause damage to cytosines by a poorly efficient radical mechanism. When the oligonucleotide displays -TT- and -TTT- sites, the three compounds photosensitize the formation of cyclobutane thymine dimers via triplet-triplet energy transfer. The behavior of these compounds toward single-stranded oligonucleotides is different, as the involvement of singlet oxygen by a Type-II mechanism appears predominant. In all cases, the efficiency of the three compounds is nearly the same suggesting that the radicals formed during the photolysis of KP and FB are not significantly involved in the formation of alkali-labile sites. Owing to the fact that the DNA damage photosensitization occurs mainly through electron transfer, as is deduced from this work, the photosensitization efficiency depends on the excited states and not on the radical formation. The quenching of the excited states of BZ and its derivatives observed in the presence of nucleic acids and attributed either to electron or energy transfer could very well slow down their photodegradation. These conclusions point out that the photosensitizing properties of these benzophenone derivatives in DNA depend mainly on the benzophenone chromophore. The problem is more complex in a biological medium, and these conclusions have not yet been extended directly to other biological targets or cells.
The ability of these compounds to photoinduce the formation of cyclobutane thymine dimers, which are at the origin of many biological lesions, is now clearly evidenced by this work. It should be noted that it is the first time that this reaction is shown with drugs, and furthermore, with commercial phototoxic drugs.
Acknowledgement-The authors thank Dr. De Montauzon for his technical assistance in electrochemistry.
(para)Posted on the website on 17 August 2001.
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Virginie Lhiaubet, Nicole Paillous and Nadia Chouini-Lalanne*
Laboratoire des Interactions Moleculaires et Reactivite Chimique et Photochimique, Universite Paul Sabatier, Toulouse, France
Received 23 April 2001; accepted 9 August 2001
*To whom correspondence should be addressed at: Laboratoire des IMRCP, Universite Paul Sabatier, 118, route de Narbonne, Toulouse, France. Fax: 5-61-55-81-55; e-mail: lalanne@chimie. ups-tlse.fr
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