Tiaprofenic Acid-photosensitized Damage to Nucleic Acids: A Mechanistic Study Using Complementary in vitro Approaches

Abstract

In order to determine whether or not tiaprofenic acid (TPA) could cause cellular DNA damage, human fibroblasts were irradiated in the presence of the drug and subsequently examined by means of the comet assay. This led to the observation that TPA actually sensitizes cellular DNA to the subsequent irradiation. When TPA was irradiated in the presence of supercoiled plasmid DNA, it produced large amounts of single-strand breaks (SSB); this is consistent with the effects observed on cellular genomic DNA by the comet assay. More importantly, low concentrations of TPA, unable to produce direct SSB, caused photo-oxidative damage to DNA as revealed by the use of excision-repair enzymes. The fact that TPA-irradiated DNA was a substrate of formamidopyrimidine glycosylase as well as endonuclease III revealed that both purine and pyrimidine bases were oxidized. This was further supported by the TPA-photosensitized oxidation of 2'-deoxyguanosine which led to a product mixture characteristic of mixed type-I/II mechanisms. Thymidine was less reactive under similar conditions, but it also decomposed to give a typical type-I product pattern. Accordingly, the TPA triplet was quenched by the two nucleosides with clearly different rate constants (10(8) vs 10(7) M-1 s-1, respectively). As cellular RNA also contains oxidizable bases, it could be the target of similar processes, thus interfering with the biosynthesis of proteins by the cells. Extraction of total RNA from TPA-irradiated human fibroblasts, followed by gel electrophoresis and PCR analysis, confirmed this hypothesis. Finally, photosensitization experiments with Saccharomyces cerevisiae showed that, in spite of an efficient drug-yeast interaction leading to cytotoxicity, neither intergenic recombination nor gene conversion took place. Thus, while TPA-photosensitized damage to nucleic acids can result in genotoxicity, the risk of mutagenicity does not appear to be significant.

Drug-biomolecule interactions in the excited states

“…With this aim, the comet assay has been used to determine whether TPA can cause DNA damage in eukaryotic cells [19]. As shown in Fig.…”

Photosensitization by drugs

“…In order to determine whether TPA is able to photosensitize cellular DNA damage, human fibroblasts have been irradiated with the drug and subsequently examined by means of the comet assay [22]. This has led to the observation that TPA actually photosensitizes DNA fragmentation inside cells.…”

“…But more importantly, lower concentrations of TPA (0.5-2.5 µM), not able to produce direct SSB under the same experimental conditions, do photosensitize oxidative damage to DNA, as revealed by the combined use of excision-repair enzymes formamidopyrimidine glycosylase (FPG) or Endo III and gel electrophoresis. The fact that TPA-irradiated DNA is substrate of the two enzymes clearly indicates that both purine and pyrimidine bases are oxidized [22].…”

“…In the case of 2'-deoxyguanosine, the product mixture is characteristic of mixed type I/type II mechanisms. Thymidine is less reactive under similar conditions, but it also decomposes to give a typical type I product pattern [22].…”

“…The results indicate that TPA, although it photosensitizes DNA oxidative damage causing genotoxicity, does not promote DNA recombination and thus does not induce adducts in the DNA of yeast cells. This, together with cell effectiveness in repairing oxidative damage, may explain the fact that no cell mutation/transformation is observed [22].…”