Applicability of the comet assay in hacat cells for the evaluation of photogenotoxicity

Abstract

The establishment of protocols to ensure the safety of pharmaceuticals and cosmetics is becoming increasingly important. Regulatory authorities require the evaluation of their phototoxic potential and, consequently, their photogenotoxicity. This study aimed to validate the comet assay as a reliable method for assessing photogenotoxicity of chemical compounds in human keratinocytes (HaCaT). It focused on analyzing sodium dodecyl sulfate (SDS) and benzophenone-3 (BZ) as negative and positive controls, respectively. The cells were incubated with the test chemicals for 1 hour, followed by irradiation with 4 J/cm² UVA for the designated time. Cell viability was measured using LDH assays, and a comet assay was performed immediately after irradiation at non-cytotoxic concentrations of the compounds. Methyl methanesulfonate (MMS) was used as a positive control for the comet assay. SDS was classified as non-phototoxic and non-photogenotoxic. In contrast, BZ was classified as phototoxic in the 24-hour MTT assay and photogenotoxic in the comet assay. The MTT assay proved to be more sensitive than the LDH assay in assessing photocytotoxicity. The comet assay conducted immediately after irradiation was determined to be the more effective for accurately evaluating DNA damage. It was concluded that a 24-hour time interval post-irradiation is not optimal, as it allows the activation of cellular repair mechanisms. Overall, the comet assay shows promise as a reliable tool for detecting photogenotoxic substances. However, to reduce variability and improve robustness, further assays are needed. This study highlights the importance of integrating additional assays to fully assess the significance of photosafety. Key words: Phototoxicity, photogenotoxocity, in vitro, HaCat, comet assay