Comparing the cytotoxicity and genotoxicity induced by compounds with different phototoxic properties

Abstract

The increased use and development of new drugs and cosmetics each year has led to an interest in the evaluation of phototoxicity. Regulatory authorities require an assessment of their phototoxic potential. A need to develop in vitro assays has arisen due new regulations emphasizing ethical considerations towards animal testing. The aim of this study was to identify a reliable in vitro assay for photocytotoxicity testing, as well as another for assessing photogenotoxicity, using a commercial human keratinocyte cell (HaCaT) line. This study has focused mainly on chlorhexidine (CHX), though four more chemicals with known phototoxic properties were also tested: chlorpromazine (CPZ), sodium dodecyl sulfate (SDS), benzophenone (BZ) and 8-methoxypsoralen (8-MOP). Cells were incubated with the test chemicals for 1 hour, following an irradiation of 4 J/cm² UVA. After irradiation, the solution with the chemicals was removed and fresh medium was added. Cell viability was measured by MTT and LDH assays, and a comet assay was performed 24 hours after irradiation at non-cytotoxic concentrations of the compounds. Methyl methanesulfonate (MMS), a known alkylating agent, was used as a positive control for the comet assay. CHX was classified as non-phototoxic and non-genotoxic, it was also the second agent with the lowest IC50. Other chemicals tested, aside from SDS, showed Photo-Irritation-Factor (PIF) values greater than 5. No increase in DNA damage was observed 24 hours after irradiation, likely due to DNA repair during this period. LDH assay showed inconsistent results due to the short exposure time of cells to the assay reagent, and further studies are needed to determine its reliability in photocytotoxicity testing. MTT and comet assay promise to be simple method to identify both photocytotoxic and photogenotoxic substances, which could improve the safety assessment of new pharmaceutical and cosmetic products.