Induction of keratinocyte apoptosis by photosensitizing chemicals plus UVA

SummaryBackgroundThe capacity of photosensitizing chemicals with ultraviolet A light (UVA) to induce apoptosis is one of the methods to assess their phototoxic and potentially photoallergic properties, since apoptotic cells may be easily presented by antigen-presenting cells.ObjectivesWe examined the photoaggravated ability to induce keratinocyte apoptosis of various chemicals that are known as causative agents of photocontact dermatitis and drug photosensitivity involving photoallergic and/or phototoxic mechanisms.MethodsHaCaT keratinocytes were incubated with 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), bithionol, diphenylhydramine, chlorpromazine, 6-methylcoumarin, sparfloxacin, and enoxacin at 10−7 to 10−4 M and irradiated with UVA at 4 J/cm2. As positive control, 8-methoxypsoralen (8-MOP) was also tested. Apoptosis and necrosis were evaluated by flow cytometric enumeration of annexin V+ 7-AAD− and annexin V+ 7-AAD+ cells, respectively. The expression of apoptosis-related molecules, caspase-3 and poly (ADP-ribose) polymerase (PARP), was tested by flow cytometric and Western blotting analyses.ResultsIn a comparison with non-irradiated cells, significant apoptosis was found in TCSA, bithionol, chlorpromazine, sparfloxacin and enoxacin at 10−4 or 10−5 M as well as 8-MOP as assessed by both annexin V and active caspase-3 stainings, while necrosis occurred in most of these chemicals at 10−4 M. Neither apoptosis nor necrosis was seen in diphenylhydramine or 6-methylcoumarin. PARP were activated in HaCaT cells phototreated with TCSA, bithionol and chlorpromazine.ConclusionsWe suggest that our method is useful for in vitro assessment of phototoxicity and potential photoallergenicity of chemicals.