Induction of keratinocyte apoptosis by photosensitizing chemicals plus UVA.

BACKGROUND

The 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.

OBJECTIVES

We 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.

METHODS

HaCaT 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 4J/cm(2). 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.

RESULTS

In 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.

CONCLUSIONS

We suggest that our method is useful for in vitro assessment of phototoxicity and potential photoallergenicity of chemicals.