Induction of keratinocyte apoptosis by photosensitizing chemicals plus UVA

Summary

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 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.

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.

Introduction

Among solar light-induced disorders, photosensitivity to exogenous agents is the most incident pathological condition and includes two diseases, photocontact dermatitis and drug photosensitivity [1]. The former disease is induced by skin application of a contactant and following exposure to ultraviolet light (UV) [2], [3], [4], [5], while the latter is evoked by oral administration of a photosensitizing drug and subsequent exposure of the skin to UV [6], [7], [8], [9], [10]. In both diseases, the action spectrum is UVA in most cases, and UVB can exceptionally evoke the photosensitivity dermatitis [1], [5], [6].

Various chemicals have been reported to cause photocontact dermatitis. Historically, the use of halogenated salicylanilide and related compounds, especially 3,3′,4′,5-tetrachlorosalicylanilide (TCSA) [2] and bithionol [3], resulted in a large number of patients with this skin disease. More recent causative agents are cosmetic or sunscreen products such as 6-methylcoumarin, musk ambtette, benzophenone, and non-steroidal anti-inflammatory drugs [4]. Likewise, there have been various culprit drugs to evoke photosensitivity, including chlorpromazine, quinolones, such as sparfloxacin (SPFX) and enoxacin (ENX), piroxicum, afloqualone, and others [1], [6], [7].

Both phototoxic and photoallergic mechanisms exist in the pathophysiology of photosensitivity to exogenous agents. The phototoxicity is mediated by reactive oxygens, in particular singlet oxygen [11], while the photoallergenicity occurs as a consequence of immunological events, where the UV-induced formation of photoantigen triggers T cell responses [8], [9], [10]. Recently, the incidence of the photoallergic type is higher than that of the phototoxic one [1], [6], [7]. Our studies have suggested that the vast majority of clinically photoallergic chemicals are photohaptens that bind covalently to protein under exposure to UV [1], [6], [7], [9].

In the photoallergic type of photocontact dermatitis and drug photosensitivity, culprit chemicals reach the epidermis directly from the outside [2], [3], [4], [5] or indirectly from the inside by diffusion [7], [8], [9], [10]. UVA irradiation induces photoconjugation of epidermal cells with photosensitive chemicals, and photoantigen-bearing epidermal Langerhans cells sensitize T cells in the draining lymph nodes [9], [10], [11], [12], [13]. When a given chemical has a strong phototoxicity, however, epidermal cells photomodified with the chemical lose viability, and the formed photoantigen on the damaged keratinocytes or Langerhans cells may be presented by other antigen-presenting cells, such as dermal macrophages or dendritic cells [13]. Since apoptotic cells are easily presented by dendritic cells [14], [15], the capacity of chemicals to evoke apoptosis may determine the feasibility of the sensitivity response. Thus, the apoptosis-inducing property of photosensitizing agents may be useful for evaluation of not only phototoxicity but also photoallergenicity.

In this study, we examined the ability to induce keratinocyte apoptosis of various chemicals that are known as causative agents of photocontact dermatitis or drug photosensitivity. Results suggest that highly photoallergic chemicals have an apoptosis-inducing activity in keratinocytes.