Cytotoxic and genotoxic effects of acitretin, alone or in combination with psoralen-ultraviolet A or narrow-band ultraviolet B-therapy in psoriatic patients.

Acitretin is currently used alone or combined with PUVA (psoralen + UVA) or with narrow-band ultraviolet B (NBUVB), to treat moderate and severe psoriasis. However, little is known about the potential genotoxic/carcinogenic risk and the cytostatic/cytotoxic effects of these treatments. Our aim was to study the cytotoxic and genotoxic effects of acitretin - alone or in combination with PUVA or NBUVB - by performing studies with blood from patients with psoriasis vulgaris who were treated with acitretin, acitretin+PUVA or acitretin+NBUVB for 12 weeks, and in vitro studies with blood from healthy volunteers, which was incubated with acitretin at different concentrations. The cytotoxic and genotoxic effects were evaluated by the cytokinesis-blocked micronucleus test and the comet assay. Our results show that psoriatic patients treated with acitretin alone or with acitretin+NBUVB, did not show genotoxic effects. In addition, these therapies reduced the rate of proliferation and induced apoptosis and necrosis of lymphocytes; the same occurred with lymphocyte cultures incubated with acitretin (1.2-20µM). The acitretin+PUVA reduced also the proliferation rate, and increased the necrotic lymphocytes. Our studies suggest that therapy with acitretin alone or combined with NBUVB, as used in psoriatic patients, does not show genotoxic effects, reduces the rate of proliferation and induces apoptosis and necrosis of lymphocytes. The combination of acitretin with PUVA also reduces the proliferation rate and increases the number of necrotic lymphocytes. However, as it induced slight genotoxic effects, further studies are needed to clarify its genotoxic potential.

Adenosine diphosphate involvement in THP-1 maturation triggered by the contact allergen 1-fluoro-2,4-dinitrobenzene.

Dendritic cells' (DC) activation is considered a key event in the adverse outcome pathway for skin sensitization elicited by covalent binding of chemicals to proteins. The mechanisms underlying DC activation by contact sensitizers are not completely understood. However, several "danger signals" are pointed as relevant effectors. Among these extra-cellular early danger signals, purines may be crucial for the development of xenoinflammation and several reports indicate their involvement in contact allergic reactions. In the present work we used the DC-surrogate monocytic cell line THP-1, cultured alone or co-cultured with the human keratinocyte cell line HaCaT, to explore the contribution of extracellular adenine nucleotides to THP-1 maturation triggered by the extreme contact sensitizer, 1-fluoro-2,4-dinitrobenzene (DNFB). We found that THP-1 maturation induced by DNFB is impaired after purinergic signaling inhibition, and that the transcription of the purinergic metabotropic receptors P2Y2 and P2Y11 is modulated by the sensitizer. We also detected that THP-1 cells only partially hydrolyse extracellular adenosine triphosphate, leading to accumulation of the mono-phosphate derivative, AMP. We detected different and non-overlapping activation patterns of mitogen activated protein kinases by DNFB and extracellular nucleotides. Overall, our results indicate that THP-1 maturation induced by DNFB is strongly modulated by extracellular adenine nucleotides through metabotropic purinergic receptors. This knowledge unveils a molecular toxicity pathway evoked by sensitizers and involved in THP-1 maturation, a DC-surrogate cell line thoroughly used in in vitro tests for the identification of skin allergens.

The in vitro and in vivo genotoxicity of isotretinoin assessed by cytokinesis blocked micronucleus assay and comet assay.

Isotretinoin is a retinoic acid frequently used in monotherapy or combined with narrow-band ultraviolet B (NBUVB) irradiation to treat patients with acne and psoriasis vulgaris. As both diseases need frequent and/or prolonged therapeutic interventions, the study of the genotoxicity of retinoids becomes important. Our aim was to study the genotoxic effects of isotretinoin alone or combined with NBUVB. In vitro studies were performed in the absence of S9 metabolic activation using blood from five healthy volunteers, incubated 72 h with isotretinoin (1.2-20 µM) (i.e., at concentrations usually achieved in blood with therapeutic doses as well as at higher concentrations). In vivo studies were also performed using blood from two patients with acne and three patients with psoriasis vulgaris treated with isotretinoin in monotherapy (8 or 20mg/day) or combined with NBUVB (20mg isotretinoin/day+NBUVB). The genotoxic effect was evaluated by the cytokinesis-blocked micronucleus and the comet assays. Our studies showed that isotretinoin alone was not genotoxic when tested in human lymphocytes in vitro and in vivo. There was no clear genotoxic effect in psoriatic patients treated with isotretinoin and NBUVB. The in vitro studies showed that isotretinoin induced apoptosis and necrosis in human lymphocytes at higher doses.