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We assessed the ability of ebselen, a glutathione peroxidase mimic, to reduce pigmentation in various models. In murine B16 melanocytes, 25 μm ebselen inhibited melanogenesis and induced a depolymerisation of actin filaments. In co-cultures of B16 melanocytes with BDVII keratinocytes, a pretreatment of melanocytes with ebselen resulted in a strong inhibition of melanosome transfer to keratinocytes, as shown under optical and electron microscopy. In reconstructed epidermis, topical 0.5% ebselen led to a twofold decrease of melanin without affecting the density of active melanocytes. A similar result was obtained with topical 0.5% ebselen in black guinea pig ears. Ebselen induced a decrease of epidermal melanin parallel to a localisation of melanin and melanosomes in the basal layer. Ebselen appears as a new depigmenting compound that inhibits melanin synthesis and melanosome transfer to keratinocytes.

Three major difficulties must be overcome to establish a quantitative method for melanosomal transfer analysis: (i) establishing a three-dimensional co-culture reassuring direct melanocyte to keratinocyte transfer, (ii) separation of melanocytes and keratinocytes following co-culture and (iii) melanosome quantification in each cell population. Melanocytes and keratinocytes are cultured on the opposite sides of the porous membrane of hanging cell inserts (1mum pores, 2x10(6) pores/cm(2) ). Cell separation is performed after 3days of co-culture by simple trypsinisation. Melanosome quantification in separated cell populations was accomplished by an ELISA-like method using gp-100 as the antigen. Melanocytes and keratinocytes come into 'direct' contact through the pores, and melanosomal transfer is accomplished without cell passage through the membrane. Cell separation by simple trypsinisation results in pure melanocyte and keratinocyte populations. Melanosome quantification by the ELISA-like method proved to be sensitive and specific to distinguish the known inhibitors and inducers of melanosomal transfer.

Monobenzylether of hydroquinone (MBEH) has long been utilized for the depigmentation therapy of patients with extensive vitiligo. In this approach, the normally pigmented areas surrounding vitiligo lesions are depigmented to achieve a uniform skin tone. One of the important disadvantages of MBEH therapy, however, is the resistance of a considerable number of vitiligo patients against the depigmenting effect of this agent. We have previously proposed that the glutathione-dependent cytoprotection of melanocytes can be impaired through the inhibition of the enzyme glutathione S-transferase by retinoic acid (RA). The combination of RA with melanocytotoxic agents could thus lead to increased susceptibility of melanocytes to such compounds. In this study we have shown, for the first time, that the melanocytotoxic and depigmenting effects of MBEH are synergistically enhanced when it is combined with RA. The treatment of black guinea pig skin with RA (0.025%) alone induced no significant changes in the number of epidermal melanocytes and no skin depigmentation. On the other hand, MBEH (10%) produced mild to moderate skin depigmentation and reduced the average number of melanocytes from 76 (+/-5)/field (magnification: x 40) in control sites, to 42 (+/-6)/field in the depigmented skin. The RA (0.025%)-MBEH (10%) combination, however, produced a complete degree of depigmentation in the majority of treated sites after 10 days of application and reduced the average number of melanocytes to only 6 (+/-6)/field. RA-MBEH combination serves as a very potent skin depigmenting formula and now awaits future assessments of its potential use for the treatment of extensive vitiligo.

The 9-cis-retinoic acid (9cRA) is an endogenous ligand of retinoid X nuclear receptors (RXRs). Although the epidermis contains five times more RXRs than RARs, little is known on the activity of topical 9cRA. In order to circumvent surface isomerization of topically applied 9cRA into all-trans-retinoic acid (atRA), we used topical 9-cis-retinaldehyde (9cRAL) as a precursor of 9cRA, hypothesizing that keratinocytes would metabolize 9cRAL into 9-cis-retinoic acid (9cRA). Retinoid content was determined by HPLC analysis of mouse tail skin that had been washed after the application of 9cRAL (0.05% for 14 days) to evaluate the metabolites produced within the epidermis. Biologic activities of 9cRAL and atRAL were analysed by assessing hyperplastic and metaplastic responses, by determining epidermal thickness and the levels of mRNAs encoding for specific keratins. atRAL and derived retinoids were found in skin treated with either atRAL or 9cRAL. The metabolite pattern obtained with 9cRAL was similar to that obtained with atRAL except the presence in 9cRAL samples of an unidentified nonpolar metabolite. However, treatment with 9cRAL yielded higher atRAL and lower retinyl ester concentrations. The biologic activities (hyperplastic and metaplastic responses) resulting from topical application of 9cRAL were lower than those induced by atRAL or atRA at similar concentrations. Taken together, these data show that topical 9cRAL does not deliver significant amounts of 9cRA and exerts less biologic activity than atRAL. Contrary to atRAL, 9cRAL does not appear therefore as a pertinent candidate for topical use in humans.

Background: Ultraviolet (UV)-induced pyrimidine dimers are an early step in skin carcinogenesis, which is accelerated in the setting of long-term immunosuppression with systemic calcineurin inhibitors. It is not known whether topical application of calcineurin inhibitors exposes to a similar risk. Objective: To assess the formation and clearance of UV-induced dipyrimidine dimers in human epidermis treated with topical pimecrolimus as compared to topical steroid, vehicle and untreated control. Methods: Pretreated buttock skin of 20 human volunteers with (10) or without (10) atopic dermatitis was exposed to two minimal erythema doses (MED) of simulated solar radiation. DNA was extracted from epidermis 1 and 24 h postirradiation. Pyrimidine dimers were visualized by immuno slot blots and quantified by chemoluminescence image analysis. Results: One-hour postirradiation, pimecrolimus-treated epidermis contains less DNA damage as compared to untreated control, but there were no statistically significant differences between pimecrolimus, triamcinolone acetonide and vehicle. Dimer levels at 24 h postirradiation showed no significant differences between different treatments. Conclusion: Treatment with pimecrolimus cream, triamcinolone acetonide cream and vehicle is not associated with increased epidermal DNA damage at 1 and 24 h post-UV exposure.

Human epidermis contains endogenous retinoids (retinol and retinyl esters) and carotenoids (mostly beta-carotene). Previous studies have shown that the enzymes involved in retinoid metabolism are present in human epidermis. There is still a controversy about the presence in the skin of the enzymes able to convert beta-carotene into vitamin A (retinol), although a recent study demonstrated the conversion of beta-carotene into retinol in human cultured epidermal cells. In this study, we addressed the question of the possible bioconversion of topical beta-carotene into vitamin A or derivatives by human and mouse skin. Surgically excised human abdominal skin was mounted on Franz perfusion chambers to assess the cutaneous penetration of topical beta-carotene as well as its metabolism, after a 24-h incubation period, whereas hairless mice received topical beta-carotene 24 h before assaying epidermal beta-carotene and retinoid concentrations. Epidermal retinoid and beta-carotene concentrations were determined by high-pressure liquid chromatography. Topical beta-carotene penetrated well into human and mouse epidermis and induced a 10-fold (human) and a threefold (mouse) increase of epidermal retinyl esters, which demonstrates that topical beta-carotene is converted into retinyl esters by human and mouse epidermis and thus appears as a precursor of epidermal vitamin A.