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Gaithersburg, MD, United States

Baldea I.,University of Medicine and Pharmacy, Cluj-Napoca | Costin G.-E.,Institute for In Vitro science Inc. IIVS | Shellman Y.,Aurora University | Kechris K.,Aurora University | And 5 more authors.
Journal of Dermatological Science | Year: 2013

Background: The effects of retinoids on melanogenesis and their mechanism as depigmenting agents in topical therapy have not been fully elucidated. Conflicting data about their impact on melanogenic pathways have been reported. Objective: To investigate the effects of all-trans-retinoic acid (ATRA) on normal human melanocytes from Caucasian subjects. Methods: We assessed ATRA's cytotoxicity by measuring viability with a cell proliferation assay, and apoptotic effects using Annexin V and γ-H2AX markers. ATRA's melanogenic activity was investigated based on spectrophotometric measurement of melanin content and tyrosinase enzymatic activity. Tyrosinase expression was assessed by Western blotting. We tested the antioxidant activity of superoxide dismutase (SOD) and catalase (CAT) in melanocytes using a spectrophotometric assay. Results: Of the concentrations tested in this 72h time-course study, the 1.0μM ATRA had a well-defined two-stage pro-melanogenic and pro-apoptotic effect on melanocytes. In the first 6h, treated cells showed significant increase (p≤0.01) of melanin content, tyrosinase, SOD, and CAT activities compared to the controls. While overall tyrosinase expression was not affected by ATRA, all other tested parameters decreased progressively beyond the short-term point of 6h. ATRA treatment of over 6h induced melanocyte apoptosis, as shown by the time-dependent decrease in cell viability, coupled with significant increase in Annexin V positive cells and nuclear accumulation of γ-H2AX foci. Conclusion: The results obtained using this testing platform show a biphasic ATRA action: immediate pro-melanogenic effect and longer-term exposure pro-apoptotic activity. These data qualify ATRA as a potent tool to better understand the mechanisms that regulate the pigmentary system. © 2013 Japanese Society for Investigative Dermatology.

Aardema M.J.,Procter and Gamble | Aardema M.J.,Marilyn Aardema Consulting LLC | Barnett B.C.,Procter and Gamble | Khambatta Z.,Procter and Gamble | And 9 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2010

Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm™ 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192-204; Mun et al., Mutat. Res. 673 (2009) 92-99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100-108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm™ 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals. © 2010 Elsevier B.V.

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