title: Biphasic pro-melanogenic and pro-apoptotic effects of all-trans-retinoic acid (ATRA) on human melanocytes: time-course study.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Physiology, University of Medicine and Pharmacy "Iuliu Hatieganu", Cluj-Napoca, Romania.
authors.forename: Ioana
authors.initials: I
authors.lastname: Baldea
authors.forename: Gertrude-Emilia
authors.initials: GE
authors.lastname: Costin
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Shellman
authors.forename: Katerina
authors.initials: K
authors.lastname: Kechris
authors.forename: Elena Diana
authors.initials: ED
authors.lastname: Olteanu
authors.forename: Adriana
authors.initials: A
authors.lastname: Filip
authors.forename: Maria Rodica
authors.initials: MR
authors.lastname: Cosgarea
authors.forename: David Albert
authors.initials: DA
authors.lastname: Norris
authors.forename: Stanca Ariana
authors.initials: SA
authors.lastname: Birlea
mesh_terms: D000287:Administration, Topical; D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis; D002374:Catalase / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D006657:Histones / Q000378:metabolism; D006801:Humans; D008543:Melanins / Q000378:metabolism; D008544:Melanocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010641:Phenotype; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology*; D055815:Young Adult
chemical_list: D017304:Annexin A5; D000975:Antioxidants; C468783:H2AX protein, human; D006657:Histones; D008543:Melanins; D014212:Tretinoin; D002374:Catalase; D014442:Monophenol Monooxygenase; D013482:Superoxide Dismutase
keywords: ATRA; Apoptosis; Melanogenesis; Proliferation; Retinoids