Molecular effects of 1-naphthyl-methylcarbamate and solar radiation exposures on human melanocytes

Carbaryl (1-naphthyl-methylcarbamate), a broad-spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, which is known to be the main etiologic factor for skin carcinogenesis. Although comprehensive and well designed, the agricultural epidemiological study was not sufficient to characterize the direct contribution of the insecticide and solar radiation in melanomagenesis. Several studies have explored the synergistic effect of certain chemicals with UV radiation, increasing its deleterious effects on the skin. We hypothesized that carbaryl exposure associated with UV solar radiation may induce melanocyte transformation. This study aimed to characterize human melanocytes after individual or combined exposure to carbaryl (100 μM) and solar radiation (375 mJ/cm2). In a microarray analysis, carbaryl, but not solar radiation, induced an important oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of Microphtalmia-associated Transcription Factor (MITF), the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Carbaryl and solar radiation induced a gene response suggestive of DNA damage and cell cycle alteration. The expression of genes in these categories was notably more intense in the combined treatment group, in a synergistic manner, for CDKN1A, BRCA1/2 and MDM2 genes. Likewise, flow cytometry assays demonstrated S-phase cell cycle arrest, reduced apoptosis levels and faster induction of cyclobutane pyrimidine dimers (CPD) lesions in carbaryl treated groups. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation. Twenty-four hours after plating, cells were at 80% confluency and were subjected to the following experimental treatment groups: Group 1: No treatment; Group 2: Irradiation and no treatment; Group 3: Carbaryl treatment; Group 4: Irradiation and carbaryl treatment; Group 5: Vehicle treatment; Group 6: Irradiation and vehicle treatment. Treatment regimen consisted of melanocyte incubation with carbaryl 100 µM (Sigma-Aldrich, St Louis, USA) for 6 hours after single dose exposure to 375mJ/cm2 of solar radiation using a solar simulator (SS2.5kW, Sciencetech Inc., Ontario, Canada) with a global air mass filter (A.M 1.5G, Sciencetech Inc, Ontario, Canada). For the irradiation assays, culture medium was replaced by PBS buffer without Ca2+ or Mg2+ (PBS-A). All experiments were performed in triplicates.