Evaluation of melanin production by melanoma cells and cytotoxicity by singlet molecular oxygen

Ultraviolet (UV) radiation can lead to serious damage to the human skin. UVB radiation (290-320 nm) mainly causes direct DNA damage and UVA radiation (320-400 nm) induces oxidative stress, cell membrane and certain types of DNA damage. Much evidence points out that melanin may act as a photoprotection agent, by absorbing UV radiation and scavenging reactive oxygen species (ROS). However, some studies have indicated that UV irradiation of melanin could generate ROS. Furthermore, cells that have a stimulated production of melanin showed higher levels of DNA damage when irradiated by UVA. Considering that UVA radiation promotes oxidative stress, involving the production of singlet molecular oxygen (¹O₂), our main interest was to investigate the specific role of ¹O₂ in DNA damage and cell death of melanin-producing cells. We now used melanoma cells B16-F10 (murine) and SK-MEL (human). The optimum conditions of melanogenesis stimulus by L-tyrosine and melanocyte-stimulating-hormone (a-MSH) were established. Also investigated was the cytotoxicity when the cells were treated with ¹O₂ generated by thermolysis of naphthalene endoperoxides (0.5 and 1 mmol/L). Cells that do not produce melanin (BHK-21, murine fibroblasts) were also used for comparison. Melanin was determined by measuring absorbance at 470 nm and the cytotoxicity was evaluated by Trypan Blue (TB) exclusion and the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The concentrations of L-tyrosine and a-MSH that showed the best response in melanin production by the cells were at 0.5 mmol/L and 30 nmol/L, respectively. After treatment with DHPNO₂ 1 mmol/L, 66% and 57% of cell viability was found for B16-F10 and SK-MEL melanoma cells, respectively. The cell viability for fibroblast cells was 90%. Interestingly, we observed more B16-F10 dead cells when evaluated by TB (34%) than MTT method (14%). Such a difference may be related with the cellular parameters considered by these two methods: TB is based on membrane integrity, while MTT is based on activity of dehydrogenases. Clearly, our results indicate that melanin-producing cells are more sensitive to ¹O₂, which may be related with the increased DNA damage detected when they were irradiated by UVA. Supported by CAPES and CNPq.