Melatonin as a noncanonical substrate in melanogenesis: Formation and photobiological properties of melanin-like hybrid pigments.

Melatonin is traditionally classified as a melanogenesis inhibitor. However, in some tissues it has been recently found to stimulate melanogenesis, paradoxically, with a parallel decrease of tyrosinase activity. This study explores this paradox by examining melatonin's potential role in pigment formation. Pigments were synthesized from DL-DOPA and melatonin via autooxidation or enzymatic reaction with tyrosinase. The resulting biopolymers were characterized using dry-mass determination, Electron Paramagnetic Resonance (EPR) spectroscopy, potentiometric titration, Dynamic Light Scattering (DLS), and spectrophotometry. The biological effect of melatonin supplementation on the mitochondrial activity of primary melanocyte cell culture (HEMn-DP) after solar-simulated illumination was assessed using the MTT assay. The presence of melatonin during autooxidation increased the overall melanin free radical signal as determined using EPR, however did not impact the signal during enzymatic synthesis. More so, enzymatic oxidation of melatonin alone yielded a detectable pigment (Melat-Mel) with about 4.4% efficiency. EPR spectra of melatonin-derived polymers and copolymers in the presence of zinc (II) ions and after thermal treatment demonstrated behavior typical to melanin pigments. UV-Vis spectrometry showed a change in the absorption profile for copolymer (DOPA/Melat-Mel) with higher UV and lower VIS absorbance compared with homopolymer (DOPA-Mel). DLS measurements confirmed the formation of supramolecular aggregation for all the examined variants. Potentiometric titration showed that copolymer with melatonin exhibits the highest buffering capacity among examined pigments. Loading of HEMn-DP cells with melatonin yielded a dose-dependent reduction of mitochondrial activity following solar-simulated light treatment with no dark toxicity observed. The study provides a chemical model that may help explain part of the "melatonin paradox" in pigmentation, while emphasizing that direct confirmation of melatonin-containing pigment formation in cells remains to be established.