Melanin-Driven Delayed CPD Formation Is Independent of Melanin Biosynthesis Pathway

Cyclobutane pyrimidine dimers (CPDs) are the major form of DNA damage induced by UVR. Although CPDs form instantaneously during UV exposure, our prior studies demonstrated that melanin chemiexcitation, in which UV-induced peroxynitrite oxidizes melanin into excited-state intermediates, continues to generate CPDs for hours after UV exposure ends. These "delayed" CPDs contribute up to 50% of the total CPD burden. Although melanin's involvement is well-established, it remains unclear whether active melanin biosynthesis is required or whether the mere presence of pigment is sufficient. To address this, we developed a depigmentation-repigmentation system using cultured melanocytes. Cells were depigmented in tyrosine-deficient medium and repigmented either by reintroducing tyrosine or by supplementing with 5,6-dihydroxyindole-2-carboxylic acid, a melanin monomer that polymerizes nonenzymatically. In both conditions, delayed CPD formation was restored. Notably, 5,6-dihydroxyindole-2-carboxylic acid treatment alone was sufficient to induce pigmentation and delayed CPD formation even in tyrosinase-deficient isogenic albino melanocytes. Pigment accumulation was also associated with nitric oxide synthase activity, a key contributor to chemiexcitation, although the mechanisms linking melanin synthesis to nitric oxide synthase activity remain to be elucidated. These findings demonstrated pigment presence, not biosynthesis, as the key determinant of delayed CPD formation, with broad implications for photobiology, pigmentation, and skin cancer risk.