Autofluorescence of melanins induced by ultraviolet radiation and near ultraviolet light. A histochemical and biochemical study.

Abstract

The induction of autofluorescence of melanins by UV radiation (330-380 nm) and near UV (400-440 nm) light (jointly called UV light) was studied in tissue sections using three commercially available mounting media. Only Immu-Mount (Shandon) was found suitable for this purpose. UV irradiation of melanins in sections mounted in this medium induced strong yellow autofluorescence irrespective of the type of the polymer (eumelanin, neuromelanin, pheomelanin and ochronotic pigment). The phenomenon of autofluorescence induction was also observed with isolated natural and in vitro prepared melanins. It was inhibited by anhydrous conditions, sodium azide and catalase. In parallel experiments, rapid degradation of melanins with an intermediate fluorescent stage was achieved in UV-irradiated sections mounted in media artificially enriched with hydrogen peroxide, or directly in aqueous solutions of H2O2, Na2O2 or HIO4. Oxidations not associated with UV light led to nonfluorogenic breakdown of melanins. These observations indicate that the common mechanism may be an oxidative attack resulting from a concerted action of hydrogen peroxide and UV light leading, through strongly fluorescent intermediates, to a complete bleaching and oxidative breakdown of melanin and melanin-like polymers. Reactive oxygen species (including ozone) are considered to be important reactants in these experiments. Lipopigments differ from melanin-like pigments by their primary autofluorescence, which mostly faded during continuous prolonged irradiation. The only regular exception was melanosis coli pigment, the autofluorescence of which was considerably augmented by UV irradiation. Our results demonstrate a novel type of fluorogen in autofluorescent pigment histochemistry. The implications of the results are discussed especially in the light of the possible presence of melanin-based fluorogens in lipopigments.