Skin Histology of Pithecopus spp. and Modulation of Glutathione Status by the Antioxidant Tryptophyllin PaT-2

Amphibians of the family Phyllomedusidae produce complex cutaneous secretions with ecological and pharmacological relevance. Here, we investigated the skin morphology of Pithecopus oreades and P. azureus and the antioxidant function of the tryptophyllin PaT-2 in microglial cells. Histological and histochemical analyses revealed a conserved glandular organization, with mucous glands rich in neutral mucopolysaccharides and serous glands containing acidic and sulfated polysaccharides, concentrated predominantly in the dorsal and cephalic regions. MALDI mass spectrometry imaging showed that PaT-2 (m/z 696.4) is heterogeneously distributed. A second ion (m/z 1626.8), assigned to a truncated form of a phylloseptin-like antimicrobial peptide, was predominantly localized in limb tissues and partially overlapped with PaT-2, suggesting co-deployment of antioxidant and antimicrobial molecules on the skin. In BV2 microglial cells, menadione (10 µM) induced oxidative stress reflected by increased oxidized glutathione (GSSG) and higher total glutathione levels, consistent with a compensatory increase in glutathione synthesis. Synthetic PaT-2 (50 µM) was associated with numerically lower GSSG accumulation and a smaller increase in total glutathione when co-administered with menadione, showing no cytotoxicity while demonstrating modulation of endogenous glutathione status. These findings advance the integumentary biology of Pithecopus, suggest differences in peptide distribution between the specimens examined, and extend the antioxidant characterization of PaT-2 to the level of glutathione status, supporting its further investigation as a candidate molecule for oxidative stress-related research.