A biologia, as glândulas de veneno e a secreção cutânea da perereca da caatinga Corythomantis greeningi: um estudo integrativo

Abstract

Corythomantis greeningi (Cg) is an endemic anuran species of the Brazilian Caatinga, which has a co-ossified head with the skull (casque-head), used as cover when the animal enters backwards in small crevices, as a defense against predators and/or desiccation, in a behavior known as fragmosis. Although the skin of this species has already been relatively well studied by our group, its morphological details and the biochemical and pharmacological characteristics of its cutaneous secretion are not yet known. Thus, this work aims to fill this gap in the knowledge of this species. Dorsal, ventral and inguinal skin fragments were processed for histology and transmission (TEM) and scanning (SEM) electron microscopy. For biochemical studies the skin secretion was analyzed by SDS-PAGE, RP-HPLC and mass spectrometry. Crude secretion and its main fractions were tested for nociceptive and edema activities in mice, and for citotoxicity and antitumor effects in cell cultures of murine melanoma (B16F10) and murine fibroblasts (L929). The results show that the surface of the skin examined by SEM has a clear sexual dimorphism, with males presenting numerous cornified spicules in the dorsum, while the females have smoother skin in this region. The skin surface in all body regions presents epidermal micro-projections which are more prominent in the ventral and inguinal regions. These projections form a network of interconnecting channels that may facilitate the uptake of water. In both genders the skin surface of the inguinal region, has prominent hydrophilic warts associated with an extensive capillary network and the absence of calcified layer; together, these features may facilitate the uptake of the substrate water. Regarding the skin glands two types of acinar mucous glands and one type of venom syncytial gland were observed. This latter gland type is particularly abundant in the dorsal region, which is more susceptible to the attack of predators. The venom granules examined by TEM showed a complex structure comprised of several distinct compartments with different electron densities. The biochemical analyzes revealed that the secretion is plenty of protein in all molecular 17 mass bands mainly in the range 55-30 kDa. When analyzed by LC-MS it shows many low weight molecules (200 to 500 Da) with masses consistent with alkaloids and steroids. The crude secretion also showed to be positive for the presence of metallopeptidases and cysteine peptidase and negative for serine peptidases. The crude skin secretion was able to induce edema in all doses tested, most of them lasting 96 hours after injection, and was highly nociceptive in all tested doses. Tests with the isolated fractions showed that several of them have the ability to induce edema activity as either nociceptive, but with different intensity levels. In addition, we observed a greater inhibition of cell growth in the B16F10 lineage than in in L929 lineage, with the effective doses (IC50) of 4.6 mg/mL and 11.9 mg/mL, respectively. These results showed a possible antitumor activity in Cg skin secretion.