Human muscarinic acetylcholine receptors are a target of the marine toxin 13-desmethyl C spirolide.

Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxication has been related to their presence in shellfish yet, the possible toxicological consequences to human health are actually unknown. The elucidation of the spirolide mechanism/s of action would help to estimate the threat to human consumers. Previous toxicological studies in mice suggested the involvement of acetylcholine receptors. In this work, the effects of the 13-desmethyl C spirolide on the activity and the expression of muscarinic acetylcholine receptors (mAChR) were analyzed using a human neuroblastoma cell model. The 13-desmethyl C spirolide inhibited the acetylcholine-induced calcium signal with a reduction of the maximum response to acetylcholine in the presence of the toxin. The 13-desmethyl C spirolide also reduced binding of the mAChR specific antagonist [(3)H]QNB to neuroblastoma cells. The effect of the 13-desmethyl C spirolide persisted after toxin removal and was inhibited by protection of the primary binding site with high concentrations of atropine suggesting an interaction of the spirolide with the orthologous binding site of mAChR. Moreover, the toxin induced a change in the characteristics of the membrane-associated M3 mAChRs, although it did not alter the total levels of M3 mAChR protein. The 13-desmethyl C spirolide targets mAChRs causing a reduction of function, a decrease of specific antagonist binding to mAChRs, and alteration of membrane-bound receptors that might have important toxicological implications.

Evaluation of various pH and temperature conditions on the stability of azaspiracids and their importance in preparative isolation and toxicological studies.

Azaspiracids (AZAs) are a group of shellfish toxins that were discovered in mussels from Irish waters in 1995. Because of the rare occurrence of poisoning incidents, the toxicity of the compounds is a continued matter of debate. Neither their mechanism of action nor their pharmacokinetic behavior has been elucidated, principally because of the lack of standards and reference tissues. Procedures to isolate AZAs from contaminated shellfish or to synthesize them have been developed; in particular, the procedures used for the preparative isolation of these toxins are currently being improved. The present paper describes the stability of AZAs in an array of pH and temperature conditions in methanolic solution, in shellfish tissue, and in aqueous mixtures of acids and shellfish tissues. Strong acids such as hydrochloric and formic acid led to rapid degradation of AZA1 at mM concentration, while the weaker acetic acid required harsher temperature conditions (70 degrees C) and greater concentrations to show similar effects. AZAs showed much greater stability in aqueous acidic mixtures with shellfish tissues, suggesting a significant protective effect of the matrix. A mechanism for the acid-catalyzed degradation is proposed, supported by mass spectral evidence from some of the degradation products. Strong bases (sodium hydroxide) also showed a detrimental effect on AZA1; however, weaker bases (ammonium hydroxide) did not lead to degradation over 24 h at room temperature. Finally, the toxic potential of acid degradation products of AZAs was found to be dramatically reduced compared to the parent compounds, as assessed through cytotoxicity.

Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti.

Ethanolic extracts from the kernels of ripe fruits from the Indian Lilac Melia azedarach and from the well-known Neem tree, Azadirachta indica were assayed against larvae of Aedes aegypti, the mosquito vector of dengue fever. The lethality bioassays were carried out according to the recommendations of the World Health Organization. Extracts were tested at doses ranging from 0.0033 to 0.05 g% in an aqueous medium for 24 and 48 h, at 25 or 30 degrees C, with or without feeding of the larvae. LC50, LC95 and LC99 were determined. Both seed extracts proved lethal for third to fourth instar larvae. Non-fed A. aegypti larvae were more susceptible to Azadirachta extracts at both temperatures. Under a more realistic environmental situation, namely with fed larvae at 25 degrees C, the death rates caused by the Melia extract were higher, although at 30 degrees C the extract of Azadirachta had an even higher lethality. Inter allia, the LC50 values for the crude extracts of these two members of the Meliaceae ranged from 0.017 to 0.034 g% while the LC99 values ranged from 0.133 to 0.189 g%. Since no downstream processing was undertaken to purify the active agents in the extracts, our findings seem very promising, suggesting that it may be possible to increase the larvicidal activity further by improving the extraction and the fractionation of the crude limonoids, for instance removing the co-extracted natural fats.