A lipidomics approach reveals new insights into Crotalus durissus terrificus and Bothrops moojeni snake venoms.

Snakebite envenomation causes > 81,000 deaths and incapacities in another 400,000 people worldwide every year. Snake venoms are complex natural secretions comprised of hundreds of different molecules with a wide range of biological functions that after injection cause local and systemic manifestations. Although several studies have investigated snake venoms, the majority have focused on the protein portion (toxins), without significant attention paid to the lipid fraction. Therefore, an untargeted lipidomic approach based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) was applied to investigate the lipid constituents of venoms of the snake species Crotalus durissus terrificus and Bothrops moojeni. Phosphatidylcholines (PC), Lyso-PCs, phosphatidylethanolamines (PE), Lyso-PE, phosphatidylserine (PS), phosphatidylinositol (PI), ceramides (Cer), and sphingomyelin (SM) species were detected in the analyzed snake venoms. The identified lipids included bioactive compounds such as platelet-activating factor (PAF) precursor, PAF-like molecules, plasmalogens, ceramides, and sphingomyelins with long fatty acid chain lengths, which may be associated with the systemic responses triggered by C. d. terrificus and B. moojeni envenomation. These responses include platelet aggregation, activation of intercellular adhesion molecule 1 (ICAM1), apoptosis, as well as the production of pro-inflammatory lipid mediators, cytokines, and reactive species. The newly proposed lipidomics strategy provided valuable information regarding the lipid profiles of viperid venoms, which could lead to increased understanding of the complex pathology promoted by snakebite envenomation.

Intravenous toxicity and toxicokinetics of an HDL mimetic, Fx-5A peptide complex, in cynomolgus monkeys.

Fx-5A peptide complex (Fx-5A), a High Density Lipoproteins (HDL) mimetic, has been shown to reduce atherosclerosis. The safety and toxicokinetics of Fx-5A administered IV by 30 min infusion at 8, 25 or 75 mg/kg body weight or vehicle, once every other day for 27 days, were assessed in cynomolgus monkeys. The Fx-5A was well tolerated at all doses. At the highest dose, there were statistically significant effects on hematology and clinical chemistry parameters that were considered non-adverse. Dose-dependent recoverable non-adverse erythrocytes morphological changes (acanthocytes, echinocytes, spherocytes, microcytes, and/or schistocytes) were observed. Fx-5A was not hemolytic in in-vitro fresh NHP or human blood assay. There were no Fx-5A-related statistically significant changes for any cardiovascular function, ECG or respiratory parameters, when compared to control. In addition, there were no Fx-5A-related effects on organ weights, macroscopic or microscopic endpoints. Finally, Fx-5A exhibited sporadic non-appreciable detection of anti-Fx-5A antibodies and a dose-dependent linear toxicokinetics with T1/2 value ranges from 2.7 to 6.2 h. In conclusion, the No Observed Adverse Effect Level was considered to be 75 mg/kg/day with associated exposures average Cmax and AUC0-last of 453 µg/mL and 2232 h µg/mL, respectively, on Day 27.

Pharmacological reversion of sphingomyelin-induced dendritic spine anomalies in a Niemann Pick disease type A mouse model.

Understanding the role of lipids in synapses and the aberrant molecular mechanisms causing the cognitive deficits that characterize most lipidosis is necessary to develop therapies for these diseases. Here we describe sphingomyelin (SM) as a key modulator of the dendritic spine actin cytoskeleton. We show that increased SM levels in neurons of acid sphingomyelinase knock out mice (ASMko), which mimic Niemann Pick disease type A (NPA), result in reduced spine number and size and low levels of filamentous actin. Mechanistically, SM accumulation decreases the levels of metabotropic glutamate receptors type I (mGluR1/5) at the synaptic membrane impairing membrane attachment and activity of RhoA and its effectors ROCK and ProfilinIIa. Pharmacological enhancement of the neutral sphingomyelinase rescues the aberrant molecular and morphological phenotypes in vitro and in vivo and improves motor and memory deficits in ASMko mice. Altogether, these data demonstrate the influence of SM and its catabolic enzymes in dendritic spine physiology and contribute to our understanding of the cognitive deficits of NPA patients, opening new perspectives for therapeutic interventions.

Pseudomonas aeruginosa cytotoxin: the influence of sphingomyelin on binding and cation permeability increase in mammalian erythrocytes.

A cytotoxic protein isolated from Pseudomonas aeruginosa damages the plasma membranes of many mammalian cells by forming pores. We studied binding of the 125I-cytotoxin and the resulting increase of cation permeability in erythrocytes of various mammalian species. The sensitivity of red blood cells was inversely related to the relative sphingomyelin content in their external surface. Thus, erythrocytes with a sphingomyelin to phosphatidylcholine ratio below 1 (dog, rat, rabbit and man) were sensitive, whereas red blood cells with a ratio above 1 (pig, cattle and sheep) were not attacked even with 100-fold higher cytotoxin concentrations. At 37 degrees C 6.8 +/- 1.2 x 10(3) molecules of 125I-cytotoxin were bound per rabbit erythrocyte (KD = 59 nM), whereas no binding occurred to cattle cells. Cleavage of sphingomyelin by sphingomyelinase C from Bacillus cereus (EC 3.1.4.12) triggered a dose-dependent enhancement in binding and permeability increase, particularly in red blood cells with a high proportion of sphingomyelin. The KDs for all animal species investigated were 53-60 nM. Pretreatment with mainly phosphatidylcholine-hydrolyzing phospholipases D from Streptomyces chromofuscus and cabbage (EC 3.1.4.4) or phospholipase C from Bacillus cereus (EC 3.1.4.3) did not influence the cytotoxin effect. The negative correlation between susceptibility and the proportion of sphingomyelin in plasma membranes suggests a binding site close to sphingomyelin.

Toxicity of sphingomyelin-containing liposomes after chronic injection into mice.

Chronic treatment (10 i.p. injections over 20 days) of Balb/c mice with SM liposomes led to 50 and 300% enlargement of the liver and spleen respectively. No such effect was observed after similar treatment with PC liposomes. Biochemical analysis of the enlarged tissues showed no significant changes in the concentrations of glycolipid, phospholipid and certain hydrolytic enzymes. However, the increase in tissue size was paralleled by an increase in protein content. Light and electron microscopy studies of the enlarged tissues revealed an increase in the number of Kupffer cells and collections of inflammatory cells in the liver and widespread granulomatous inflammation in the spleen. We conclude that SM liposomes, although probably toxic for use as a drug carrier, may serve as a model agent in the study of tissue granulomatous inflammation.