Dissecting the Enantioselective Neurotoxicity of Isocarbophos: Chiral Insight from Cellular, Molecular, and Computational Investigations.

Chiral organophosphorus pollutants are found abundantly in the environment, but the neurotoxicity risks of these asymmetric chemicals to human health have not been fully assessed. Using cellular, molecular, and computational toxicology methods, this story is to explore the static and dynamic toxic actions and its stereoselective differences of chiral isocarbophos toward SH-SY5Y nerve cells mediated by acetylcholinesterase (AChE) and further dissect the microscopic basis of enantioselective neurotoxicity. Cell-based assays indicate that chiral isocarbophos exhibits strong enantioselectivity in the inhibition of the survival rates of SH-SY5Y cells and the intracellular AChE activity, and the cytotoxicity of (S)-isocarbophos is significantly greater than that of (R)-isocarbophos. The inhibitory effects of isocarbophos enantiomers on the intracellular AChE activity are dose-dependent, and the half-maximal inhibitory concentrations (IC50) of (R)-/(S)-isocarbophos are 6.179/1.753 µM, respectively. Molecular experiments explain the results of cellular assays, namely, the stereoselective toxic actions of isocarbophos enantiomers on SH-SY5Y cells are stemmed from the differences in bioaffinities between isocarbophos enantiomers and neuronal AChE. In the meantime, the modes of neurotoxic actions display that the key amino acid residues formed strong noncovalent interactions are obviously different, which are related closely to the molecular structural rigidity of chiral isocarbophos and the conformational dynamics and flexibility of the substrate binding domain in neuronal AChE. Still, we observed that the stable "sandwich-type π-π stacking" fashioned between isocarbophos enantiomers and aromatic Trp-86 and Tyr-337 residues is crucial, which notably reduces the van der Waals' contribution (ΔGvdW) in the AChE-(S)-isocarbophos complexes and induces the disparities in free energies during the enantioselective neurotoxic conjugations and thus elucidating that (S)-isocarbophos mediated by synaptic AChE has a strong toxic effect on SH-SY5Y neuronal cells. Clearly, this effort can provide experimental insights for evaluating the neurotoxicity risks of human exposure to chiral organophosphates from macroscopic to microscopic levels.

Serum components stimulate pericardial tissue contraction.

BACKGROUND AND AIMS OF THE STUDY: Previous experiments have demonstrated the retraction and fibrosis of vital autologous pericardial flap implants in the descending aorta of sheep. An in-vitro model of pericardial tissue contraction was developed that showed healing reactions similar to those observed in the fresh in-vivo flap. Here, the component(s) of serum that stimulate tissue contraction were partially characterized. The molecular weight range and stability (heat and protease resistance) of the serum component(s) are described. Tissue contraction also requires de-novo protein synthesis. METHODS: Sections (1 cm2) of sheep pericardium were incubated with fractionated, heat-treated, or protease-treated fetal bovine serum for 14 days. In addition, SDS-PAGE protein profiles were generated using tissues incubated with and without cycloheximide for up to 12 days. RESULTS: Tissue contraction was observed in molecular weight serum fractions > or =5 kDa, as well as in samples incubated with heat and protease-treated serum. SDS-PAGE showed the appearance of a protein band after day 4 during the process of tissue contraction that was absent in samples incubated with cycloheximide. CONCLUSION: Serum fractions > or =5 kDa stimulated protein synthesis and pericardial tissue contraction. The active component(s) was shown to be heat stable, but partially sensitive to protease. The addition of cycloheximide to the culture medium, shown previously to prevent pericardial tissue contraction, inhibited de-novo synthesis of the protein that appeared during the process of tissue contraction.