Campanha Helen M, Carvalho Félix, Schlosser Paul M
Rutgers, New Jersey Medical School-Graduate School of Biomedical Sciences, 185 South Orange Avenue, MSB H609, Newark, NJ 07103, United States.
REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal.
Toxicol Lett. 2014 Oct 15;230(2):122-31. doi: 10.1016/j.toxlet.2014.03.012. Epub 2014 Mar 27.
The classical enzymatic role of acetylcholinesterase (AChE) is to terminate impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine (ACh). Inactivation of this enzyme's catalytic site is the primary mechanism of acute toxicity of OP insecticides (e.g. parathion, chlorpyrifos). There is now sufficient evidence to suggest that AChE has a neurotrophic function that may be altered by organophosphate (OP) exposure, resulting in defects of neuronal growth and development, though the clarification of the mechanisms involved require further in vitro investigation. In the present study, the mouse neuroblastoma×rat glioma hybrid NG108-15 cell line was used to investigate the differential effects between inhibition of the catalytic site and peripheral anionic site (PAS) of acetylcholinesterase (AChE) on cell adhesion, proliferation and neuritogenesis, in the presence and absence of human red blood cell (hRBC) AChE (ED3.1.1.7). AChE active-site inhibitor paraoxon (PO; 0.1-1.0μM), when added to NG108-15 cells grown on AChE-coated plates, had no effect on cell proliferation, but exerted a significant reduction in strongly adherent viable cells accompanied by mostly short process formations, with 18% of cells considered to be neuritogenic, similar to that observed on uncoated plates. In contrast, PO had no significant effect on cell adhesion and proliferation of NG108-15 cells on uncoated plates. The PAS-ligand thioflavin-T (Th-T; 0.5-25μM), however, decreased cell adhesion and proliferation, on both uncoated and ACh-E coated plates, with less magnitude on AChE-coated plates. Taken together, these results suggest that strong cell adherence and neuritogenesis are sensitive to PO in this cell culture model, with no impact on proliferation, in the presence of membrane bound AChE-coating, while there is no sensitivity to PO on uncoated plates. On the other hand, binding of Th-T directly to the PAS affects both cell adherence and proliferation, with less magnitude in the presence of membrane-bound AChE. The current study indicates that PO is deleterious in neural development during critical periods of strong cell adhesion and differentiation, interfering with AChE trophic function.
乙酰胆碱酯酶（AChE）的经典酶促作用是通过快速水解乙酰胆碱（ACh）来终止胆碱能突触处的冲动传递。该酶催化位点的失活是有机磷（OP）杀虫剂（如对硫磷、毒死蜱）急性毒性的主要机制。现在有足够的证据表明，AChE具有神经营养功能，可能会因有机磷（OP）暴露而改变，导致神经元生长和发育缺陷，不过对其中涉及的机制的阐明还需要进一步的体外研究。在本研究中，使用小鼠神经母细胞瘤×大鼠胶质瘤杂交NG108-15细胞系，在有和没有人红细胞（hRBC）AChE（ED3.1.1.7）的情况下，研究抑制乙酰胆碱酯酶（AChE）的催化位点和外周阴离子位点（PAS）对细胞黏附、增殖和神经突形成的不同影响。当将AChE活性位点抑制剂对氧磷（PO；0.1 - 1.0μM）添加到在AChE包被的平板上生长的NG108-15细胞中时，对细胞增殖没有影响，但会使强黏附的活细胞显著减少，同时大多形成短突起，只有18%的细胞被认为具有神经突形成能力，这与在未包被平板上观察到的情况相似。相比之下，PO对未包被平板上的NG108-15细胞的黏附和增殖没有显著影响。然而，PAS配体硫黄素-T（Th-T；0.5 - 25μM）在未包被和平板和ACh-E包被的平板上均会降低细胞黏附和增殖，在AChE包被的平板上影响程度较小。综上所述，这些结果表明，在这种细胞培养模型中，在存在膜结合AChE包被的情况下，强细胞黏附和神经突形成对PO敏感，对增殖无影响，而在未包被的平板上对PO不敏感。另一方面，Th-T直接与PAS结合会影响细胞黏附和增殖，在存在膜结合AChE的情况下影响程度较小。当前研究表明，在细胞强黏附和分化的关键时期，PO对神经发育有害，会干扰AChE的营养功能。
