Involvement of superoxide in malaoxon-induced toxicity in primary cultures of cortical neurons.

Organophosphorus compounds (OP) represent a class of insecticides that are used most globally. The neurotoxic effects are attributed mainly to acetylcholinesterase (AChE) enzyme inhibition, which is responsible for cholinergic manifestations in individuals acutely exposed to OP. However, AChE inhibition alone cannot account for the wide range of symptoms that were reported following OP exposures. In agreement with this, evidence shows that non-cholinergic events may be mechanistically linked to OP-induced neurotoxicity. The aim of this study was to investigate the potential occurrence of oxidative stress as a critical step in the toxicity induced by the OP malaoxon(MAL) using primary cultures of mouse cortical neurons, as well as to distinguish MAL-induced oxidative stress and cell toxicity from an action on AChE blockade. Primary cultures of mouse cortical neurons were treated with MAL (0.01; 0.1; 1; 10; or 100 µM) at varying time points (1, 3, 6, 24, 48, or 144 hr) and the following biochemical parameters determined including cell viability, AChE activity, and superoxide production. MAL significantly reduced cell viability in a concentration- and time-dependent manner. Of note, 1 µM MAL significantly inhibited (approximately 75%) AChE activity after 48 hr incubation. Pralidoxime (PRAL) (600 µM), a classical AChE reactivator, significantly protected against MAL-induced AChE blockade; however, PRAL did not affect MAL-mediated fall in cellular viability, indicating that AChE inhibition is not necessarily correlated with insecticide-induced decrease in cell survival. MAL-induced diminished cell viability was preceded by a significant increase in superoxide anion production. The antioxidant agent ascorbic acid (AA) (200 µM), which significantly protected against MAL-induced superoxide anion production, did not alter MAL-induced AChE inhibition and significantly prevented insecticide-mediated fall in cell survival. Data show that increased superoxide anion production is an event that precedes MAL-induced cell toxicity in primary cultures of mouse cortical neurons. Based on the preventative effects of AA against MAL-mediated superoxide anion production and reduced cell viability, evidence indicates that oxidative stress represents an important step mediating MAL-induced toxicity in neurons and that AChE inhibition is not necessarily correlated with lowered cell survival noted in insecticide-exposed cells.

GABAergic pharmacological activity of propofol related compounds as possible enhancers of general anesthetics and interaction with membranes.

Phenol compounds, such as propofol and thymol, have been shown to act on the GABAA receptor through interaction with specific sites of this receptor. In addition, considering the high lipophilicity of phenols, it is possible that their pharmacological activity may also be the result of the interaction of phenol molecules with the surrounding lipid molecules, modulating the supramolecular organization of the receptor environment. Thus, in the present study, we study the pharmacological activity of some propofol- and thymol-related phenols on the native GABAA receptor using primary cultures of cortical neurons and investigate the effects of these compounds on the micro viscosity of artificial membranes by means of fluorescence anisotropy. The phenol compounds analyzed in this article are carvacrol, chlorothymol, and eugenol. All compounds were able to enhance the binding of [(3)H]flunitrazepam with EC50 values in the micromolar range and to increase the GABA-evoked Cl(-) influx in a concentration-dependent manner, both effects being inhibited by the competitive GABAA antagonist bicuculline. These results strongly suggest that the phenols studied are positive allosteric modulators of this receptor. Chlorothymol showed a bell-type effect, reducing its positive effect at concentrations >100 µM. The concentrations necessary to induce positive allosteric modulation of GABAA receptor were not cytotoxic. Although all compounds were able to decrease the micro viscosity of artificial membranes, chlorothymol displayed a larger effect which could explain its effects on [(3)H]flunitrazepam binding and on cell viability at high concentrations. Finally, it is suggested that these compounds may exert depressant activity on the central nervous system and potentiate the effects of general anesthetics.