Molecular Physicochemical Properties of Selected Pesticides as Predictive Factors for Oxidative Stress and Apoptosis-Dependent Cell Death in Caco-2 and HepG2 Cells.

Abstract

In this work, three pesticides of different physicochemical properties: glyphosate (GLY, herbicide), imidacloprid (IMD, insecticide), and imazalil (IMZ, fungicide), were selected to assess their cytotoxicity against Caco-2 and HepG2 cells. Cell viability was assessed by the Alamar Blue assay, after 24 and 48 h exposure to different concentrations, and IC50 values were calculated. The mechanisms underlying toxicity, namely cellular reactive oxygen species (ROS), glutathione (GSH) content, lipid peroxidation, loss of mitochondrial membrane potential (MMP), and apoptosis/necrosis induction were assessed by flow cytometry. Cytotoxic profiles were further correlated with the molecular physicochemical parameters of pesticides, namely: water solubility, partition coefficient in an n-octanol/water (Log Pow) system, topological polar surface area (TPSA), the number of hydrogen-bonds (donor/acceptor), and rotatable bonds. In vitro outputs resulted in the following toxicity level: IMZ (Caco-2: IC50 = 253.5 ± 3.37 μM, and HepG2: IC50 = 94 ± 12 μM) > IMD (Caco-2: IC50 > 1 mM and HepG2: IC50 = 624 ± 24 μM) > GLY (IC50 >>1 mM, both cell lines), after 24 h treatment, being toxicity time-dependent (lower IC50 values at 48 h). Toxicity is explained by oxidative stress, as IMZ induced a higher intracellular ROS increase and lipid peroxidation, followed by IMD, while GLY did not change these markers. However, the three pesticides induced loss of MMP in HepG2 cells while in Caco-2 cells only IMZ produced significant MMP loss. Increased ROS and loss of MMP promoted apoptosis in Caco-2 cells subjected to IMZ, and in HepG2 cells exposed to IMD and IMZ, as assessed by Annexin-V/PI. The toxicity profile of pesticides is directly correlated with their Log Pow, as affinity for the lipophilic environment favours interaction with cell membranes governs, and is inversely correlated with their TPSA; however, membrane permeation is favoured by lower TPSA. IMZ presents the best molecular properties for membrane interaction and cell permeation, i.e., higher Log Pow, lower TPSA and lower hydrogen-bond (H-bond) donor/acceptor correlating with its higher toxicity. In conclusion, molecular physicochemical factors such as Log Pow, TPSA, and H-bond are likely to be directly correlated with pesticide-induced toxicity, thus they are key factors to potentially predict the toxicity of other compounds.