Effects of imidacloprid on viability and increase of reactive oxygen and nitrogen species in HepG2 cell line.

Imidacloprid (IMD) is a neonicotinoid insecticide used in large quantities worldwide in both veterinary and agronomic applications. Several studies have shown adverse effects of IMD on non-target organisms, with the liver being identified as the main affected organ. This study aimed to evaluate the effects of IMD on human hepatoblastoma (HepG2) cells. HepG2 were exposed to IMD (0.25-2.0 mM) for 24 and 48 h. IMD treatment resulted in cytotoxicity in the HepG2, inhibiting cell proliferation in a dose- and time-dependent manner, starting at concentrations of 0.5 mM (24 h) and 0.25 mM (48 h), and reducing cell viability from 0.5 mM onwards (24 and 48 h). IMD significantly decreased the mitochondrial membrane potential at both time points investigated (2.0 mM), and also induced damage to the cell membrane, demonstrated by significant dose and time-dependent increases in lactate dehydrogenase (LDH) release from concentrations of 1.0 mM (24 h) and 0.5 mM (48 h) upwards. IMD treatment also increased the production of reactive oxygen and nitrogen species (ROS/RNS) at rates above 50% following 0.5 mM (24 h) or 0.25 mM (48 h) concentrations, and caused a significant decrease in reduced/oxidized glutathione ratio (GSH/GSSG), indicating oxidative stress. Furthermore, the antioxidant dithiothreitol, which reacts with ROS/RNS and acts as a thiol reducing agent, inhibited the cytotoxic effect of IMD. In addition, the metabolite IMD-olefin was more toxic than IMD. Our results indicate that IMD induces cytotoxicity in HepG2 cells and that this effect may be associated with an increase in the generation of ROS/RNS.

Diazinon impairs bioenergetics and induces membrane permeability transition on mitochondria isolated from rat liver.

Diazinon (DZN) is a broad-spectrum insecticide extensively used to control pests in crops and animals. Several investigators demonstrated that DZN produced tissue toxicity especially to the liver. In addition, the mitochondrion was implicated in DZN-induced toxicity, but the precise role of this organelle remains to be determined. The aim of this study was thus to examine the effects of DZN (50 to 150 µM) on the bioenergetics and mitochondrial permeability transition (MPT) associated processes in isolated rat liver mitochondria. DZN inhibited state-3 respiration in mitochondria energized with glutamate plus malate, substrates of complex I, and succinate, substrate of complex II of the respiratory chain and decreased the mitochondrial membrane potential resulting in inhibition of ATP synthesis. MPT was estimated by the extent of mitochondrial swelling, in the presence of 10 µM Ca2+. DZN elicited MPT in a concentration-dependent manner, via a mechanism sensitive to cyclosporine A, EGTA, ruthenium red and N-ethylmaleimide, which was associated with mitochondrial Ca2+ efflux and cytochrome c release. DZN did not result in hydrogen peroxide accumulation or glutathione oxidation, but this insecticide oxidized endogenous NAD(P)H and protein thiol groups. Data suggest the involvement of mitochondria, via apoptosis, in the hepatic cytotoxicity attributed to DZN.