Study on the Effects of Malathion on Hepatic Mitochondrial Function and Glucose Metabolism

Abstract

Concept Organophosphate compounds (OPs) are cholinesterase inhibiting chemicals used as pesticides and nerve agents. Exposure to OPs causes a significant number of poisoning and death each year. The extensive use of pesticides to protect agricultural crops can result in the transfer of these compounds into the atmosphere and their diffusion toward urban areas. Inhibition of the enzyme acetylcholinesterase (AChE) resulting in an accumulation of the neurotransmitter acetylcholine and the continued stimulation of cholinergic receptors is the main mechanism of toxicity. Other mechanisms have been proposed for the toxicity of OPs in animals and human, particularly oxidative stress. Furthermore, one of their adverse effects seen in human is hyperglycemia, so that animal studies have indicated disrupted glucose homeostasis following acute and subchronic exposure to these compounds. Liver, as the most important organ involved in detoxification of xenobiotics, as well as regulation of glucose metabolism and related enzymes can deviate from its normal function in different imbalanced situations like oxidative stress and antioxidant depletion, so it is considered as a potential target for OP-induced damages.Objective this study was to evaluate hepatotoxic effects of the most common used OP, malathion, in acute and subchronic exposure by using both in vitro and in vivo models. It has been enforced on the role of reactive oxygen species (ROS), mitochondrial dysfunction and cellular antioxidant glutathione (GSH) in malathion-induced toxicity toward hepatocytes. Just as, following them in animal study, the influence of subchronic exposure to malathion on hepatic glucose metabolism in relation to AChE inhibition, oxidative stress and inflammatory response has been investigated. Methods hepatocytes were isolated from male rats by liver perfusion with collagenase solution. Cell suspensions were incubated under atmosphere of 95% O2 + 5% CO2. Malathion, 1-bromoheptane (to deplete cellular GSH) and N-Acetylcysteine (NAC; GSH precursor) were added in related protocols, and after sampling at the indicated time intervals (1, 2 and 3 hours), cell viability, ROS formation and mitochondrial membrane potential (MMP) were determined by using trypan blue exclusion, fluorogenic probe, 2', 7' -dichlorofluorescin diacetate (DCFH-DA) and Rhodamine 123 fluorescense methods, respectively.To study in vivo, adult male rats received malathion via oral gavage at dose of 25, 50 and 100 mg/kg/day for 32 days. At the end of specified treatment, the fasted animals were anesthetized with pentobarbital and then blood and liver samples were removed and kept at -70C for related tests.Results malathion significantly decreased cell viability and MMP and increased ROS formation at concentrations more than 0.5 mM in freshly isolated hepatocytes, as such, cytotoxicity was further intensified by administration of 1-bromoheptane. Despite the protective effect of NAC on cell viability and MMP in both groups treated with malathion 1and 1.5 mM, its efficacy against ROS formation was only seen in the first group. The data obtained from animal study indicated fasting hyperglycemia in line with increased activity of hepatic phosphoenolpyruvate carboxykinase, glucose 6-phosphatase, and tumor necrosis factor alpha (TNF). In addition to the impaired glucose tolerance and inhibition of AChE activity in a dose-dependent manner, there was a significant increase in hepatic lipid peroxidation, carbonyl groups and 8-deoxyguanosine as the biomarkers of reactive oxygen species mediated damage to lipid, protein and DNA, respectively.Discussion and conclusion results of the study in vitro, not only confirmed occurrence of oxidative stress and mitochondrial dysfunction beside GSH depletion in malathion-induced hepatotoxicity, but also proposed other mechanisms in addition to ROS scavenging for beneficial effects of NAC against OP toxicity. In animal experiments, impaired glucose tolerance and fasting hyperglycemia in line with increased activity of gluconeogenesis are indicators of elevated hepatic glucose production. Considering significant oxidative damages to lipids, proteins and DNA as well as increased level of TNF, the results suggest the possibility of malathion-induced insulin resistance in the liver through oxidative and inflammatory signaling pathways.