The organophosphorus pesticide dimethoate decreases cell viability and induces changes in different biochemical parameters of rat pancreatic stellate cells

Highlights

• Dimethoate inhibited certain esterases enzymatic activity in PSCs.

• Cell viability was reduced in the presence of the pesticide.

• Dimethoate induced a slight increase in reactive oxygen species production.

• In the presence of dimethoate a slow and progressive increase in [Ca²⁺]_c was observed.

Abstract

In the present study we employed cultured pancreatic stellate cells to study the effect of the organophosphorus insecticide dimethoate on pancreatic cell physiology. Esterase activity, cell viability, reactive oxygen species generation and Ca²⁺ mobilization were examined. Our results show that dimethoate (0.1, 1 and 10 μM) induced a concentration-dependent inhibition of cholinesterase enzymatic activity at all concentrations tested. A drop in carboxylesterase activity was noted in the presence of 10 μM dimethoate. In the presence of the pesticide a decrease in cell viability was detected. The clearer effect could be observed when the cells had been incubated during 96 h in the presence of dimethoate. The pesticide induced a slight but statistically significant increase in the production of reactive oxygen species in the mitochondria. Incubation of cells with dimethoate, in the presence of Ca²⁺ in the extracellular medium, led to a slow and progressive increase in [Ca²⁺]_c towards an elevated value over the prestimulation level. A similar behavior was observed in the absence of extracellular Ca²⁺, indicating that dimethoate releases Ca²⁺ from the intracellular stores. Our results suggest that dimethoate might alter intracellular pathways that are critical for pancreatic physiology, creating a situation potentially leading to dysfunction in the exocrine pancreas.

Introduction

Organophosphorus compounds (OPs) are widespread used pesticides because of their benefits to mankind in the agricultural, industrial, and health areas. However, the OPs exert toxicities in both humans and animals. It is generally accepted that exposure to OPs can lead to toxicity, which is based on the impairment of cell physiology and, hence, acute and chronic effects (Rush et al., 2010). The main toxicological effects are related to irreversible inactivation of esterases (WHO, 1986). The OPs act as potent inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) activity, a critical enzyme in the functioning of cholinergic synapses at the central and peripheral nervous systems (Fukuto, 1990). The cholinesterases (ChE) consist of a ubiquitous class of enzymes that catalyze the hydrolysis of the ester bond of acetylcholine (ACh). In vertebrates the isoforms AChE and butyrylcholinesterase (BChE, EC 3.1.1.8) have been described. AChE inactivates acetylcholine, and a consequence of its inhibition is the accumulation of this neurotransmitter in the synaptic cleft in animals exposed to OP agrochemicals. This leads to a subsequent over-excitation of the postsynaptic cell by ACh. Besides, it has been hypothesized that BChE acts as a scavenging molecule for various xenobiotics such as OPs (Masson and Lockridge, 2010; Massoulié et al., 1993).

Carboxylesterases (CbEs, EC 3.1.1.1) belong to the same group of serine hydrolases, they present binding affinity to OPs and their inhibition could be used as a potential biomarker of exposition to pesticides. Despite the physiological roles of BChE and CbE remain uncertain, their sensitivity to inhibition by binding to OPs can be regarded to as a pivotal action in line with a detoxification mechanism of pesticides (Sogorb and Vilanova, 2002; Wheelock et al., 2008; Wheelock and Nakagawua, 2010).

Dimethoate is a systemic OP insecticide effective against a broad range of insects and mites, which is approved for use in the European Union and in other parts of the world. It is moderately toxic to mammals and may have serious health implications (for humans as well as for livestock and companion animals) as it is an AChE inhibitor. Upon exposure, dimethoate can enter the blood and reach kidney, brain, myocardium, liver, lung, skeletal muscle, and other organs (Tarbah et al., 2007).

In the body, the pancreas can be a target organ for toxicity of pesticides. The implications in health of a deregulation of its function by pesticides are based on the role of this gland in hormone secretion (for example insulin and glucagon, which play a pivotal role in glucose metabolism) and also its role in the secretion of digestive enzymes (Karami-Mohajeri and Abdollahi, 2011).

Isolated pancreatic cells are a useful model to investigate the direct effects of certain drugs. Cell culture techniques have been developed to study the effect of long term exposure to drugs in vitro. Most cellular models available for studies on the exocrine pancreas are derived from tumors, i.e., are based on transformed cells. To date, it is not possible to culture pancreatic acinar cells or, at least, to reliably maintain a viable culture of these cells beyond 48 h. Pancreatic stellate cells (PSCs) represent a cellular, non-tumoral, population present in the exocrine pancreas, whose physiology is expected not to be transformed as it occurs in tumors. PSCs can be cultured and, hence, can be useful for drug tests in the laboratory. Within the pancreas, PSCs have particular interest because of their role in maintenance of regular extracellular matrix (ECM). This is carried out through to synthesis and secretion of proteins as well as matrix degrading enzymes (Phillips et al., 2003). Following insult to the pancreas, PSCs become activated thus paying an important role in the fibrosis that occurs in pancreatitis and cancer (Gryshchenko et al., 2016). In addition, it has been suggested that rat PSCs synthesize AChE and secrete ACh, which could signal neighboring pancreatic cells (Phillips et al., 2010).

Studies of esterases´ properties and their characterization are of great interest because they can be used as biomarkers of exposure to OPs products. The analysis of the effects of OPs using cellular models can be useful for a better understanding of the responses evoked in organisms.

In the present study, cultured PSCs were incubated in the presence of dimethoate, esterase activities were analyzed and enzymatic characterization of AChE, BChE and CbEs was performed. Cell viability, reactive oxygen species generation and intracellular calcium (Ca²⁺) mobilization were also examined. The results obtained employing cultures of PSCs will be useful to analyze the effects of different drugs, including pesticides, acting directly on the cells. Our objective was to shed more light on the mechanisms involved in the actions of dimethoate on the exocrine pancreatic function and its putative role in the induction of disease.