The organophosphorus pesticide dimethoate decreases cell viability and induces changes in different biochemical parameters of rat pancreatic stellate cells
Graphical abstract
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.
Section snippets
Animals and chemicals
In the present study, newborn Wistar rats (one week) were used. Animals were obtained from the animal house of the University of Extremadura (Caceres, Spain). Animals were humanely handled and sacrificed in accordance to the institutional Bioethical Committee. Collagenase CLSPA was obtained from Worthington Biochemical Corporation (Lakewood, NJ, U.S.A.). Ethylene glycol-bis(2-aminoethylether)-N,N,N´N´-tetraacetic acid (EGTA) and thapsigargin were obtained from Sigma Chemicals Co. (Madrid,
Characterization of esterase activity in pancreatic stellate cells
The activities of ChEs and CbE in extracts obtained from PSCs were determined respectively by the hydrolysis of the substrates ATCI and BTCI for ChEs and α-NA for CbE. The concentration used for each one was calculated from the kinetic profiles, adjusting the data to Michaelis-Menten eq. Y = Vmax × X/(Km + X). The kinetic parameters Vmax and Km for the three enzymes were calculated and are shown in Table 1. With the information obtained we chose the concentration of 10 mM for ATCI, 60 mM for
Discussion
Dimethoate is an OP insecticide that is used worldwide in agriculture and household practices. This compound exerts side effects, which are derived from its toxicity. This matter has resulted in a series of environmental impacts on non-target organisms. The OP may enter the body by oral ingestion, inhalation or after cutaneous exposure. The major mechanism of action of OP is the inhibition of AChE activity that mainly, but not exclusively, occurs in the nervous system (Mostafalou and Abdollahi,
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgments
Funding for this study was provided by Ministerio de Economía y Competitividad (UNEX13-1E-1608, BFU2016-79259-R). The funding source(s) had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication. The authors would like to thank Mrs. Ana Maria Moreno and Mercedes Gomez for their valuable technical assistance.
References (45)
- et al.
Sub-chronic exposure to the insecticide dimethoate induces a proinflammatory status and enhances the neuroinflammatory response to bacterial lypopolysaccharide in the hippocampus and striatum of male mice
Toxicol. Appl. Pharmacol.
(2013) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Molar absorption coefficients for the reduced Ellman reagent: reassessment
Anal. Biochem.
(2003) - et al.
Generation of ROS in response to CCK-8 stimulation in mouse pancreatic acinar cells
Mitochondrion
(2004) - et al.
A new generation of Ca2+ indicators with greatly improved fluorescence properties
J. Biol. Chem.
(1985) - et al.
Calcium signalling in pancreatic stellate cells: Mechanisms and potential roles
Cell Calcium
(2016) - et al.
A biochemical, histochemical, and ultrastructural evaluation of the effect of dimethoate intoxication on rat pancreas
Toxicol. Lett.
(2002) - et al.
Altered glucose homeostasis and oxidative impairment in pancreas of rats subjected to dimethoate intoxication
Toxicology
(2007) - et al.
Inhibitory effects of biocides on transcription and protein activity of acetylcholinesterase in the intertidal copepod Tigriopus japonicus
Comp. Biochem. Physiol. Part C Toxicol.
(2015) - et al.
New insights on molecular interactions of organophosphorus pesticides with esterases
Toxicology
(2017)
Butyrylcholinesterase for protection from organophosphorus poisons: Catalytic complexities and hysteretic behavior
Arch. Biochem. Biophys.
Structure and functions of acetylcholinesterase and butyrylcholinesterase
Prog. Brain Res.
Galectin-1-driven upregulation of SDF-1 in pancreatic stellate cells promotes pancreatic cancer metastasis
Cancer Lett.
Mechanisms of chlorpyrifos and diazinon induced neurotoxicity in cortical culture
Neuroscience
Effects of acute dimethoate administration on antioxidant status of liver and brain of experimental rats
Toxicology
Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis
Toxicol. Lett.
Distribution of dimethoate in the body after a fatal organphosphate intoxication
Forensic Sci. Int.
Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos
Aquat. Toxicol.
Biochemical changes in primary culture of skeletal muscle cells following dimethoate exposure
Toxicology
Sex differences and effects of estrogenic compounds on the expression of inflammatory molecules by astrocytes exposed to the insecticide dimethoate
Neurotox. Res.
Pancreatic stellate cell: Pandora's box for pancreatic disease biology
World J. Gastroenterol.
Melatonin induces calcium release from CCK-8-and thapsigargin-sensitive cytosolic stores in pancreatic AR42J cells
J. Pineal Res.
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