Mitochondrial Impact of Organophosphate Pesticide-Induced Cardiotoxicity: An In Silico and In Vitro Study.

Organophosphate pesticides are widely used; however, their use is limited due to neurotoxicity and, to a lesser extent, cardiotoxicity in humans. Given the high energy demands of cardiac muscle, which is characterized by a dense population of mitochondria, any damage to these organelles can exacerbate cardiotoxicity. This study aims to elucidate whether the cardiotoxic effects of organophosphate pesticides originate from mitochondrial dysfunction. To investigate this, in silico toxicogenomic analyses were performed using various tools, such as the Comparative Toxicogenomic Database, GeneMANIA, STRING, and Cytoscape. Results revealed that 11 out of the 13 WHO-recommended Class Ia organophosphate pesticides target genes associated with cardiotoxicity. Notably, three of these genes were mitochondrial, with catalase (CAT) being the common differentially expressed gene among parathion, methyl parathion, and phorate. Furthermore, protein-protein interaction analysis indicated a strong association between CAT and superoxide dismutase 2, mitochondrial (SOD2). Subsequently, isolated heart mitochondria were utilized to assess CAT and superoxide dismutase (SOD) activities in vitro. The findings demonstrated that at a concentration of 7.5 ng/µL, both methyl parathion and phorate significantly decreased CAT activity by approximately 35%. Moreover, phorate reduced total SOD and SOD2 activities by 17% and 19%, respectively, at the same concentration. In contrast, none of the three organophosphate pesticides induced the opening of the mitochondrial permeability transition pore. These results suggest that the reduction in CAT and SOD2 activities, critical antioxidant enzymes, leads to the accumulation of reactive oxygen species within mitochondria, ultimately resulting in mitochondrial damage. This mechanism likely underlies the observed cardiotoxicity induced by these organophosphate pesticides.

Proteomic analysis of Burkholderia zhejiangensis CEIB S4-3 during the methyl parathion degradation process.

Methyl parathion is an organophosphorus pesticide widely employed worldwide to control pests in agricultural and domestic environments. However, due to its intensive use, high toxicity, and environmental persistence, methyl parathion is recognized as an important ecosystem and human health threat, causing severe environmental pollution events and numerous human poisoning and deaths each year. Therefore, identifying and characterizing microorganisms capable of fully degrading methyl parathion and its degradation metabolites is a crucial environmental task for the bioremediation of pesticide-polluted sites. Burkholderia zhejiangensis CEIB S4-3 is a bacterial strain isolated from agricultural soils capable of immediately hydrolyzing methyl parathion at a concentration of 50 mg/L and degrading the 100% of the released p-nitrophenol in a 12-hour lapse when cultured in minimal salt medium. In this study, a comparative proteomic analysis was conducted in the presence and absence of methyl parathion to evaluate the biological mechanisms implicated in the methyl parathion biodegradation and resistance by the strain B. zhejiangensis CEIB S4-3. In each treatment, the changes in the protein expression patterns were evaluated at three sampling times, zero, three, and nine hours through the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the differentially expressed proteins were identified by mass spectrometry (MALDI-TOF). The proteomic analysis allowed the identification of 72 proteins with differential expression, 35 proteins in the absence of the pesticide, and 37 proteins in the experimental condition in the presence of methyl parathion. The identified proteins are involved in different metabolic processes such as the carbohydrate and amino acids metabolism, carbon metabolism and energy production, fatty acids ß-oxidation, and the aromatic compounds catabolism, including enzymes of the both p-nitrophenol degradation pathways (Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase), as well as the overexpression of proteins implicated in cellular damage defense mechanisms such as the response and protection of the oxidative stress, reactive oxygen species defense, detoxification of xenobiotics, and DNA repair processes. According to these data, B. zhejiangensis CEIB S4-3 overexpress different proteins related to aromatic compounds catabolism and with the p-nitrophenol  degradation pathways, the higher expression levels observed in the two subunits of the enzyme Hydroquinone dioxygenase, suggest a preferential use of the Hydroquinone metabolic pathway in the p-nitrophenol degradation process. Moreover the overexpression of several proteins implicated in the oxidative stress response, xenobiotics detoxification, and DNA damage repair reveals the mechanisms employed by B. zhejiangensis CEIB S4-3 to counteract the adverse effects caused by the methyl parathion and p-nitrophenol exposure.

The Effect of Organophosphate Exposure on Neuronal Cell Coenzyme Q10 Status.

Organophosphate (OP) compounds are widely used as pesticides and herbicides and exposure to these compounds has been associated with both chronic and acute forms of neurological dysfunction including cognitive impairment, neurophysiological problems and cerebral ataxia with evidence of mitochondrial impairment being associated with this toxicity. In view of the potential mitochondrial impairment, the present study aimed to investigate the effect of exposure to commonly used OPs, dichlorvos, methyl-parathion (parathion) and chloropyrifos (CPF) on the cellular level of the mitochondrial electron transport chain (ETC) electron carrier, coenzyme Q10 (CoQ10) in human neuroblastoma SH-SY5Y cells. The effect of a perturbation in CoQ10 status was also evaluated on mitochondrial function and cell viability. A significant decreased (P < 0.0001) in neuronal cell viability was observed following treatment with all three OPs (100 µM), with dichlorvos appearing to be the most toxic to cells and causing an 80% loss of viability. OP treatment also resulted in a significant diminution in cellular CoQ10 status, with levels of this isoprenoid being decreased by 72% (P < 0.0001), 62% (P < 0.0005) and 43% (P < 0.005) of control levels following treatment with dichlorvos, parathion and CPF (50 µM), respectively. OP exposure was also found to affect the activities of the mitochondrial enzymes, citrate synthase (CS) and mitochondrial electron transport chain (ETC) complex II+III. Dichlorvos and CPF (50 µM) treatment significantly decreased CS activity by 38% (P < 0.0001) and 35% (P < 0.0005), respectively compared to control levels in addition to causing a 54% and 57% (P < 0.0001) reduction in complex II+III activity, respectively. Interestingly, although CoQ10 supplementation (5 µM) was able to restore cellular CoQ10 status and CS activity to control levels following OP treatment, complex II+III activity was only restored to control levels in neuronal cells exposed to dichlorvos (50 µM). However, post supplementation with CoQ10, complex II+III activity significantly increased by 33% (P < 0.0005), 25% (P < 0.005) and 35% (P < 0.0001) in dichlorvos, parathion and CPF (100 µM) treated cells respectively compared to non-CoQ10 supplemented cells. In conclusion, the results of this study have indicated evidence of neuronal cell CoQ10 deficiency with associated mitochondrial dysfunction following OP exposure. Although CoQ10 supplementation was able to ameliorate OP induced deficiencies in CS activity, ETC complex II+III activity appeared partially refractory to this treatment. Accordingly, these results indicate the therapeutic potential of CoQ10 supplementation in the treatment of OP poisoning. However, higher doses may be required to engender therapeutic efficacy.

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish.

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25 mg/L of MP and 2.3 mg/L of C in mixture (one-third of LC50 value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p < 0.005). LDH activity increased significantly (p < 0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p < 0.005) decrease in AChE activity after initial 24 h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP + C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.

Acute toxicity of the insecticide methyl parathion and its hydrolytic product p-nitrophenol to the native Australian cladoceran Daphnia carinata.

The toxicity of an organophosphorus (OP) insecticide, methyl parathion (MP), and its hydrolysis product, p-nitrophenol (PNP), to the native Australian cladoceran species, Daphnia carinata, was assessed. Both MP and PNP were stable in cladoceran water during the test period. D. carinata was sensitive to both MP and PNP; however, the parent compound was more toxic than its metabolite. This is the first study that demonstrated the acute toxicity of MP and PNP towards an Australian daphnid species. The present investigation emphasizes the need for including the native taxa as non-target test organisms while evaluating the toxicity of environmental pollutants.

Renal damage induced by the pesticide methyl parathion in male Wistar rats.

Little information is apparently available regarding the nephrotoxic effects induced by pesticides. The aim of this study was to examine the influence of low doses of methyl parathion (MP) on the structure and function of the kidney of male Wistar rats. A corn oil (vehicle) was administered to control rats, whereas treated rats received MP at 0.56 mg/kg orally (1/25 of LD50), every third day, for 8 weeks. At the end of each week following MP exposure, creatinine and glucose levels were measured in plasma, while glucose, inorganic phosphate, total proteins, albumin, and activity of γ-glutamyltranspeptidase (GGT) were determined in urine. Kidney histological study was also performed. Compared with control rats, MP significantly increased plasma glucose and creatinine levels accompanied by decreased urinary flow rate and elevated urinary excretion rates of glucose, phosphate, and albumin. Further, the activity of GGT in urine was increased significantly. The proximal cells exhibited cytoplasmic vacuolization, positive periodic acid Schiff inclusions, and brush border edge loss after 2 or 4 weeks following MP treatment. Finally, renal cortex samples were obtained at 2, 4, 6, and 8 weeks of MP treatment, and the concentrations of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity were measured. The mRNA expression levels of BAX and tumor necrosis factor-α (TNF-α) were also determined (RT-PCR). MP significantly decreased renal GSH levels, increased GPx activity, as well as downregulated the mRNA expression of TNF-α and BAX. Densitometry analysis showed a significant reduction in TNF-α and BAX mRNA expression levels at 2 and 4 weeks following MP treatment. Low doses of MP produced structural and functional damage to the proximal tubules of male rat kidney.

Downregulation of human paraoxonase 1 (PON1) by organophosphate pesticides in HepG2 cells.

Paraoxonase 1 (PON1) is a calcium-dependent esterase synthesized primarily in the liver and secreted into the plasma where it is associated with high-density lipoproteins (HDL). PON1 hydrolyzes and detoxifies some toxic metabolites of organophosphorus compounds (OPs) such as methyl parathion and chlorpyrifos. Thus, PON1 activity and expression levels are important for determining susceptibility against OPs poisoning. Some studies have demonstrated that OPs can modulate gene expression through interactions with nuclear receptors. In this study, we evaluated the effects of methyl parathion and chlorpyrifos on the modulation of PON1 in Human Hepatocellular Carcinoma (HepG2) cells by real-time PCR, PON1 activity assay, and western blot. The results showed that the treatments with methyl parathion and chlorpyrifos decreased PON1 mRNA and immunoreactive protein and increased inflammatory cytokines in HepG2 cells. The effects of methyl parathion and chlorpyrifos on the downregulation of PON1 gene expression in HepG2 cells may provide evidence of OPs cytotoxicity related to oxidative stress and an inflammatory response. A decrease in the expression of the PON1 gene may increase the susceptibility to OPs intoxication and the risk of diseases related to inflammation and oxidative stress. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 490-500, 2017.

Muscarinic receptors mediate the endocrine-disrupting effects of an organophosphorus insecticide in zebrafish.

The glucocorticoid cortisol, the end product of hypothalamus-pituitary-interrenal axis in zebrafish (Danio rerio), is synthesized via steroidogenesis and promotes important physiological regulations in response to a stressor. The failure of this axis leads to inability to cope with environmental challenges preventing adaptive processes in order to restore homeostasis. Pesticides and agrichemicals are widely used, and may constitute an important class of environmental pollutants when reach aquatic ecosystems and nontarget species. These chemical compounds may disrupt hypothalamus-pituitary-interrenal axis by altering synthesis, structure or function of its constituents. We present evidence that organophosphorus exposure disrupts stress response by altering the expression of key genes of the neural steroidogenesis, causing downregulation of star, hsp70, and pomc genes. This appears to be mediated via muscarinic receptors, since the muscarinic antagonist scopolamine blocked these effects.

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure.

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6.

Impaired brain StAR and HSP70 gene expression in zebrafish exposed to Methyl-Parathion based insecticide.

Fish production ponds and natural water body areas located in close proximity to agricultural fields receive water with variable amounts of agrochemicals, and consequently, compounds that produce adverse effects may reach nontarget organisms. The aim of this study was to investigate whether waterborne methyl-parathion-based insecticide (MPBI) affected gene expression patterns of brain glucocorticoid receptor (GR), steroidogenic acute regulatory protein (StAR), and heat shock protein 70 (hsp70) in adult zebrafish (Danio rerio) exposed to this chemical for 96 h. Treated fish exposed to MPBI-contaminated water showed an inhibition of brain StAR and hsp70 gene expression. Data demonstrated that MPBI produced a decrease brain StAR and hsp70 gene expression.

Effects of Exposure to Methyl Parathion on Egg Hatchability and Eggshell Chemical Composition in Podocnemis expansa (Testudines, Podocnemididae).

Eggshell evaluation may serve as an indicator of the effect of substances released in the environment, which may change eggshell shape, size, structure, and/or chemical composition. Additionally, exposure may interfere with hatching rates in contaminated eggs. The objective of this study was to better understand how exposure to the insecticide methyl parathion interferes with chemical changes in eggshells of Podocnemis expansa throughout their artificial incubation, as well as with egg hatchability. A total of 343 P. expansa eggs were collected in a natural reproduction area for the species. These eggs were transferred to and artificially incubated in the Wild Animal Teaching and Research Laboratory at Universidade Federal de Uberlândia. On the first day of artificial incubation, 0, 35, 350, and 3500 ppb of methyl parathion were incorporated to the substrate. Eggs were collected every three days for chemical analysis of eggshells. Hatchability was evaluated as the number of hatchlings in each treatment, for the eggs that were not used in the chemical analysis. Student's T-test was used for data on eggshell chemical composition, and the Binomial Test for Two Proportions was used in the hatchability analysis, at a 5% significance level. It was observed that the incorporation of methyl parathion to the substrate on the first day of artificial incubation of P. expansa eggs reduced the levels of total fat in the shells throughout their incubation, besides reducing egg hatchability.

Oxidative Stress, Cytotoxicity, and Genotoxicity Induced by Methyl Parathion in Human Gingival Fibroblasts: Protective Role of Epigallocatechin-3-Gallate.

Organophosphorous (OP) compounds are pesticides frequently released into the environment because of extensive use in agriculture. Among these, methyl parathion (mPT) recently received attention as a consequence of illegal use. The predominant route of human exposure to mPT is via inhalation, but inadvertent consumption of contaminated foods and water may also occur. The goal of this study was to investigate the in vitro effects of mPT on cells in the oral cavity and evaluate the potential protective role of epigallocathechin-3-gallate (EGCG) on these effects. Human gingival fibroblasts (HGF) were exposed to 10, 50, or 100 µ g/ml mPT for 24 h and assessed for oxidative stress, as evidenced by reactive generation of oxygen species (ROS), induction of apoptotic cell death, DNA damage (comet assay and cytochinesis-block micronucleus test), and nitric oxide (NO) production. The results showed that mPT produced significant oxidative stress, cytotoxicity, and genotoxicity and increased NO levels through stimulation of inducible NO synthase expression. Finally, data demonstrated that EGCG (10, 25, or 50 µ M) was able to inhibit the pesticide-induced effects on all parameters studied. Data indicate that cytotoxic and genotoxic effects may be associated with oxidative stress induced by mPT observed in HGF cultures and that EGCG plays a protective role via antioxidant activities.

In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes.

Organophosphate (OP) pesticides are widely used for agricultural and household pest control. We studied the genotoxicity of the commonly used OP pesticides chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT), individually and in combination, in Wistar rat peripheral blood lymphocytes in vitro. DNA single-strand and double-strand breaks were measured by single cell gel electrophoresis (SCGE; comet assay). To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes formamidoaminopyrimidineglycosylase (Fpg) and endonuclease (Endo III), which convert base damages to strand breaks, were used. Significant increases in strand breaks and in levels of the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide were observed in lymphocytes treated with pesticides. MPT exposure caused the greatest DNA damage and ROS production, followed by CPF and ML. Our results demonstrate genotoxic potential of these OP pesticides.

Comparison of biosorption and phytoremediation of cadmium and methyl parathion, a case-study with live Lemna gibba and Lemna gibba powder.

Heavy metals and pesticides can be adsorbed by several biomasses such as living or non-living aquatic plants. In this study adsorption properties of live Lemna gibba and Lemna gibba powder were investigated with regard to cadmium and methyl parathion (MP). Toxicity data (IC50) on live L. gibba indicated that the period of four days was adequate for phytoremediation. Initial adsorption studies showed that both adsorbents were capable of removing cadmium and methyl parathion. Cadmium and methyl parathion adsorption onto L. gibba powder was fast and equilibrium was attained within 120min. The adsorption data could be well interpreted by the Freundlich model. The KF were: 7.8963 (Cd(2+)/ live Lemna); 0.7300 (MP/live Lemna); 11.5813 (Cd(2+)/Lemna powder); 1.1852 (MP/Lemna powder) indicating that Cd(2+) was more efficiently removed by both biosorbents than MP. Adsorption kinetics for cadmium and methyl parathion in both systems and rate constants were determined for each contaminant. It was found that the overall adsorption process was best described by pseudo-second-order kinetics. Boyd model and external mass-transfer expression were tested. It was concluded that cadmium and methyl parathion sorption onto Lemna powder is governed by film diffusion.

Prediction of the joint toxicity of five organophosphorus pesticides to Daphnia magna.

The individual toxicities of five organophosphorus pesticides (dichlorvos, parathion, methyl parathion, malathion and dimethoate) to Daphnia magna were investigated in 24-h immobilization experiments. Using these toxicity data, their combined toxicities were measured in pesticide mixtures designed using either 'equivalent-effect concentration ratios' or 'uniform-design concentration ratios'. The toxicities of mixtures of similarly or dissimilarly acting toxicants are often predicted from the individual toxicities of the component compounds, using one of two distinct biometric models: concentration addition (CA) or independent action (IA). The relative accuracies of the CA and IA models were assessed using the model deviation rate (MDR), which represents the difference between the effect predicted from the individual pesticide concentrations and the observed effect. The mean MDR value of CA was 0.93 (range 0.75-1.31) and the mean value obtained by IA was 3.13 (range 2.52-4.37). We conclude that the CA model is better able to predict the joint toxicities of mixtures of organophosphorus pesticides to D. magna.

Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.

Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandem mass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2ß- and 6ß-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme.

Evaluation of DNA damage and cytotoxicity induced by three commonly used organophosphate pesticides individually and in mixture, in rat tissues.

Organophosphate pesticides are among the most widely used synthetic chemicals for controlling a wide variety of pests. Chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) are among the most extensively used organophosphate (OP) pesticides. The main target of action of OP compounds is the central and peripheral nervous system, although it has also been postulated that these compounds in both acute and chronic intoxication, disturb the redox processes and thus induce oxidative stress. The excessive generation of reactive oxygen species (ROS) causes damage to all vital macromolecules including lipids, proteins, and DNA. This study was aimed to investigate the genotoxicity and cytotoxicity of CPF, MPT, and MLT when given singly or in combination. The DNA damage was measured by alkaline single-cell gel electrophoresis or comet assay and expressed as DNA damage index. The results showed that both acute and chronic exposure with CPF, MPT, and MLT, caused significantly marked DNA damage in rat tissues namely, liver, brain, kidney, and spleen, when measured 24 hour posttreatment. It was also observed that MPT caused highest level of DNA damage and brain was maximally affected by these OP compounds. When these pesticides were given in mixture, the damage was not the sum of damage caused by individual pesticide, confirming that these pesticides do not potentiate the toxicity of each other. When the DNA damage was measured 48 and 72 hour posttreatment, the damage was partially repaired. Pesticide exposure also caused histopathological changes in rat tissues.

Involvement of oxidative stress in methyl parathion and parathion-induced toxicity and genotoxicity to human liver carcinoma (HepG2) cells.

Methyl parathion (C8H10NO5PS) and parathion (C10H14 NO5PS) are both organophosphate insecticides (OPI) widely used for household and agricultural applications. They are known for their ability to irreversibly inhibit acetylcholinesterase which often leads to a profound effect on the nervous system of exposed organisms. Many recently published studies have indicated that human exposure to OPI may be associated with neurologic, hematopoietic, cardiovascular, and reproductive adverse effects. Studies have also linked OPI exposure to a number of degenerative diseases including Parkinson's, Alzheimer's, and amyotrophic lateral sclerosis. Also, oxidative stress (OS) has been reported as a possible mechanism of OPI toxicity in humans. Hence, the aim of the present investigation was to use human liver carcinoma (HepG2) cells as a test model to evaluate the role of OS in methyl parathion- and parathion-induced toxicity. To achieve this goal, we performed the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay for cell viability, lipid peroxidation assay for malondialdehyde (MDA) production, and Comet assay for DNA damage, respectively. Results from MTT assay indicated that methyl parathion and parathion gradually reduce the viability of HepG2 cells in a dose-dependent manner, showing 48 h-LD50 values of 26.20 mM and 23.58 mM, respectively. Lipid peroxidation assay resulted in a significant increase (P < 0.05) of MDA level in methyl parathion- and parathion-treated HepG2 cells compared with controls, suggesting that OS plays a key role in OPI-induced toxicity. Comet assay indicated a significant increase in genotoxicity at higher concentrations of OPI exposure. Overall, we found that methyl-parathion is slightly less toxic than parathion to HepG2 cells. The cytotoxic effect of these OPI was found to be associated, at least in part, with oxidative cell/tissue damage.

Comparing toxicity endpoints on Lecane quadridentata (Rotifera: Monogononta) exposed to two anticholinesterases pesticides.

Toxicity tests were performed on the freshwater rotifer Lecane quadridentata exposed to the pesticides carbaryl and methyl parathion (lethal, sublethal, and chronic) to compare the sensitivity between different endpoints: (a) 48-h mortality; (b) 30-min in vivo inhibition of esterase activity; (c) 5-day inhibition of the instantaneous growth rate. The emphasis of this work was to find the most appropriate endpoint to evaluate the toxicity of these pesticides in view of their sensitivity, duration, and ecological relevance. The comparison between the three toxicity tests show that the 5-day chronic tests have the lowest EC50 (2.22 and 6.6 mg/L), lowest-observed-effect concentration (2.5 and 2.5 mg/L), and no-observed-effect concentration (1.0 and 1.2 mg/L) values for carbaryl and methyl parathion, respectively. This indicates that the estimate of the instantaneous rate of natural increase r is the most sensitive endpoint regarding the toxicity of these pesticides. This sensitivity might be due to the effect on reducing the growth potential form the first generation on. Lethal and sublethal tests are closely related, suggesting that the immediate effect after inhibition of esterases is death. In general, the sensitivity of L. quadridentata is similar to other species of rotifers exposed to methyl parathion. Therefore, the 5-day chronic toxicity test with the freshwater rotifer L. quadridentata should be considered a good candidate to evaluate the effect of anticholinesterase pesticides, due to its high sensitivity and ecological relevance.

Concentration-mortality responses of Myzus persicae and natural enemies to selected insecticides.

The toxicity of six insecticides was determined for the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae), and some of its natural enemies - the predatory beetles Cycloneda sanguinea (Coccinellidae) and Acanthinus sp. (Anthicidae), and the wasp parasitoid Diaeretiella rapae (Aphidiidae). Natural enemies from these groups are important natural biological control agents in a number of agroecosystems, and insecticides potentially safe to these non-target organisms should be identified using standardized tests. Thus, concentration-mortality bioassays were carried out with both the aphid and its natural enemies to assess the toxicity and selectivity of acephate, deltamethrin, dimethoate, methamidophos, methyl parathion, and pirimicarb. The latter insecticide was highly selective to all natural enemies tested, and its LC(90) for M. persicae was 14-fold lower than the field rate recommended for control of the aphid in brassica crops. Methyl parathion also showed selectivity to C. sanguinea and Acanthinus sp., but not to D. rapae. Acephate was the least potent insecticide against M. persicae and was equally or more toxic to the natural enemies relative to the aphid. Pirimicarb and methyl parathion were efficient against M. persicae and selective in favor of two of the natural enemies tested. Acanthinus sp. and C. sanguinea were more tolerant to the insecticides than was the parasitoid D. rapae. This study shows that there are selective insecticides that may be compatible with conservation of natural enemies in brassica crops, which is important practical information to improve integrated pest management systems in these crops.