Peripheral T3 signaling is the target of pesticides in zebrafish larvae and adult liver.

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in 'de novo lipogenesis' and ß-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

Identification of metabolite biomarkers in serum of rats exposed to chlorpyrifos and cadmium.

Chlorpyrifos (CPF) and cadmium (Cd) are widespread environmental pollutants, which are often present in drinking water and foods. However, the combined effects of CPF and Cd were not entirely clear at present. There was also no biomarker available to diagnose the poisoning of the two chemicals at low dose for long-term exposures. In this study, we investigated the change of serum metabolites of rats with subchronic exposure to CPF, Cd, and CPF plus Cd using gas chromatography-mass spectrometer-based metabolomics approach. We performed a stepwise optimization algorithm based on receiver operating characteristic to identify serum metabolite biomarkers for toxic diagnosis of the chemicals at different doses after 90-day exposure. We found that aminomalonic acid was the biomarker for the toxicity of Cd alone administration, and serine and propanoic acid were unique biomarkers for the toxicities of CPF plus Cd administrations. Our results suggest that subchronic exposure to CPF and Cd alone, or in combination at their low doses, could cause disturbance of energy and amino acid metabolism. Overall, we have shown that analysis of serum metabolomics can make exceptional contributions to the understanding of the toxic effects following long-term low-dose exposure of the organophosphorus pesticide and heavy metal.

Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum ) in the presence of pesticides.

Three phosphate solubilizing bacteria were isolated and identified by 16S rRNA sequencing as Pseudomonas putida, Pseudomonas sp and Pseudomonas fulva . The strains were subjected to plant biochemical testing and all the PGPR attributes were checked in the presence of pesticides (chlorpyrifos and pyriproxyfen). The phosphate solubilizing index of strain Ros2 was highest in NBRIP medium i.e 2.23 mm. All the strains showed acidic pH (ranges from 2.5-5) on both medium i.e PVK and NBRIP. Strain Ros2 was highly positive for ammonia production as well as siderophore production while strain Rad2 was positive for HCN production. The results obtained by the strains Rad1, Rad2 and Ros2 for auxin production were 33.1, 30.67 and 15.38 µg ml-1, respectively. Strain Rad1 showed 16% increase in percentage germination in comparison to control in the presence of pesticide stress. Most promising results for chlorophyll content estimation were obtained in the presence of carotenoids upto 6 mgg-1 without stress by both strains Rad1 and Rad2. Study suggests that especially strain Ros2 can enhance plant growth parameters in the pesticide stress.

Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum ) in the presence of pesticides / Impacto das bactérias solubilizantes de fosfato no trigo (Triticum aestivum ) na presença de pesticidas

Abstract Three phosphate solubilizing bacteria were isolated and identified by 16S rRNA sequencing as Pseudomonas putida, Pseudomonas sp and Pseudomonas fulva . The strains were subjected to plant biochemical testing and all the PGPR attributes were checked in the presence of pesticides (chlorpyrifos and pyriproxyfen). The phosphate solubilizing index of strain Ros2 was highest in NBRIP medium i.e 2.23 mm. All the strains showed acidic pH (ranges from 2.5-5) on both medium i.e PVK and NBRIP. Strain Ros2 was highly positive for ammonia production as well as siderophore production while strain Rad2 was positive for HCN production. The results obtained by the strains Rad1, Rad2 and Ros2 for auxin production were 33.1, 30.67 and 15.38 µg ml-1, respectively. Strain Rad1 showed 16% increase in percentage germination in comparison to control in the presence of pesticide stress. Most promising results for chlorophyll content estimation were obtained in the presence of carotenoids upto 6 mgg-1 without stress by both strains Rad1 and Rad2. Study suggests that especially strain Ros2 can enhance plant growth parameters in the pesticide stress.

Resumo Três bactérias solubilizantes de fosfato foram isoladas e identificadas por seqüenciamento de rRNA 16S como Pseudomonas putida, Pseudomonas sp e Pseudomonas fulva. As estirpes foram submetidas a testes bioquímicos de plantas e todos os atributos PGPR foram verificados na presença de pesticidas (clorpirifos e piriproxifeno). O índice de solubilização de fosfato da estirpe Ros2 foi mais elevado no meio NBRIP, isto é, 2,23 mm. Todas as estirpes apresentaram um pH ácido (varia de 2,5-5) em ambos os meios, isto é PVK e NBRIP. A estirpe Ros2 foi altamente positiva para a produção de amoníaco, bem como a produção de sideróforos enquanto a estirpe Rad2 foi positiva para a produção de HCN. Os resultados obtidos pelas estirpes Rad1, Rad2 e Ros2 para a produção de auxina foram 33,1, 30,67 e 15,38 μg ml-1 , respectivamente. A deformação Rad1 mostrou aumento de 16% na germinação percentual em comparação com o controlo na presença de stress de pesticida. Os resultados mais promissores para a estimativa do teor de clorofila foram obtidos na presença de carotenóides até 6 mgg-1 sem estresse por ambas as cepas Rad1 e Rad2. Estudo sugere que especialmente a estirpe Ros2 pode melhorar parâmetros de crescimento de plantas no estresse de pesticidas.

Disruption of Kidney Metabolism in Rats after Subchronic Combined Exposure to Low-Dose Cadmium and Chlorpyrifos.

Cadmium (Cd) and chlorpyrifos (CPF) often coexist in the environment and induce combined toxicity to organisms. Here we studied the combined nephrotoxicity of environmentally relevant low doses of Cd and CPF. We treated the mice for 90 days with different doses of Cd and CPF and their mixtures via oral gavage. Then histopathological evaluation and biochemical analysis for kidney tissues were carried out. The change of metabolites in kidney was detected by using a metabolomics approach using GC-MS. We found that Cd, CPF, and their mixtures caused oxidative damage as well as disturbance of renal amino acid metabolism. We identified potential metabolite biomarkers in kidney, which included acetic acid for CPF treatment, glycerol and carboxylic acid for Cd treatment, and l-ornithine for the mixture of CPF and Cd treatment, respectively. In addition, we found that Cd promoted the metabolism of CPF in kidney. This may contribute to the result that the toxicity of the mixtures was lower than the sum of the toxicities of Cd and CPF alone. In conclusion, our results indicated that CPF and Cd could disrupt the kidney metabolism in rats even when they were exposed to a very low dose of CPF and Cd.

Evaluation of chlorpyrifos toxicity through a 28-day study: Cholinesterase activity, oxidative stress responses, parent compound/metabolite levels, and primary DNA damage in blood and brain tissue of adult male Wistar rats.

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Exposure of amateur gardeners to pesticides via the non-gloved skin per day.

To predict a risk to gardeners not wearing protective gloves, the dermal absorption of three active insecticides was assessed in vitro using porcine ear-skin simulating 1-h handling of diluted plant protection products. Acetamiprid and Pirimicarb were found in the receptor fluid immediately after 1-h skin exposure, whereas Chlorpyrifos-methyl absorbed in the skin was not released into the receptor fluid even after 23 hours. The Estimated Gardener Exposure Level (EGEL) at 23 hours after 1-h exposure for two worst-case scenarios (i) non-gloved hands; (ii) non-gloved hands/uncovered forearms, was (i) 0.002, 0.042, and 0.057; (ii) 0.006, 0.101, and 0.135 mg/kg bw/day for Acetamiprid, Pirimicarb, and Chlorpyrifos-methyl, respectively, although the systemically available Chlorpyrifos-methyl amount, due to retention in the skin, is probably lower than determined. The Gardener Exposure Risk (GER), as a ratio of Acceptable Operator Exposure Level (databased values) to EGEL, for Acetamiprid was (i) 35 and 12-fold higher than the limit 1, so the risk via the skin is assumed to be low. Based on the GER values of (i) 0.83 and 0.18; (ii) 0.34 and 0.07 (i.e.<1) for Pirimicarb and Chlorpyrifos-methyl, respectively there is a level of concern regarding the health risk to gardeners handling pesticide products without skin protection.

The protective role of alpha-lipoic acid on long-term exposure of rats to the combination of chlorpyrifos and deltamethrin pesticides.

The study was aimed at evaluating the protective role of α-lipoic acid (ALA) on long-term exposure of rats to the combination of chlorpyrifos (CPF) and deltamethrin (DLT). Forty-two (42) male Wistar rats were divided into 6 exposure groups with 7 animals in each group: (I) soya oil (2 ml kg-1), (II) ALA (60 mg kg-1), (III) DLT (6.25 mg kg-1), (IV) CPF (4.75 mg kg-1), (V) (CPF + DLT) DLT (6.25 mg kg-1) and CPF (4.75 mg kg-1; 1/20th of the previously determined median lethal dose) and (VI) (ALA + CPF + DLT) pretreated with ALA (60 mg kg-1) and then co-exposed to CPF and DLT, 45 min later. The regimens were administered by gavage once daily for a period of 16 weeks. Sera obtained from blood collected at the end of the experimental period were used for the evaluation of serum glucose, total protein, albumin, urea, creatinine and the activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase and acetylcholinesterase. The liver homogenate was used to assay for the activities of superoxide dismutase and glutathione peroxidase and the concentrations of malondialdehyde, cytokine and tumour necrotic factor α. The result showed that the combination of CPF and DLT resulted in marked alterations of these biochemical parameters in most cases compared to either of the pesticides singly, supplementation with ALA ameliorated these alterations.

Metabolomics analysis and biomarker identification for brains of rats exposed subchronically to the mixtures of low-dose cadmium and chlorpyrifos.

Cadmium (Cd) and chlorpyrifos (CPF) are widespread harmful environmental pollutants with neurotoxicity to mammals. Although the exposure to Cd and CPF at the same time may pose a significant risk to human health, the subchronic combined neurotoxicity of these two chemicals at low levels in the brain is poorly understood. In this study, we treated rats with three doses (low, middle, and high) of Cd, CPF, or their mixture for 90 days. No obvious symptom was observed in the treated animals except those treated with high-dose CPF. Histological results showed that middle and high doses of the chemicals caused neuronal cell damage in brains. GC-MS-based metabonomics analysis revealed that energy and amino acid metabolism were disturbed in the brains of rats exposed to the two chemicals and their combinations even at low doses. We further identified the unique brain metabolite biomarkers for rats treated with Cd, CPF, or both. Two amino acids, tyrosine and l-leucine, were identified as the biomarkers for Cd and CPF treatment, respectively. In addition, a set of five unique biomarkers (1,2-propanediol-1-phosphate, d-gluconic acid, 9H-purine, serine, and 2-ketoisovaleric acid) was identified for the mixtures of Cd and CPF. Therefore, the metabolomics analysis is more sensitive than regular clinical observation and pathological examination for detecting the neurotoxicity of the individual and combined Cd and CPF at low levels. Overall, these results identified the unique biomarkers for Cd and CPF exposure, which provide new insights into the mechanism of their joint toxicity.

Chlorpyrifos and cypermethrin induce apoptosis in human neuroblastoma cell line SH-SY5Y.

Our previous in vivo studies showed that chlorpyrifos (CPF) and cypermethrin (CM) in a mixture dermally administered, strongly inhibited cholinesterase activity in plasma and the brain and were very toxic to the rat central nervous system. In this work, the mechanisms of neurotoxicity have not been elucidated. We used human undifferentiated SH-SY5Y cells to study mechanisms of pesticide-induced neuronal cell death. It was found that chlorpyrifos (CPF) and its mixture with cypermethrin (CPF+CM) induced cell death of SH-SY5Y cells in a dose- and time-dependent manner, as shown by MTT assays. Pesticide-induced SH-SY5Y cell death was characterized by concentration-dependent down-regulation of Bcl-2 and Bcl-xL as well as an increase in the caspase 3 activation. Pan-caspase inhibitor Q-VD-OPh produced a slight but significant reversal effect of pesticide-induced toxicity indicating that the major caspase pathways are not integral to CPF- and CPF+CM-induced cell death. Furthermore, signal transduction inhibitors PD98059, SL-327, SB202190, SP600125 and mecamylamine failed to attenuate pesticides effect. Atropine exhibited minimal ability to reverse toxicity. Finally, it was shown that inhibition of TNF-α by pomalidomide attenuated CPF-/CPF+CM-induced apoptosis. Overall, our data suggest that FAS/TNF signalling pathways may participate in CPF and CPF+CM toxicity.

Effect of chlorpyrifos on the profile of subpopulations immunocompetent cells B, T and NK in in vivo model.

BACKGROUND: Current studies have indicated many environmental factors, such as pesticides, that cause immune system disorders through inducing changes in humoral and cellular responses thereby increasing the risk of contracting infectious diseases and cancer. The literature suggests that low exposures to certain organophosphorus pesticides stimulate the immune system, whilst high exposures result in decreased function. Precise mechanisms for the fall in immunocompetence are often unclear, however it can be predicted that the intimate interaction between the nervous and immune systems can potentially lead to toxicity. OBJECTIVES: To determine the effects of organophosphorus pesticide, chlorpyrifos that is often used in Poland, on selected immunological responses, such as immune-competent cell proportions formed experimentally in-vivo by cells of Wistar rats during subchronic exposures after 45 and 90 days. MATERIALS AND METHODS: The test was carried out on ten male and ten female Wistar rats in each of three test groups, who received 3 chlorpyrifos doses for 90 days intragastrically, according to OECD guidelines (No. 401). Two control groups were given olive oil. After completion, the animals were deeply anaesthetised by a mixture of ketamine (Vetaketam) and xylazine (Vetaxym). Immuno-competent cells were profiled by a commercial monoclonal antibody method. In order to measure the dynamics of any changes, the aforementioned immunological responses were investigated after 45 days using the same procedures for obtaining the relevant biological test material. RESULTS: Test animals exposed to chlorpyrifos had altered number of white bood cells which were either increased or decreased relative to controls after 45 and 90 days for all exposure levels used. CONCLUSIONS: The study demonstrated changes in white-blood cell (lymphocyte) response profiles, reflecting an immunomodulation although such changes were equivocal, where both suppression and stimulation were observed. KEY WORDS: immunomodulation, immune system, lymphocyte, organophosphorus pesticides, chlorpyrifos.

A single high dose of chlorpyrifos reduces long-term basal c-fos expression in the rat arcuate hypothalamic nucleus / La administración de una dosis aguda de clorpirifós reduce a largo plazo la expresión basal de c-fos en el núcleo arqueado del hipotálamo

Chlorpyrifos (CPF) is an organophosphate compound used worldwide as a pesticide in agriculture that, after subcutaneous injection, keeps acetylcholinesterase (AChE) activity inhibited within an organism for months. Ample clinical and experimental evidence shows that CPF exposure induces relevant and persistent neurobehavioral deficits in humans and animals, even after one single episode/injection. Additionally, clinical and epidemiological studies evidence that a significant percentage (60%) of Gulf War veterans as well as agricultural workers suffering from acute OP intoxication may have developed intolerance to nicotine and ethanol-containing beverages. Consistent with it, administration of a single high dose of CPF to adult Wistar rats elicited long-lasting reduced voluntary ethanol drinking and increased sedation to ethanol without evidence of altered ethanol metabolism, which indicates that CPF-ethanol neurobiological interactions may exist. In this study, we explore whether CPF exposure induces significant disturbances in basal and/or ethanol-evoked neural activity in a set of cholinoceptive brain regions critically involved with neurobiological responses to ethanol. For this aim, brain regional c-fos expression in response to acute ethanol (1.5 or 3.0 g/kg, i.p.) or saline was assessed in adult male Wistar rats previously injected with either a single high dose of CPF (250 mg/kg, sc) or vehicle. We found that CPF administration reduces long-term basal, but not ethanol-evoked, c-fos expression in the arcuate hypothalamic nucleus. Because independent brain pathways may modulate acute responses to ethanol and voluntary ethanol consumption, we do not rule out the contribution of basal neural disturbances observed in the Arc to CPF-elicited ethanol avoidance.

El clorpirifós (CPF) es un compuesto organofosforado utilizado como plaguicida en todo el mundo. Después de ser inyectado de manera subcutánea, mantiene la actividad de la enzima acetilcolinesterasa (AChE) inhibida durante meses. Estudios clínicos y experimentales muestran que la exposición al CPF induce déficits neuroconductuales persistentes en seres humanos y animales, incluso después de un solo episodio/inyección. Además, estudios epidemiológicos evidencian que un porcentaje significativo (60%) de los veteranos de la Guerra del Golfo, así como los agricultores que sufren una intoxicación aguda por organofosforados, desarrollan intolerancia a la nicotina y las bebidas que contienen etanol. Datos experimentales mostraron que la administración de una sola dosis alta de CPF en ratas Wistar adultas provoca una reducción a largo plazo del consumo voluntario de etanol y un incremento en la sedación provocada por etanol sin evidencias de alteración del metabolismo de esta sustancia, lo que indica que pueden existir interacciones neurobiológicas entre CPF-etanol. En este estudio, se explora si la exposición a CPF induce alteraciones significativas en la actividad neuronal basal o evocada por el etanol en un conjunto de regiones colinoceptivas del cerebro involucradas en las respuestas neurobiológicas al etanol. Para ello, se evaluó la expresión de c-fos en respuesta a una dosis de etanol aguda (1.5 o 3.0 g/kg, ip) o solución salina en ratas Wistar macho adultas previamente inyectados con dosis aguda de CPF (250 mg/kg, sc) o un vehículo. Encontramos que la administración de CPF redujo la expresión basal de c-fos a largo plazo, pero no la evocada por el etanol en el núcleo arqueado del hipotalámo. Debido a que vías cerebrales independientes podrían modular las respuestas agudas al etanol y el consumo voluntario del mismo, no se descarta la contribución de las alteraciones neuronales basales observadas en el Arc en la evitación del consumo de etanol provocado por CPF.

Chlorpyrifos induced toxicity in reproductive organs of female Wistar rats.

Chlorpyrifos (CPF) is an organophosphate (OP) insecticide extensively used in agricultural and domestic settings. Healthy adult female albino rats were divided into three groups of six rats in each. Two groups were dosed orally with CPF in vegetable oil (0.1 and 2.5 mg/kg/day) and third group was given vegetable oil for 8 weeks. Non-significant changes were observed for body weight and feed intake. A disruption in estrous cyclicity was observed with a prolonged metestrous. Erythrocyte osmotic fragility and lipid peroxidation levels increased significantly. Mammary gland whole mounts revealed a significant (P<0.05-0.0001) increase in the ductal thickness, number of branches, alveolar and terminal end bud number and terminal end bud diameter. A significant increase in ovarian surface epithelium height, follicular diameter and follicular atresia was observed in treated rats (P<0.05-0.0001). A similar significant increase in the uterine surface epithelium height, endometrial gland epithelium height and myometrium thickness in higher dose group was recorded (P<0.05-0.0001). Luminal epithelium height and endometrial gland diameter was increased significantly in both the treated groups (P<0.05-0.0001). The results indicate that sub-chronic exposure of CPF causes oxidative stress and negative effects on the reproductive organs of female rats, which may be a pointer towards beginning of cancer incidence.

Chronic chlorpyrifos exposure does not promote prostate cancer in prostate specific PTEN mutant mice.

Environmental factors are likely to interact with genetic determinants to influence prostate cancer progression. The Agricultural Health Study has identified an association between exposure to organophosphorous pesticides including chlorpyrifos, and increased prostate cancer risk in pesticide applicators with a first-degree family history of this disease. Exploration of this potential gene-environment interaction would benefit from the development of a suitable animal model. Utilizing a previously described mouse model that is genetically predisposed to prostate cancer through a prostate-specific heterozygous PTEN deletion, termed C57/Luc/Ptenp+/-, we used bioluminescence imaging and histopathological analyses to test whether chronic exposure to chlorpyrifos in a grain-based diet for 32 weeks was able to promote prostate cancer development. Chronic exposure to chlorpyrifos in the diet did not promote prostate cancer development in C57/Luc/Ptenp+/- mice despite achieving sufficient levels to inhibit acetylcholinesterase activity in plasma. We found no significant differences in numbers of murine prostatic intraepithelial neoplasia lesions or disease progression in chlorpyrifos versus control treated animals up to 32 weeks. The mechanistic basis of pesticide-induced prostate cancer may be complex and may involve other genetic variants, multiple genes, or nongenetic factors that might alter prostate cancer risk during pesticide exposure in agricultural workers.

Influence of chlorpyrifos on the profile of subpopulations of immunoactive cells and their phagocytic activity in an experimental in vivo model.

Many environmental factors, including pesticides, cause immunological system disorders by inducing changes in humoral and cellular response. They may stimulate or trigger immunological autoaggression, hypersensitivity and allergy, as well as lead to immunosuppression, thus increasing the incidence of infectious diseases and cancers. Such activity is also attributed to organophosphorus compounds used in agriculture as insecticides, and commonly in households as biocides. The aim of the study was to define possible mechanisms of the immunotoxic activity of the chlorpyrifos (an organophosphorus compound) on experimental animals following their exposure to the compound via the oral route. The present study attempts to define the influence of chlorpyrifos on the profile of subpopulations of immunoactive cells: B, T, CD4+, CD8+, and NK, and on their phagocytic activity in an experimental in vivo model. For this purpose, the Wistar rats, were exposed orally to increasing doses of chlorpyrifos: 0.1 LD(50), 0.15 LD(50), 0.2 LD(50), 0.3 LD(50) and 0.4 LD(50) for 28 days. In the study animals, we failed to demonstrate a statistically significant decrease in the phagocytic activity of the granulocyte.

Evaluation of micronuclei induction capacity and mutagenicity of organochlorine and organophosphate pesticides.

The genotoxic and mutagenic effects of two commonly used organochlorine pesticides, lindane (LND) and endosulfan (ENS), and two commonly used organophosphate pesticides, chlorpyrifos (CPF) and monocrotophos (MCP) were assessed using in vivo mouse bone marrow micronucleus test and in vitro Ames Salmonella/ microsome mutagenicity test. The results showed that these pesticides alone or in combination, induced significantly high frequency of micronuclei (MN) formation that increased with concentration of pesticides. All these four pesticides produced significant increase in the frequencies of micronucleated-polychromatic erythrocytes (MN-PCE) and decrease infrequencies of PCE in dose-dependent manner. The results indicate the suppression of proliferative activity of the bone marrow and increase in the extent of cell death. ENS and MCP showed mutagenic potential in Salmonella/ microsome assay. ENS induced mutagenic and nontoxic response only in TA98 tester strain of S.typhimurium at the dose of 500 µg/plate and in the absence of metabolic activation. MCP showed weak mutagenic and nontoxic effect only in TA100 tester strain at the dose of 5000 µg/plate in both assays, with or without metabolic activation when compared with negative control. MCP was toxic in TA98 tester strain at the dose of 5000 µg/plate in absence of metabolic activation while reduction in toxicity was seen on addition of S9 mixture. The study clearly showed the genotoxic potential of all these four pesticides and mutagenic response of endosulfan and monocrotophos.

Effects of the organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflammatory molecule production.

Epidemiological data have associated environmental organophosphate insecticide (OP) exposure during pregnancy with fetal growth deficits. To better understand OP injury that may adversely affect pregnancy, we used the JEG-3 choriocarcinoma cell line, which provide a recognized in vitro model to study placental function. The effects of the OP phosmet (Pm) and chlorpyrifos (Cp) on JEG-3 cells viability, proliferation, cell cycle and inflammatory molecule production were evaluated. Both insecticides affected cellular viability in a concentration- and time-dependent manner, inducing apoptosis and decreasing [(3)H]-thymidine incorporation. However, only Pm reduced DNA synthesis independently of cellular death and decreased the cell percentage at the S-phase. Unlike apoptosis, TNFα production varied with the concentration tested, suggesting that other TNFα independent mechanisms might trigger cell death. No induction of the inflammatory molecule nitric oxide was detected. The mRNA levels of pro-inflammatory IL-6, IL-17 and the anti-inflammatory IL-13 cytokines were differentially modulated. These findings show that Pm and Cp generate a specific toxicity signature, altering cell viability and inducing an inflammatory cytokine profile, suggesting that trophoblasts may represent a possible target for OP adverse effects.

Effects of sub-lethal neurite outgrowth inhibitory concentrations of chlorpyrifos oxon on cytoskeletal proteins and acetylcholinesterase in differentiating N2a cells.

Previous work in our laboratory has shown that sub-lethal concentrations (1-10 µM) of chlorpyrifos (CPF), diazinon (DZ) and diazinon oxon (DZO) inhibit the outgrowth of axon-like neurites in differentiating mouse N2a neuroblastoma cells concomitant with altered levels and/or phosphorylation state of axonal cytoskeleton and growth-associated proteins. The aim of the present work was to determine whether chlorpyrifos oxon (CPO) was capable of inhibiting N2a cell differentiation in a similar manner. Using experimental conditions similar to our previous work, sub-lethal concentrations (1-10 µM) of CPO were found to inhibit N2a cell differentiation. However, unlike previous studies with DZ and DZO, there was a high level of sustained inhibition of acetylcholinesterase (AChE) in CPO treated cells. Impairment of neurite outgrowth was also associated with reduced levels of growth associated protein-43 and neurofilament heavy chain (NFH), and the distribution of NFH in cells stained by indirect immunofluorescence was disrupted. However, in contrast to previous findings for DZO, the absolute level of phosphorylated NFH was unaffected by CPO exposure. Taken together, the findings suggest that sub-lethal concentrations of CPO inhibit axon outgrowth in differentiating N2a cells and that this effect involves reduced levels of two proteins that play key roles in axon outgrowth and maintenance. Although the inhibition of neurite outgrowth is unlikely to involve AChE inhibition directly, further work will help to determine whether the persistent inhibition of AChE by CPO can account for the different effects induced by CPO and DZO on the levels of total and phosphorylated NFH.

Cytochrome P450-specific human PBPK/PD models for the organophosphorus pesticides: chlorpyrifos and parathion.

Organophosphorus pesticides (OPs) remain a potential concern to human health because of their continuing use worldwide. Phosphororthioate OPs like chlorpyrifos and parathion are directly activated and detoxified by various cytochrome P450s (CYPs), with the primary CYPs involved being CYP2B6 and CYP2C19. The goal of the current study was to convert a previously reported human pharmacokinetic and pharmacodynamic (PBPK/PD) model for chlorpyrifos, that used chlorpyrifos metabolism parameters from rat liver, into a human CYP based/age-specific model using recombinant human CYP kinetic parameters (V(max), K(m)), hepatic CYP content and plasma binding measurements to estimate new values for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition and to use the model as a template for the development of a comparable parathion PBPK/PD model. The human CYP based/age-specific PBPK/PD models were used to simulate single oral exposures of adults (19 year old) and infants (1 year) to chlorpyrifos (10,000, 1000 and 100 µg/kg) or parathion (100, 25 and 5 µg/kg). Model simulations showed that there is an age dependency in the amount of blood cholinesterase inhibition observed, however additional age-dependent data are needed to further optimize age-specific human PBPK/PD modeling for these OP compounds. PBPK/PD model simulations estimated that a 4-fold increase or decrease in relative CYP2B6 and CYP2C19 content would produce a 9-22% inhibition in blood AChE activity following exposure of an adult to chlorpyrifos (1000 µg/kg). Similar model simulation produced an 18-22% inhibition in blood AChE activity following exposure of an adult to parathion (25 µg/kg). Individuals with greater CYP2B6 content and lower CYP2C19 content were predicted to be most sensitive to both OPs. Changes in hepatic CYP2B6 and CYP2C19 content had more of an influence on cholinesterase inhibition for exposures to chlorpyrifos than parathion, which agrees with previously reported literature that these CYPs are more reaction biased for desulfurization (activation) and dearylation (detoxification) of chlorpyrifos compared to parathion. The data presented here illustrate how PBPK/PD models with human enzyme-specific parameters can assist ongoing risk assessment efforts and aid in the identification of sensitive individuals and populations.