Comparison of two in vitro activation systems for protoxicant organophosphorous esterase inhibitors.
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In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests. (+info)
Developmental cholinotoxicants: nicotine and chlorpyrifos.
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The stimulation of cholinergic receptors in target cells during a critical developmental period provides signals that influence cell replication and differentiation. Accordingly, environmental agents that promote cholinergic activity evoke neurodevelopmental damage because of the inappropriate timing or intensity of stimulation. Nicotine evokes mitotic arrest in brain cells possessing high concentrations of nicotinic cholinergic receptors. In addition, the cholinergic overstimulation programs the expression of genes that evoke apoptosis and delayed cell loss. Effects of cholinesterase inhibitors exhibit many similarities to those of nicotine. Chlorpyrifos administered to developing rats in doses that do not evoke signs of overt toxicity decreased DNA synthesis and caused shortfalls in cell numbers in brain regions enriched in cholinergic innervation. In embryo cultures, chlorpyrifos also evoked apoptosis during neurulation. However, chlorpyrifos also evokes noncholinergic disruption of cell development by interfering with cell signaling via adenylyl cyclase, leading to widespread disruption that is not limited to cholinergic systems. We have tested this hypothesis in vitro with PC12 cells, which lack the enzymes necessary to produce chlorpyrifos oxon, the metabolite that inhibits cholinesterase. Chlorpyrifos inhibited DNA synthesis in undifferentiated PC12 cells, which have relatively few cholinergic receptors. Furthermore, chlorpyrifos was more effective than nicotine and its effects were not blocked by cholinergic antagonists. When cells were allowed to differentiate in the presence of chlorpyrifos, cell replication was inhibited even more profoundly and cell acquisition was arrested. At higher concentrations, chlorpyrifos also inhibited neuritic outgrowth. Thus, chlorpyrifos elicits damage by both noncholinergic and cholinergic mechanisms extending from early stages of neural cell replication through late stages of axonogenesis and terminal differentiation. Accordingly, the window of developmental vulnerability to chlorpyrifos is likely to extend from the embryonic period into postnatal life. (+info)
Dermal transfer of chlorpyrifos residues from residential surfaces: comparison of hand press, hand drag, wipe, and polyurethane foam roller measurements after broadcast and aerosol pesticide applications.
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Indoor residential pesticide applications present the potential for human exposures, particularly for small children. Personal contact with target and nontarget surfaces can result in transfer of pesticides to the skin, but the magnitude of such transfer is uncertain. This research compared surface sampling techniques [wipe and polyurethane foam (PUF) roller] with the removal ability of human skin following broadcast and total aerosol release applications of Dursban (Dow Elanco, Midland, MI), a residential formulation containing the insecticide chlorpyrifos. Hands were washed immediately after surface contact, following a protocol that included a laboratory-generated adjustment factor to account for incomplete removal of chlorpyrifos from skin. Chlorpyrifos transfer was similar for hand press and hand drag techniques, averaging approximately 1-6 ng/cm2 of carpet contacted. These amounts represented < 1% of the amount of chlorpyrifos deposited on the surfaces 3.5 hr earlier. Chlorpyrifos transfer from carpet to skin was 23-24 times lower than for wipe sampling and 33-36 times lower than for PUF roller sampling (p = 0.0007 and p = 0.0006 for broadcast and aerosol applications, respectively). Hand press sampling removed approximately 4.5 times less chlorpyrifos from nontarget furniture surfaces (12 ng/cm2) than did wipe sampling (56 ng/cm2; p = 0.009). Chlorpyrifos residues on carpet were substantially higher after broadcast applications than after aerosol applications, but residues on such nontarget surfaces as furniture were substantially higher for the aerosol application. This study indicates that human skin removes substantially less residue from carpets and furniture than either conventional wipe or PUF roller sampling methods following residential pest control applications of chlorpyrifos. Although this paper focuses on quantifying residue transfer from surface to skin using different surface sampling techniques, no attempt is made to quantify the amount of chlorpyrifos residue that is subsequently absorbed. (+info)
Oral and dermal absorption of chlorpyrifos: a human volunteer study.
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OBJECTIVES: To determine the kinetics of elimination of urinary dialkylphosphate metabolites after oral and dermally applied doses of the organophosphate pesticide chlorpyrifos to human volunteers and to determine whether these doses affected plasma and erythrocyte cholinesterase activity. METHOD: Five volunteers ingested 1 mg (2852 nmol) of chlorpyrifos. Blood samples were taken over 24 hours and total void volumes of urine were collected over 100 hours. Four weeks later 28.59 mg (81567 nmol) of chlorpyrifos was administered dermally to each volunteer for 8 hours. Unabsorbed chlorpyrifos was washed from the skin and retained for subsequent measurement. The same blood and urine sampling regime was followed as for the oral administration. Plasma and erythrocyte cholinesterase concentrations were determined for each blood sample. The concentration of two urinary metabolites of chlorpyrifos--diethylphosphate and diethyl-thiophosphate--was determined for each urine sample. RESULTS: The apparent elimination half life of urinary dialkylphosphates after the oral dose was 15.5 hours and after the dermal dose it was 30 hours. Most of the oral dose (mean (range) 93% (55-115%)) and 1% of the applied dermal dose was recovered as urinary metabolites. About half (53%) of the dermal dose was recovered from the skin surface. The absorption rate through the skin, as measured by urinary metabolites was 456 ng/cm2/h. Blood plasma and erythrocyte cholinesterase activity did not fall significantly during either dosing regime. CONCLUSION: An oral dose of chlorpyrifos was readily absorbed through the skin and almost all of the dose was recovered as urinary dialkylphosphate metabolites. Excretion was delayed compared with the oral dose. Only a small proportion of the applied dose was recovered during the course of the experiment. The best time to collect urine samples for biological monitoring after dermal exposure is before the shift the next day. The amounts of chlorpyrifos used did not depress acetyl cholinesterase activity but could be readily detected as urinary dialkylphosphate metabolites indicating that the urinary assay is a more sensitive indicator of exposure. (+info)
Pitfalls when determining tissue distributions of organophosphorus chemicals: sodium fluoride accelerates chemical degradation.
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This paper describes the tissue distributions of dichlorvos, an organophosphate, and chlorpyrifos-methyl, an organophosphorothioate, in a male individual who died after ingesting an insecticidal preparation containing these chemicals and the results of an in vitro stability study on dichlorvos and chlorpyrifos-methyl in blood and buffers. Tiny amounts of dichlorvos, 0.067 and 0.027 mg/L, were detected in the vitreous humor and cerebrospinal fluid, respectively. Although dichlorvos (0.082-8.99 mg/L or mg/kg) was detected in the thoracic aortic blood, thoracic inferior vena caval blood, pericardial fluid, bile, and spleen, it was strongly suggested that it had diffused postmortem from the stomach, which contained 879 mg, because no dichlorvos was detected in the other blood samples and tissues tested. Substantial amounts (0.615-4.15 mg/L) of chlorpyrifos-methyl were detected in all blood samples, and the order of its concentrations was as follows: pulmonary vessel blood > thoracic inferior vena caval blood > blood in the right cardiac chambers > blood in the left cardiac chambers approximately thoracic aortic blood > right femoral venous blood. The total amount of chlorpyrifos-methyl in the stomach was 612 mg. However, it was strongly suggested that virtually no chlorpyrifos-methyl diffused from the stomach into surrounding fluids and tissues postmortem because no chlorpyrifos-methyl was detected in the bile and little was found in the pericardial fluids. Neither compound was detected in the urine. In vitro experiments showed that dichlorvos (10 mg/L) almost disappeared from fresh (pH 7.4) and acidified (pH 6.2) blood samples within 24 and 72 h, respectively. However, 53 and 77% of the original amount of dichlorvos in 0.05M phosphate buffers at pH 7.4 and 6.2 were detected 72 h later. Chlorpyrifos-methyl (1 mg/L) was very stable in blood samples, regardless of the pH, during the 72-h study period, but in the pH 7.4 and 6.2 phosphate buffers, approximately 80% of the original amount had degraded after 72 h. These results indicate that organophosphates are degraded more rapidly by esterase activities than by chemical mechanisms and that organophosphorothioates are hydrolyzed chemically in aqueous solutions but are very stable in biological specimens and not metabolized by esterases. When sodium fluoride was added to blood samples, dichlorvos degraded completely within 15 min, and chlorpyrifos-methyl became very unstable. Thus, when analyzing samples to detect organophosphorus chemicals, this common preservative should not be added to fluid specimens. (+info)
Changes in rat brain cholinesterase activity and muscarinic receptor density during and after repeated oral exposure to chlorpyrifos in early postnatal development.
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The effects of repeated oral exposures to the organophosphorus insecticide chlorpyrifos (CPS) on brain muscarinic receptor densities, together with cholinesterase (ChE) activity, were studied in early postnatal rats. Initially, the effects on esterases from lactational exposure to CPS were investigated in young rats by administering CPS (100, 150, or 200 mg/kg subcutaneously in corn oil) to dams 1 day postpartum, yielding a significant body burden of CPS in the dams for possible excretion in the milk. Brain ChE inhibition in pups was less severe than in dams, whereas liver carboxylesterase (CbxE) inhibition in pups was at the same level as in dams. Because of the limited brain ChE inhibition obtained following lactation, pups were exposed to CPS directly by gavage, using 3 dosing regimens to yield a dose response. The rats were gavaged with CPS in corn oil on alternate days from postnatal day (PND) 1 through PND 21. Rats in the low-dosage group received 11 treatments at 3 mg/kg, those in the medium-dosage group received 3 treatments at 3 mg/kg and 8 at 6 mg/kg, and those in the high dosage group received 3 treatments at 3 mg/kg, 4 at 6 mg/kg, and 4 at 12 mg/kg. ChE activity in brain homogenates were inhibited significantly by 29% and 63% in the low- and high-dosage groups, respectively, on PND 22 and by 17% in the high dosage group on PND 40. Muscarinic receptor densities in brain synaptosomes were reduced using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB) as ligands, with the effects more prominent from 3H-NMS. Densities of both ligands recovered to the control level several days after terminating treatment. The results indicate that pups are apparently exposed to only limited amounts of chlorpyrifos and/or its oxon through the milk when dams are exposed to extremely high chlorpyrifos levels. In addition, repeated direct oral exposures of early postnatal rats to CPS will result in persistent brain ChE inhibition and will transiently reduce muscarinic receptor density. (+info)
Gestational exposure to chlorpyrifos: dose response profiles for cholinesterase and carboxylesterase activity.
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This study investigates the in vivo dose response profiles of the target enzyme cholinesterase (ChE) and the detoxifying enzymes carboxylesterase (CaE) in the fetal and maternal compartments of pregnant rats dosed with chlorpyrifos [(O,O'-diethyl O-3,5,6-trichloro-2-pyridyl) phosphorothionate], a commonly used organophosphorus insecticide. Pregnant rats were dosed daily (po) with chlorpyrifos in corn oil (0, 3, 5, 7, or 10 mg/kg) on gestational days (GD) 14-18. Animals were sacrificed 5 h after the last chlorpyrifos dose (time of maximum brain cholinesterase inhibition) for analysis of ChE and CaE activity in maternal blood, liver, brain, placenta, and fetal liver and brain. The in vitro sensitivity (i.e., IC50, 30 min, 26 degrees C) of CaE also was determined by assaying the activity remaining after incubation with a range of chlorpyrifos-oxon concentrations. In vivo exposure to 10 mg/kg chlorpyrifos from GD14-18 caused overt maternal toxicity, with dose-related decreases in ChE activity more notable in maternal brain than fetal brain. Dose-related effects were also seen with chlorpyrifos-induced inhibition of fetal liver ChE and maternal brain CaE activities. Gestational exposure caused no inhibition of placental ChE or CaE, fetal brain CaE, or maternal blood CaE. ChE activities in the maternal blood and liver, as well as fetal and maternal liver CaE, however, were maximally inhibited by even the lowest dosage of chlorpyrifos. The in vitro sensitivity profiles of CaE to chlorpyrifos-oxon inhibition were valuable in predicting and verifying the in vivo CaE response profiles. Both the in vivo and in vitro findings indicated that fetal liver CaE inhibition was an extremely sensitive indicator of fetal chlorpyrifos exposure. (+info)
Studies of toxicity of dermally-absorbed nurelle D 550 EC preparations.
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The aim of the study was the evaluation of the toxic effect of a two-component, preparation Nurelle D 550 EC (500 g of chlorpyrifos and 50 g cypermethrin per 1 l), administrated dermally. Toxicity was evaluated from histological and ultrastructural studies of the internal organs and immunotoxic effects (evaluation of phagocytical and bactericidal activity of neutrophils). The preparation for dermal application was applied in 2 concentrations (200 mg/kg/day of chlorpyrifos plus 20 mg/kg/day of cypermethrin or 1000 mg/kg/day of chlorpyrifos, plus 100 mg/kg/day of cypermethrin). The preparation was administrated on the tail skin of female Wistar rats for 4 hours daily for a period of 4 weeks. After 28 days of the experiment, the animals were anaesthetised and blood was taken from the heart to evaluate the granulocyte system. The following organs were taken for histological studies: liver, kidney, lung, heart, spleen, thymus and lymph nodes. Ultrastructural studies were carried out on the lung, liver, kidney and heart. The results of the study showed that dermal application of the pesticide Nurelle D 550 EC resulted in slight morphological and ultrastructural changes in the liver, kidney, lung and heart. The preparation examined slightly elevated the bactericidal activity of neutrophils. The differences, however, were not statistically significant. The phagocytic reaction in animals of both experimental groups did not differ from that observed in control group (+info)