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A variety of chemicals, such as organophosphate (OP) and carbamate pesticides, nerve agents, and industrial chemicals, inhibit acetylcholinesterase (AChE) leading to overstimulation of the cholinergic nervous system. The resultant neurotoxicity is similar across mammalian species; however, the relative potencies of the chemicals across and within species depend in part on chemical-specific metabolic and detoxification processes. Carboxylesterases and A-esterases (paraoxonases, PON) are two enzymatic detoxification pathways that have been widely studied. We used an in vitro system to measure esterase-dependent detoxification of 15 AChE inhibitors. The target enzyme AChE served as a bioassay of inhibitor concentration following incubation with detoxifying tissue. Concentration-inhibition curves were determined for the inhibitor in the presence of buffer (no liver), rat liver plus calcium (to stimulate PONs and thereby measure both PON and carboxylesterase), and rat liver plus EGTA (to inhibit calcium-dependent PONs, measuring carboxylesterase activity). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (chlorpyrifos oxon, paraoxon, methyl paraoxon, malaoxon) were in agreement with the literature, serving to support the use of this assay. Only a few other inhibitors showed slight or a trend towards detoxification via carboxylesterases or PONs (mevinphos, aldicarb, oxamyl). There was no apparent PON- or carboxylesterase-mediated detoxification of the remaining inhibitors (carbofuran, chlorfenvinphos, dicrotophos, fenamiphos, methamidophos, methomyl, monocrotophos, phosphamidon), suggesting that the influence of esterases on these chemicals is minimal. Thus, generalizations regarding these metabolic pathways may not be appropriate. As with other aspects of AChE inhibitors, their metabolic patterns appear to be chemical-specific., (Published by Elsevier Ireland Ltd.)

This study aimed to model long-term subtoxic human exposure to an organophosphorus pesticide, chlorpyrifos, and to examine the influence of that exposure on the response to intermittent high-dose acute challenges. Adult Long-Evans male rats were maintained at 350 g body weight by limited access to a chlorpyrifos-containing diet to produce an intake of 0, 1, or 5 mg/kg/day chlorpyrifos. During the year-long exposure, half of the rats in each dose group received bi-monthly challenges (spikes) of chlorpyrifos, and the other half received vehicle. Rats were periodically tested using a neurological battery of evaluations and motor activity to evaluate the magnitude of the acute response (spike days) as well as recovery and ongoing chronic effects (non-spike days). Effects of the spikes differed as a function of dietary level for several endpoints (e.g., tremor, lacrimation), and in general, the high-dose feed groups showed greater effects of the spike doses. Animals receiving the spikes also showed some neurobehavioral differences among treatment groups (e.g., hypothermia, sensory and neuromotor differences) in the intervening months. During the eleventh month, rats were tested in a Morris water maze. There were some cognitive deficits observed, demonstrated by slightly longer latency during spatial training, and decreased preference for the correct quadrant on probe trials. A consistent finding in the water maze was one of altered swim patterning, or search strategy. The high-dose feed groups showed more tendency to swim in the outer annulus or to swim very close to the walls of the tank (thigmotaxic behavior). Overall, dietary exposure to chlorpyrifos produced long-lasting neurobehavioral changes and also altered the response to acute challenges.

More than 30 years ago, scientists recognized that, at a given dosage, the young rat was more sensitive than the adult to the toxicity of many organophosphorus, anticholinesterase pesticides. This observation went basically unexamined until recently. Renewed interest has emerged in scrutinizing this age-related sensitivity to pesticides, especially in light of the many new pesticides which are currently marketed. Our laboratory has been involved in comparing the age-related sensitivity of young and adult rats to chlorpyrifos [Dursban, Lorsban; O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] and methamidophos (Monitor; O,S-dimethyl phosphoamidothioate). Using chlorpyrifos, there is marked age-related sensitivity: direct oral dosing of the preweanling rat (postnatal day 17; PND17) with chlorpyrifos causes a toxic response (defined either behaviorally or biochemically) at a approximately 5-fold lower dosage than in adults (oral, maximum tolerated dose for the PND17 is 20 mg/kg versus 100 mg/kg for the adult). Other studies have indicated that the rat detoxifies chlorpyrifos and its oxon by binding to carboxylesterases and hydrolysis by A-esterases. The young rat is deficient in both these detoxification enzymes, which may explain the increased sensitivity of the young to chlorpyrifos toxicity. The age-related pattern for methamidophos is distinctly different: the oral, maximum tolerated dose is the same (8 mg/kg) whether the animal is 17 days old or an adult. We present data which indicate that methamidophos is not detoxified appreciably either in vivo or in vitro by A-esterases or carboxylesterases. Therefore, we submit the following hypothesis: organophosphorus pesticides, like chlorpyrifos, which are detoxified via A-esterases or carboxylesterases are more likely to exhibit age-related differences in sensitivity than pesticides which are not detoxified via these routes.

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.

Young rats are more sensitive than adults to a single oral dose of chlorpyrifos, an organophosphorus pesticide. A direct comparison of chlorpyrifos effects in young (postnatal day 17; PND17), adolescent (PND27), and adult (70 days) Long-Evans rats was conducted to determine quantitative and possibly qualitative differences in sensitivity in terms of behavioral changes and cholinesterase (ChE; total cholinesterase activity) inhibition at these three ages. Male and female rats were administered chlorpyrifos orally at one of two doses (PND17, 5 or 20 mg/kg; PND27, 20 or 50 mg/kg; adult, 20 or 80 mg/kg) and tested at either 3.5 or 6.5 h after dosing. Behavioral testing included observational evaluations and measurements of motor activity and was followed immediately by tissue collection for ChE determination in brain and blood. For both behavioral changes and ChE inhibition, peak effects occurred at 3.5 h in adult male and PND27 rats (both sexes) and at 6.5 h in adult female and PND17 rats (both sexes). Comparisons of the 20 mg/kg dose across ages showed generally less ChE inhibition and fewer behavioral effects with increasing age, except that the adult females were similar to the PND27 rats. The high dose used for each age group produced similar brain ChE inhibition (80-90%) and generally similar behavioral effects. Interestingly, a few end-points in the young rats were less affected than in adults at this level of ChE inhibition. The degree of ChE inhibition in the brain more closely paralleled the blood inhibition in the younger rats, compared to the adults. Carboxylesterase (CaE) and A-esterase are known to play an important role in the detoxification of organophosphates and may be partially responsible for these sensitivity differences. Liver and plasma CaE and A-esterase activities were measured in untreated male rats on PND1, 4, 7, 12, 17, and 21 and in adults of both sexes (82-92 days old). Preweanling rats had considerably less activity of both enzymes, and adult females had less liver CaE activity than males. These differences in detoxifying enzymes correlate with the age-related differences in behavioral and biochemical effects, as well as the gender differences seen in adult rats, and thus may be a major influence on the differential sensitivity to chlorpyrifos.

Previous studies have shown that, in general, young, postnatal animals are more sensitive than adults to the toxic effects of anticholinesterase (antiChE) pesticides. Paradoxically, often fetal brain cholinesterase (ChE) is less inhibited than maternal brain after gestational exposure to an antiChE, presumably due to placental and fetal detoxification of the antiChE. The present investigation was designed to study selected toxicokinetic and toxicodynamic factors surrounding the toxicity of chlorpyrifos (CPF; [O,O'-diethyl O-3,5,6-trichloro-2-pyridyl] phosphorothionate) in pregnant rats dosed repeatedly or singly during late gestation. Dams were dosed daily (po) with CPF in corn oil (0 or 7 mg/kg) on gestational days (GD) 14 to 18. Animals were euthanized at 2 to 120 h after the last dose and tissues were collected for enzyme analysis. Using this dosing regimen, we found that (1) the time of maximal ChE inhibition was the same (i.e., 5-10 h after dosing) for both maternal and fetal brain, (2) the degree of fetal brain ChE inhibition was 4.7 times less than maternal brain inhibition, and (3) the detoxification potential (i.e., carboxylesterase and chlorpyrifos-oxonase) of the fetal tissues was very low compared to the maternal tissues. A separate group of experiments showed that if pregnant dams received only one oral dose of 7 or 10 mg/kg CPF on GD18, the degree of ChE inhibition in the fetal brain was comparable to the maternal brain ChE inhibition. Taking into consideration the net increase (more than fourfold) in fetal brain ChE activity from GD14 to 18 in control animals, and the fact that maternal brain ChE was inhibited more than fetal brain ChE only in a repeated-dosing regimen, we conclude that the fetus is not genuinely protected from the toxic effects of a given dose of CPF. We propose that fetal brain ChE is simply able to recover more fully between each dose as compared to maternal brain ChE, giving the illusion that the fetal compartment is less affected than the maternal compartment.

It is well known that young animals are generally more sensitive to lethal effects of cholinesterase-inhibiting pesticides, but there are sparse data comparing less-than-lethal effects. We compared the behavioral and biochemical toxicity of chlorpyrifos in young (postnatal Day 17; PND17) and adult (about 70 days old) rats. First, we established that the magnitude of the age-related differences decreased as the rat matures. Next, we evaluated the time course of a single oral dose of chlorpyrifos in adult and PND17 male and female rats. Behavioral changes were assessed using a functional observational battery (with age-appropriate modifications for pre-weanling rats) and an evaluation of motor activity. Cholinesterase (ChE) activity was measured in brain and peripheral tissues and muscarinic receptor binding assays were conducted on selected tissues. Rats received either vehicle (corn oil) or chlorpyrifos (adult dose: 80 mg/kg; PND17 dose: 15 mg/kg); these doses were equally effective in inhibiting ChE. The rats were tested, and tissues were then taken at 1, 2, 3.5, 6.5, 24, 72, 168, or 336 h after dosing. In adult rats, peak behavioral changes and ChE inhibition occurred in males at 3.5 h after dosing, while in females the onset of functional changes was sooner, the time course was more protracted and recovery was slower. In PND17 rats, maximal behavioral effects and ChE inhibition occurred at 6.5 h after dosing, and there were no gender-related differences. Behavioral changes showed partial to full recovery at 24 to 72 h, whereas ChE inhibition recovered markedly slower. Blood and brain ChE activity in young rats had nearly recovered by 1 week after dosing, whereas brain ChE in adults had not recovered at 2 weeks. Muscarinic-receptor binding assays revealed apparent down-regulation in some brain areas, mostly at 24 and 72 h. PND17 rats generally showed more receptor down-regulation than adults, whereas only adult female rats showed receptor changes in striatal tissue that persisted for 2 weeks. Thus, compared to adults (1) PND17 rats show similar behavioral changes and ChE inhibition although at a five-fold lower dose; (2) the onset of maximal effects is somewhat delayed in the young rats; (3) ChE activity tended to recover more quickly in the young rats; (4) young rats appear to have more extensive muscarinic receptor down-regulation, and (5) young rats show no gender-related differences.

Behavioral changes and tissue cholinesterase (ChE) inhibition were examined in animals treated with the commonly used insecticide chlorpyrifos. Adult male rats were dosed by gavage with 0, 10, 30, 60, or 100 mg/kg chlorpyrifos. Rats (n = 20/dose group) were evaluated using a functional observational battery (FOB) and an automated measure of motor activity. All rats were tested the day before dosing and at 3.5 h (the time of peak effect) after dosing; half of these (n = 10/dose) were sacrificed immediately after testing for tissue collection. The remaining rats were tested again at 24 h, followed by sacrifice. The following tissues were collected from each animal: half brain, individual brain areas from the other half of the brain (frontal cortex, hippocampus, striatum, hypothalamus, cerebellum, pons/medulla), retina, liver, heart, diaphragm, quadriceps femoris muscle, and blood (separated into whole blood, plasma, and erythrocytes). ChE activity was measured in all tissues, and muscarinic receptor density was assessed as quinuclidinyl benzilate (QNB) binding in all brain regions, heart, and retina. The lowest dose produced no behavioral effects but did produce significant ChE inhibition in most tissues at 3.5 h. Higher doses produced more ChE inhibition and cholinergic signs of toxicity. Partial recovery from behavioral effects was evident at 24 h, with little or no corresponding recovery of ChE activity. Apparent downregulation of muscarinic receptor density was noted only in striatum and pons/medulla of rats treated with the highest dose of chlorpyrifos. Correlations for behavioral and biochemical effects were generally poor because: a) the low-dose effects on ChE inhibition were not reflected in behavioral signs, and b) behavioral signs showed recovery at 24 h, whereas ChE activity did not. Examination of data for individual rats indicated that > 60% of brain ChE inhibition was reached before neurobehavioral effects were evident.

Organophosphate (OP) pesticides can bind to carboxylesterase (CaE), which may lower the concentration of OPs at the target site enzyme, acetylcholinesterase (ChE). It is unclear from the literature whether it is the CaE's affinity for the OP and/or the number of CaE molecules which is the dominant factor in determining the protective potential of CaE. We undertook a detailed, in vitro and in vivo survey of both CaE and ChE to ascertain if in vitro sensitivity of CaE and ChE predicted the pattern of inhibition seen after in vivo dosing with chlorpyrifos (CPF; 80 mg/kg, p.o.) in male or female adult Long-Evans rats. For the brain, the in vitro sensitivity to CPF-oxon did predict the in vivo patterns of inhibition: In vitro, brain ChE was approximately 25 times more sensitive to the active metabolite, CPF-oxon, than brain CaE, and in vivo brain ChE was more inhibited than brain CaE. In contrast, the in vitro sensitivity of plasma ChE and CaE did not correlate well with the in vivo pattern of inhibition: In vitro, plasma ChE was approximately 6.5 times less sensitive to CPF-oxon than plasma CaE, but in vivo, plasma ChE was more inhibited than CaE. In order to understand the role of CaE in protecting the brain ChE from inhibition by CPF-oxon in vitro, adult rat striatal tissue was incubated in the presence and absence of adult rat liver tissue and IC50s of CPF-oxon were determined. The increase in the striatal CPF-oxon IC50 value noted for ChE in the presence of liver suggested that CaE was binding the CPF-oxon and limiting its access to ChE. Male liver CaE, which has the same affinity for binding CPF-oxon as female liver CaE but has twice as many binding sites, caused a greater increase in the striatal CPF-oxon IC50 than female liver, suggesting that the number of binding sites does play a role in the detoxification potential of a tissue. In summary, we found that (1) there are tissue and gender-related differences for basal ChE and CaE activity; (2) the in vitro sensitivity of CaE or ChE to CPF-oxon is highly tissue-specific; (3) the pattern of ChE and CaE inhibition after in vivo dosing with CPF is not necessarily predictable from the in vitro IC50 for these same enzymes, and (4) the number of CaE molecules may play a role in modifying the toxicity of CPF.

The clinical signs of intoxication produced by cholinesterase inhibitors, many of which are used as pesticides, are considered important information for regulatory purposes. We conducted acute studies of cholinesterase inhibitors to compare their effects as determined by a functional observational battery (FOB) and motor activity. The acute effects of two carbamates (carbaryl, aldicarb) and five organophosphates (OP) (chlorpyrifos, diazinon, parathion, fenthion, and diisopropyl fluorophosphate, or DFP) were evaluated on the day of dosing at the time of peak effect, at 1 and 3 days, and 1 week after dosing (oral gavage, in corn oil). A high dose was selected that produced clear cholinergic signs, and lower doses were chosen to produce a range of effects. Generally all cholinesterase inhibitors produced autonomic signs of cholinergic overstimulation (salivation, lacrimation, and miosis), hypothermia, mild tremors and mouth-smacking (chewing motions), lowered motor activity, decreased tail-pinch response, and altered neuromuscular function (gait changes and increased foot splay). The measures generally found to be most sensitive on the day of dosing were body temperature, motor activity, gait, and the presence of mouth-smacking and fine tremors. However, no single measure was the most sensitive across all compounds; for example, the lowest dose of fenthion decreased motor activity by 86% but did not alter the tail-pinch response, whereas the lowest dose of parathion did not lower activity but did decrease the tail-pinch response. For some measures, differences in the slopes of the dose-response curves were evident. Many effects were still observed at 24 h, but recovery was apparent for all compounds. Interestingly, residual effects at 72 h were obtained with the carbamates (carbaryl, aldicarb) as well as with the Op fenthion, but not with the other compounds. Thus, the overall clinical picture of toxicity was similar for these cholinesterase inhibitors, but compound-specific differences emerged in terms of the individual measures, dose-response, and time course.

When given to rats, O,O'-diethyl-O-[3,5,6-trichloro-2-pyridyl]- phosphorothionate (chlorpyrifos), a common insecticide, causes an unusually lengthy dose-dependent fall in the activity of brain acetylcholinesterase (AChE; EC 3.1.1.7). To determine whether the slow recovery involves impaired AChE synthesis, experiments were designed to measure AChE activity, immunoreactive AChE protein (AChE-IR) and AChE mRNA. Male, Long-Evans rats, maintained at 350 +/- 5 g, were dosed (s.c.) weekly for 4 weeks with 0, 15, 30, or 60 mg/kg chlorpyrifos in peanut oil. Brain tissue was harvested 1, 3, 5, 7 and 9 weeks after treatment began. AChE activity was measured by Ellman assay, and AChE-IR was estimated by two-site ELISA using monoclonal antibodies to rat brain AChE. While AChE activity fell significantly at all times and doses, AChE-IR increased at 3 and 5 weeks in the two higher dosage groups. Larger increases of AChE-IR were observed after chlorpyrifos was administered for 4 weeks by the oral route. Northern blots quantified with reference to cyclophilin were consistent with stable levels of AChE mRNA. Overall, it appears that chronically reduced brain AChE activity after chlorpyrifos reflects sustained enzyme inhibition, not loss of enzyme protein or suppression of AChE message.