Virginia C. Moser

Comparison of chlordimeform and carbaryl using a functional observational battery

The effects of the formamidine pesticide chlordimeform (CDM), and the carbamate carbaryl (CAR) were compared using a functional observational battery (FOB). The FOB, a series of observations and measurements that can be rapidly administered to toxicant-treated rats, includes home-cage and open-field observations, neuromuscular and sensorimotor tests, and physiological measures. Evaluations were made according to U.S. Environmental Protection Agency testing guidelines so as to determine dose-, time-, and sex-related toxicant effects. Long-Evans hooded rats of both sexes were tested initially and then dosed ip with either vehicle, CDM (1, 25, 56 mg/kg) or CAR (3, 10, 30 mg/kg), and tested at various times after dosing (for CDM 1, 5, 24 hr; for CAR 0.5, 3, 24, 48 hr). Both compounds affected general activity (home-cage and open-field), equilibrium, CNS excitability, and sensory responsiveness. Whereas similar decreases were obtained on rearing, gait, and arousal, there were important qualitative differences in the effects of CAR and CDM on reactions to handling and the reflex tests in that CDM increased excitability and enhanced responses to several stimuli but CAR either had no effect or decreased these measures. Only CDM produced an increase in muscle tone as measured by grip strength, and only CAR produced cholinergic autonomic signs of intoxication. Body weight and temperature were decreased by both compounds. Thus, the profiles of effect produced by these two pesticides could be clearly differentiated using the FOB.

Utility of a neurobehavioral screening battery for differentiating the effects of two pyrethroids, permethrin and cypermethrin

The ability of a neurobehavioral screening battery to differentiate the effects of two pyrethroids, permethrin and cypermethrin, was assessed in this experiment. Although the structures of these pesticides differ only in the alpha-cyano group, the behavioral syndromes associated with the Type I and II pyrethroids are quite different. The tests included a functional observational battery which is a series of subjective and quantitative measures of neurological function and behavior, and an automated measure of motor activity. Our results verified previous reports in the literature describing these different syndromes, i.e., aggressive sparring behavior, fine to whole-body tremor, hyperthermia, and decreased motor activity for the Type I pyrethroid permethrin, and pawing, burrowing, salivation, whole body tremor to choreoathetosis, hypothermia, and lowered motor activity for the Type II pyrethroid cypermethrin. In addition, we report that permethrin produced decreased grip strengths, increased resistance to capture, increased reactivity to a click stimulus, and induced head and forelimb shaking and agitated behaviors, whereas cypermethrin produced pronounced neuromuscular weakness and equilibrium changes, retropulsion, lateral head movements, alterations in responses to various stimuli, and increased urination. Although there were similarities in some effects (e.g., decreased motor activity), the pesticides differed sufficiently in their overall behavioral profiles, and severity and time course of effects, to discriminate these two compounds. Thus, this type of screening approach is sensitive enough to differentiate these pyrethroids for hazard identification purposes.

Age- and Gender-Related Differences in Sensitivity to Chlorpyrifos in the Rat Reflect Developmental Profiles of Esterase Activities

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.

Developmental Exposure to a Commercial PBDE Mixture, DE-71: Neurobehavioral, Hormonal, and Reproductive Effects

Developmental effects of polybrominated diphenyl ethers (PBDEs) have been suspected due to their structural similarities to polychlorinated biphenyls (PCBs). This study evaluated neurobehavioral, hormonal, and reproductive effects in rat offspring perinatally exposed to a widely used pentabrominated commercial mixture, DE-71. Pregnant Long-Evans rats were exposed to 0, 1.7, 10.2, or 30.6 mg/kg/day DE-71 in corn oil by oral gavage from gestational day 6 to weaning. DE-71 did not alter maternal or male offspring body weights. However, female offspring were smaller compared with controls from postnatal days (PNDs) 35-60. Although several neurobehavioral endpoints were assessed, the only statistically significant behavioral finding was a dose-by-age interaction in the number of rears in an open-field test. Developmental exposure to DE-71 caused severe hypothyroxinemia in the dams and early postnatal offspring. DE-71 also affected anogenital distance and preputial separation in male pups. Body weight gain over time, reproductive tissue weights, and serum testosterone concentrations at PND 60 were not altered. Mammary gland development of female offspring was significantly affected at PND 21. Congener-specific analysis of PBDEs indicated accumulation in all tissues examined. Highest PBDE concentrations were found in fat including milk, whereas blood had the lowest concentrations on a wet weight basis. PBDE concentrations were comparable among various brain regions. Thus, perinatal exposure to DE-71 leads to accumulation of PBDE congeners in various tissues crossing blood-placenta and blood-brain barriers, causing subtle changes in some parameters of neurobehavior and dramatic changes in circulating thyroid hormone levels, as well as changes in both male and female reproductive endpoints. Some of these effects are similar to those seen with PCBs, and the persistence of these changes requires further investigation.

Comparisons of the acute effects of cholinesterase inhibitors using a neurobehavioral screening battery in rats

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.

Screening Approaches to Neurotoxicity: A Functional Observational Battery

Observational tests to assess the behavioral and neurologic integrity of laboratory rats have been advanced for use in the primary screening of chemicals to detect neurotoxic potential. To examine the use of such an approach, we are systematically using a functional observational battery (FOB) to characterize known neurotoxicants and to assess its selectivity, reliability, specificity, and sensitivity. Our FOB is a series of tests that includes home cage and open field observations, neuromuscular and sensorimotor tests, and physiologic measures. Described in this article are the compounds we have tested to date and how the data have been integrated to produce a profile of effects for each. We have found the FOB to be sensitive to a variety of neurotoxicants, whose effects can be distinguished using a type of profile analysis as illustrated herein. This research effort provides important information about both the execution and the interpretation of FOB studies.

Interlaboratory comparison of motor activity experiments: implications for neurotoxicological assessments.

Motor activity is an important functional measure used in neurotoxicology. The effects of chemicals on motor activity, however, may depend on variables such as type of measurement apparatus, physical and environmental testing conditions, and many other experimental protocol and organismic variables. Due to the increasing use of motor activity in neurotoxicology, a major question concerns the potential for differences in experimental findings due to variations in sensitivity and reliability between different laboratories and devices used to measure motor activity. This study examined historical data from a number of laboratories that employed different devices and experimental protocols to measure motor activity. Four aspects of the motor activity data were compared: 1) within-laboratory control variability across time; 2) within-laboratory replicability of control data; 3) between-laboratory variability in the effects of chemicals; and 4) between-laboratory comparison of the control rates of habituation. The analyses indicated that there was a relatively restricted range of within-laboratory variability and reliability in control values, and that these ranges were comparable across laboratories. Similar profiles of habituation were also seen across the different laboratories. Moreover, in virtually every case, all laboratories were capable of detecting qualitatively similar changes in motor activity following acute exposure to a variety of chemicals. These data indicate a high degree of comparability in the data generated by the different devices and experimental protocols.

The relationship of oral chlorpyrifos effects on behavior, cholinesterase inhibition, and muscarinic receptor density in rat

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.

A multidisciplinary approach to toxicological screening: III. Neurobehavioral toxicity

The neurobehavioral effects of 10 known toxicants were examined as part of a multidisciplinary screening battery. The toxicants included carbaryl (CAR), triadimefon (TDM), heptachlor (HEP), chlordane (CDN), diethylhexyl phthalate (DEHP), carbon tetrachloride (CCl4), phenol, trichloroethylene (TCE), tetrachloroethylene (PER or perchlorethylene), and dichloromethane (DCM or methylene chloride). A functional observational battery and motor activity measurements were conducted before exposure, at specified times after an acute exposure, and during and after 14-d exposure. Severity scoring analysis was used to generate profiles of effect. The pesticides, CAR, TDM, HEP, and CDN, displayed the most acute neurotoxicity and were active at lower proportions of their respective acute LD50 values than were the solvents or the industrial chemicals. Although CAR and TDM showed little or no neurobehavioral effects with repeated dosing, cumulative neurotoxicity and lethality were evident with HEP and CDN. Phenol produced acute convulsive effects, and the most prominent finding with repeated exposure was lethality. DEHP displayed no neurobehavioral toxicity. The organic solvents, TCE, PER, CCl4, and DCM, produced various degrees of general nervous system depression following acute administration of high dose levels. Repeated dosing produced little or no effect with TCE or PER, marked physiological changes with CCl4, and cumulative toxicity and lethality with DCM. Some results of these studies were unexpected and should provide impetus for further research. Overall, these findings illustrate the utility of these screening methods.

Dose–response and time-course of neurobehavioral changes following oral chlorpyrifos in rats of different ages ☆

Young rats have been shown in several laboratories to be more sensitive to the neurotoxic effects of acute exposure to chlorpyrifos. To examine the neurobehavioral effects of chlorpyrifos as a function of age and dose, we conducted dose-response and time-course assessments in rats of three different ages (postnatal day, or PND, 17, 27, and adults). Doses were selected to span the effective dose range in each age group: PND17 - 4, 10, 20 mg/kg; PND27 - 10, 25, 50 mg/kg; adult - 10, 50, 100 mg/kg. Rats were tested at the time of peak effect on the day of dosing, and again at 1 and 3 days, and at 1 and 2 weeks after a single oral dose. There were age- and sex-related differences in the recovery of these behavioral effects; the adult males recovered from the behavioral effects more quickly than the other age groups, and the adult females showed the slowest recovery (up to at least 3 days). Although these doses had been shown previously to produce a similar degree of cholinesterase inhibition, the neurobehavioral alterations fell into the following three patterns of effect as a function of age. (1) Some endpoints (e.g., gait abnormalities, tremor) showed a dose-response curve that was shifted to the right in the older animals. Calculated ED50 values indicated that the PND17 rats were three- to five-fold more sensitive than the adults. (2) Some measures showed less effect in the youngest rats; for example, maximal motor activity decreases were half as great as with adults. (3) A few effects that were typically observed in adults, e.g., salivation, were not seen at all in the PND17 rats. Thus, differential responses on these neurobehavioral endpoints were observed as a function of age. These data suggest that, for some endpoints, young rats are more sensitive to a range of chlorpyrifos doses; however, the magnitude of age-related differences depends on the specific endpoint and time of assessment, as well as age and sex of the test subject.