Prenatal ozone exposure programs a sexually dimorphic susceptibility to high-fat diet in adolescent Long Evans rats.

Altered fetal growth, which can occur due to environmental stressors during pregnancy, may program a susceptibility to metabolic disease. Gestational exposure to the air pollutant ozone is associated with fetal growth restriction in humans and rodents. However, the impact of this early life ozone exposure on offspring metabolic risk has not yet been investigated. In this study, fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone on gestation days 5 and 6 (4 hr/day) in Long Evans rats. To uncover any metabolic inflexibility, or an impaired ability to respond to a high-fat diet (HFD), a subset of peri-adolescent male and female offspring from filtered air or ozone exposed dams were fed HFD (45% kcal from fat) for 3 days. By 6 weeks of age, male and female offspring from ozone-exposed dams were heavier than offspring from air controls. Furthermore, offspring from ozone-exposed dams had greater daily caloric consumption and reduced metabolic rate when fed HFD. In addition to energy imbalance, HFD-fed male offspring from ozone-exposed dams had dyslipidemia and increased adiposity, which was not evident in females. HFD consumption in males resulted in the activation of the protective 5'AMP-activated protein kinase (AMPKα) and sirtuin 1 (SIRT1) pathways in the liver, regardless of maternal exposure. Unlike males, ozone-exposed female offspring failed to activate these pathways, retaining hepatic triglycerides following HFD consumption that resulted in increased inflammatory gene expression and reduced insulin signaling genes. Taken together, maternal ozone exposure in early pregnancy programs impaired metabolic flexibility in offspring, which may increase susceptibility to obesity in males and hepatic dysfunction in females.

Impacts of a perinatal exposure to manganese coupled with maternal stress in rats: Tests of untrained behaviors.

Manganese (Mn), an element that naturally occurs in the environment, has been shown to produce neurotoxic effects on the developing young when levels exceed physiological requirements. To evaluate the effects of this chemical in combination with non-chemical factors pregnant Long-Evans rats were treated with 0, 2, or 4 mg/mL Mn in their drinking water from gestational day (GD) 7 to postnatal day (PND) 22. Half of the dams received a variable stress protocol from GD13 to PND9, that included restraint, small cage with reduced bedding, exposure to predator odor, intermittent intervals of white noise, lights on for 24 h, intermittent intervals of lights on during dark cycle and cages with grid floors and reduced bedding. One male and one female offspring from each litter were tested to assess untrained behavior. Ultrasonic vocalizations (USV) were recorded from PND13 pups while they were isolated from the litter. Locomotor activity (MA) was measured in figure-eight mazes at PND 17, 29, and 79 (different set of rats at each time point). Social approach (SA) was tested at PND48. Acoustic startle response (ASR) and pre-pulse inhibition (PPI) were measured starting at PND58. At PND53 a sweetness preference for a chocolate flavored milk solution was assessed. There were sex related differences on several parameters for the USVs. There was also a Mn by stress by sex interaction with the females from the 4 mg/mL stressed dams having more frequency modulated (FM) call elements than the 4 mg/mL non-stressed group. There was an effect of Mn on motor activity but only at PND29 with the 2 mg/mL group having higher counts than the 0 mg/mL group. The social approach test showed sex differences for both the habituation and test phase. There was an effect of Mn, with the 4 mg/mL males having a greater preference for the stimulus rat than did the 0 mg/mL males. There was also a stress by sex interaction. The ASR and PPI had only a sex effect. Thus, with only the FM call elements having a Mn by stress effect, and the PND29 MA and SA preference index having a Mn effect but at different doses requires further investigation.

In vivo neurophysiological assessment of in silico predictions of neurotoxicity: Citronellal, 3,4-dichloro-1-butene, and benzyl bromoacetate.

Neurotoxicants may be widespread in the environment and can produce serious health impacts in the human population. Screening programs that use in vitro methods have generated data for thousands of chemicals. However, these methods often do not evaluate repeated or prolonged exposures, which are required for many neurotoxic outcomes. Additionally, the data produced by such screening methods may not include mechanisms which play critical biological roles necessary for in vivo neurotoxicity. The Hard and Soft Acids and Bases (HSAB) in silico model focuses on chemical structure and electrophilic properties which are important to the formation of protein adducts. A group of structurally diverse chemicals have been evaluated with an in silico screening approach incorporating HSAB parameters. However, the predictions from the expanded chemical space have not been evaluated using in vivo methods. Three chemicals predicted to be cumulative toxicants were selected for in vivo neurotoxicological testing. Adult male Long-Evans rats were treated orally with citronellal (CIT), 3,4-dichloro-1-butene (DCB), or benzyl bromoacetate (BBA) for 8 weeks. Behavioral observations were recorded weekly to assess motor function. Peripheral neurophysiological measurements were derived from nerve excitability (NE) tests which involved compound muscle action potentials (CMAPs) in the tail and foot, and mixed nerve action potentials (MNAPs) in the tail. Compound nerve action potentials (CNAPs) and nerve conduction velocity (NCV) in the tail were also quantified. Peripheral inputs into the central nervous system were examined using somatosensory evoked potentials recorded from the cortex (SEPCTX) and cerebellum (SEPCEREB). CIT or BBA did not result in significant alterations to peripheral nerve or somatosensory function. DCB reduced grip-strength and altered peripheral nerve function. The MNAPs required less current to reach 50% amplitude and had a lower calculated rheobase, suggesting increased excitability. Increased CNAP amplitudes and greater NCV were also observed. Novel changes were found in the SEPCTX with an abnormal peak forming in the early portion of the waveforms of treated rats, and decreased latencies and increased amplitudes were observed in SEPCEREB recordings. These data contribute to testing an expanded chemical space from an in silico HSAB model for predicting cumulative neurotoxicity and may assist with prioritizing chemicals to protect human health.

Impacts of a perinatal exposure to manganese coupled with maternal stress in rats: Maternal somatic measures and the postnatal growth and development of rat offspring.

Psychological stress experienced by the mother during pregnancy has been associated with emotional and cognitive disorders in children such as depression and anxiety. Socioeconomically disadvantaged populations are vulnerable to adverse life experiences and can also be disproportionally exposed to environmental contaminants. To better understand the neurodevelopmental impacts of an environmental toxicant coupled with elevated psychological stress, we exposed pregnant rats to a series of perinatal stressors. Manganese (Mn), a neurotoxicant at excessive concentrations was delivered through drinking water (0, 2, or 4 mg/mL) from gestational day (GD) 7 to postnatal day (PND) 22. A variable stress paradigm was applied to half of the animals from GD13 to PND9. Measurements of somatic development and behavior were examined in the offspring at different developmental stages. No evidence of overt maternal toxicity was observed although the 4 mg/mL Mn-exposed dams gained less body weight during gestation compared to the other dams. Stress also reduced gestational maternal weight gain. Daily fluid consumption normalized for body weight was decreased in the Mn-exposed dams in a dose-dependent manner but was not altered by the stress paradigm. Maternal stress and/or Mn exposure did not affect litter size or viability, but pup weight was significantly reduced in the 4 mg/mL Mn-exposed groups on PNDs 9 through 34 when compared to the other offspring groups. The efficacy of the manipulations to increase maternal stress levels was determined using serum corticosterone as a biomarker. The baseline concentration was established prior to treatment (GD7) and levels were low and similar in all treatment groups. Corticosterone levels were elevated in the perinatal-stress groups compared to the no-stress groups, regardless of Mn exposure, on subsequent time points (GD16, PND9), but were only significantly different on GD16. An analysis of tissue concentrations revealed Mn was elevated similarly in the brain and blood of offspring at PND2 and at PND22 in a significant dose-dependent pattern. Dams also showed a dose-dependent increase in Mn concentrations in the brain and blood; the addition of stress increased the Mn concentrations in the maternal blood but not the brain. Perinatal stress did not alter the effects of Mn on the maternal or offspring somatic endpoints described here.

Impacts of maternal diet and exercise on offspring behavior and body weights.

Human and animal studies indicate that maternal obesity can negatively impact aspects of metabolism and neurodevelopment in the offspring. Not known, however, is whether maternal exercise can alter these adverse outcomes. In this study, Long-Evans female rats were provided a high fat (60%; HFD) or control diet (CD) 44days before mating and throughout gestation and lactation. Running wheels were available to half of each diet group during the gestational period only, resulting in four conditions: CD diet with (CDRW) or without (sedentary; CDSED) exercise, and HFD with (HFRW) or without (HFSED) exercise. Only male offspring (one per litter) were available for this study: they were put on control diet two weeks after weaning and examined using behavioral evaluations up to four months of age. Before weaning, offspring of CDRW dams weighed less than offspring from CDSED or HFD dams. After weaning, the lower weight in CDRW offspring generally persisted. Adult offspring from HFSED dams performed worse than the HFRW group in a Morris water maze during initial spatial training as well as reversal learning; memory was not impacted. No differences between groups were seen in tests of novel object recognition, social approach, or chocolate milk preference. Thus, maternal diet and exercise produced differential effects on body weights and cognitive behaviors in the offspring, and the data demonstrate a positive impact of maternal exercise on the offspring in that it ameliorated some deleterious behavioral effects of a maternal high fat diet.

Locomotor activity and tissue levels following acute administration of lambda- and gamma-cyhalothrin in rats.

Pyrethroids produce neurotoxicity that depends, in part, on the chemical structure. Common behavioral effects include locomotor activity changes and specific toxic syndromes (types I and II). In general these neurobehavioral effects correlate well with peak internal dose metrics. Products of cyhalothrin, a type II pyrethroid, include mixtures of isomers (e.g., λ-cyhalothrin) as well as enriched active isomers (e.g., γ-cyhalothrin). We measured acute changes in locomotor activity in adult male rats and directly correlated these changes to peak brain and plasma concentrations of λ- and γ-cyhalothrin using a within-subject design. One-hour locomotor activity studies were conducted 1.5h after oral gavage dosing, and immediately thereafter plasma and brains were collected for analyzing tissue levels using LC/MS/MS methods. Both isomers produced dose-related decreases in activity counts, and the effective dose range for γ-cyhalothrin was lower than for λ-cyhalothrin. Doses calculated to decrease activity by 50% were 2-fold lower for the γ-isomer (1.29mg/kg) compared to λ-cyhalothrin (2.65mg/kg). Salivation, typical of type II pyrethroids, was also observed at lower doses of γ-cyhalothrin. Administered dose correlated well with brain and plasma concentrations, which furthermore showed good correlations with activity changes. Brain and plasma levels were tightly correlated across doses. While γ-cyhalothrin was 2-fold more potent based on administered dose, the differences based on internal concentrations were less, with γ-cyhalothrin being 1.3- to 1.6-fold more potent than λ-cyhalothrin. These potency differences are consistent with the purity of the λ-isomer (approximately 43%) compared to the enriched isomer γ-cyhalothrin (approximately 98%). Thus, administered dose as well as differences in cyhalothrin isomers is a good predictor of behavioral effects.

Neurotoxicological and thyroid evaluations of rats developmentally exposed to tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) and tris(2-chloro-2-ethyl)phosphate (TCEP).

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) and tris(2-chloro-2-ethyl)phosphate (TCEP) are organophosphorous flame retardants with widespread usage and human exposures through food, inhalation, and dust ingestion. They have been detected in human tissues including urine and breast milk. Reports of disrupted neural growth in vitro, abnormal development in larval zebrafish, and altered thyroid hormones in several species have raised concern for neurodevelopmental toxicity. This is especially the case for TDCIPP, which is more potent and has more activity in those assays than does TCEP. We evaluated the potential for developmental neurotoxicity of TDCIPP and TCEP in a mammalian model. Pregnant Long-Evans rats were administered TDCIPP (15, 50, or 150 mg/kg/day) or TCEP (12, 40, 90 mg/kg/day) via oral gavage from gestational day 10 to weaning. Corn oil was the vehicle control in both studies. Body weight and righting reflex development were monitored in all pups. A subset of offspring at culling and weaning, and dams at weaning, were sacrificed for serum and organ collection for measurement of brain, liver, and thyroid weights, serum thyroid levels, and serum and brain acetylcholinesterase activities. Brain weights were also measured in a group of adult TDCIPP-treated offspring. One male and one female from each litter were allocated for behavioral testing at several ages: standard locomotor activity (preweaning, postweaning, adults), locomotor activity including a lighting change mid-way (postweaning, adults), elevated zero maze (postweaning, adults), functional observational battery (FOB; postweaning, adults), and Morris water maze (place learning, reference and working memory; adults). Neither chemical produced changes in maternal body weight or serum thyroid hormones, but relative liver weight was increased at the high doses of both TDCIPP and TCEP. In offspring, there were no effects on viability, litter size, or birth weight. With TDCIPP, absolute liver weights were lower at weaning and weight gain was lower in the high-dose offspring until about two months of age. Thyroid hormones and brain weights were not altered and acetylcholinesterase (both brain and serum) was not inhibited by either chemical. TDCIPP-treated offspring showed slight differences in floating in the water maze, hindlimb grip strength, and altered activity habituation, whereas TCEP-treated rats showed differences in quadrant time (probe) and middle-zone preference in the water maze. Regarding these few changes, the effects were minimal, mostly not related to dose, and did not appear treatment-related or biologically significant. Overall, these data do not support the potential for thyrotoxicity or developmental neurotoxicity produced by TDCIPP or TCEP.

Toxicological assessments of rats exposed prenatally to inhaled vapors of gasoline and gasoline-ethanol blends.

The primary alternative to petroleum-based fuels is ethanol, which may be blended with gasoline in the United States at concentrations up to 15% for most automobiles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol and the lack of information about the neurodevelopmental toxicity of ethanol-blended fuels prompted the present work. Pregnant Long-Evans rats were exposed for 6.5h/day on days 9-20 of gestation to clean air or vapors of gasoline containing no ethanol (E0) or gasoline blended with 15% ethanol (E15) or 85% ethanol (E85) at nominal concentrations of 3000, 6000, or 9000 ppm. Estimated maternal peak blood ethanol concentrations were less than 5mg/dL for all exposures. No overt toxicity in the dams was observed, although pregnant dams exposed to 9000 ppm of E0 or E85 gained more weight per gram of food consumed during the 12 days of exposure than did controls. Fuel vapors did not affect litter size or weight, or postnatal weight gain in the offspring. Tests of motor activity and a functional observational battery (FOB) administered to the offspring between post-natal day (PND) 27-29 and PND 56-63 revealed an increase in vertical activity counts in the 3000- and 9000-ppm groups in the E85 experiment on PND 63 and a few small changes in sensorimotor responses in the FOB that were not monotonically related to exposure concentration in any experiment. Neither cell-mediated nor humoral immunity were affected in a concentration-related manner by exposure to any of the vapors in 6-week-old male or female offspring. Systematic concentration-related differences in systolic blood pressure were not observed in rats tested at 3 and 6 months of age in any experiment. No systematic differences were observed in serum glucose or glycated hemoglobin A1c (a marker of long-term glucose homeostasis). These observations suggest a LOEL of 3000 ppm of E85 for vertical activity, LOELs of 9000 ppm of E0 and E85 for maternal food consumption, and NOELs of 9000 ppm for the other endpoints reported here. The ethanol content of the vapors did not consistently alter the pattern of behavioral, immunological, or physiological responses to the fuel vapors. The concentrations of the vapors used here exceed by 4-6 orders of magnitude typical exposure levels encountered by the public.

Assessment of biochemical and behavioral effects of carbaryl and methomyl in Brown-Norway rats from preweaning to senescence.

Factors impacting life stage-specific sensitivity to chemicals include toxicokinetic and toxicodynamic changes. To evaluate age-related differences in the biochemical and behavioral impacts of two typical N-methyl carbamate pesticides, we systematically compared their dose-response and time-course in preweanling (postnatal day, PND, 18) and adult male Brown Norway rats (n=9-10/dose or time) ranging from adolescence to senescence (1, 4, 12, 24 mo). Carbaryl was administered orally at 3, 7.5, 15, or 22.5mg/kg and data were collected at 40 min after dosing, or else given at 3 or 15 mg/kg and data collected at 30, 60, 120, and 240 min. Methomyl was studied only in adult and senescent rat (4, 12, 24 mo) in terms of dose-response (0.25. 0.6, 1.25, 2.5mg/kg) and time-course (1.25mg/kg at 30, 60, 120, 240 min). Motor activity as well as brain and erythrocyte (RBC) cholinesterase (ChE) activity were measured in the same animals. In the carbaryl dose-response, PND18 rats were the most sensitive to the brain ChE-inhibiting effects of carbaryl, but 12- and 24-mo rats showed more motor activity depression even at similar levels of brain ChE inhibition. We have previously reported that brain ChE inhibition, but not motor activity effects, closely tracked carbaryl tissue levels. There were no age-related differences in methomyl-induced ChE inhibition across doses, but greater motor activity depression was again observed in the 12- and 24-mo rats. Carbaryl time-course data showed that motor activity depression reached a maximum later, and recovered slower, in the 12- and 24-mo rats compared to the younger ages; slowest recovery and maximal effects were seen in the 24-mo rats. Acetylcholinesterase sensitivity (concentration-inhibition curves) was measured in vitro using control tissues from each age. Inhibitory concentrations of carbaryl were somewhat lower in PND18, 12-, and 24-mo tissues compared to 1- and 4-mo, but there were no differences with methomyl-treated tissues. Thus, in the dose-response and time-course, there were dissociations between brain ChE inhibition and the magnitude as well as recovery of motor activity changes. The explanation for this dissociation is unclear, and is likely due to early development followed by aging-related decline in both kinetic parameters and neurological responsiveness.

Use of electroencephalography (EEG) to assess CNS changes produced by pesticides with different modes of action: effects of permethrin, deltamethrin, fipronil, imidacloprid, carbaryl, and triadimefon.

The electroencephalogram (EEG) is an apical measure, capable of detecting changes in brain neuronal activity produced by internal or external stimuli. We assessed whether pesticides with different modes of action produced different changes in the EEG of adult male Long-Evans rats. The EEG was recorded using two montages (visual cortex referenced to the cerebellum and to the frontal cortex) in unrestrained rats at the time of peak behavioral effects. Pesticides included: permethrin and deltamethrin (Type I and Type II pyrethroids; 2 h), fipronil (single and repeated doses; phenylpyrazole; 6 h), imidacloprid (neonicotinoid; 2 h), carbaryl (carbamate; 0.5 h), and triadimefon (triazole; 1 h), using dosages that produced approximately an ED30 or an ED50-ED80 change in motor activity. Permethrin (43, 100 mg/kg) increased amplitudes or areas (delta, alpha, or gamma bands) in the EEG. Deltamethrin (2.5, 5.5 mg/kg) reduced the amplitudes or areas of the delta, theta, alpha, beta, and gamma bands, but the changes were not dose-related. A single treatment with fipronil (25, 50 mg/kg, but not 5, 10 mg/kg) decreased gamma band area. Additional changes in the delta, theta, and gamma bands were observed when fipronil (5, 10 mg/kg) was administered for 14 days. Imidacloprid (50, 100 mg/kg) did not alter the EEG. Carbaryl (10, 50 mg/kg) decreased theta area, and decreased delta and increased beta frequency. Triadimefon (75, 150 mg/kg) produced minimal changes in the EEG. The results show that the EEG is affected differently by approximately equipotent doses of pesticides with different modes of action.

Toxicological outcomes in rats exposed to inhaled ethanol during gestation.

Recent legislation has encouraged replacing petroleum-based fuels with renewable alternatives including ethanol, which is typically blended with gasoline in the United States at concentrations up to 10%, with allowances for concentrations up to 85% for some vehicles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol, and the lack of information about its toxicity by inhalation prompted the present work on its potential developmental effects in a rat model. Pregnant Long-Evans rats were exposed for 6.5h/day on days 9-20 of gestation to clean air or ethanol vapor at concentrations of 5000, 10,000, or 21,000 ppm, which resulted in estimated peak blood ethanol concentrations (BECs) of 2.3, 6.7, and 192 mg/dL, respectively. No overt toxicity in the dams was observed. Ethanol did not affect litter size or weight, or postnatal weight gain in the pups. Motor activity was normal in offspring through postnatal day (PND) 29. On PND 62, the 5000 and 21,000 ppm groups were more active than controls. On PND 29 and 62, offspring were tested with a functional observational battery, which revealed small changes in the neuromuscular and sensorimotor domains that were not systematically related to dose. Cell-mediated and humoral immunity were not affected by ethanol exposure in 6-week-old offspring. Systolic blood pressure was increased by 10,000 ppm ethanol in males at PND 90 but not at PND 180. No differences in lipoprotein profile, liver function, or kidney function were observed. In summary, prenatal exposure to inhaled ethanol caused some mild changes in physiological and behavioral development in offspring that were not clearly related to inhaled concentration or BEC, and did not produce detectable changes in immune function. This low toxicity of inhaled ethanol may result from the slow rise in BEC by the inhalation route.

Carbaryl and 1-naphthol tissue levels and related cholinesterase inhibition in male Brown Norway rats from preweaning to senescence.

Studies incorporating both toxicokinetic and dynamic factors provide insight into chemical sensitivity differences across the life span. Tissue (brain, plasma, liver) levels of the N-methyl carbamate carbaryl, and its metabolite 1-naphthol, were determined and related to brain and RBC cholinesterase (ChE) inhibition in the same animals. Dose-response (3, 7.5, 15, or 22.5 mg/kg, 40-45 min postdosing) and time course (3 or 15 mg/kg at 30, 60, 120, or 240 min postdosing) of acute effects of carbaryl (oral gavage) in preweanling (postnatal day [PND] 18) and adult male Brown Norway rats from adolescence to senescence (1, 4, 12, 24 mo) were compared. At all ages there were dose-related increases in carbaryl and 1-naphthol in the dose-response study, and the time-course study showed highest carbaryl levels at 30 min postdosing. There were, however, age-related differences in that the 1- and 4-mo rats showed the lowest levels of carbaryl and 1-naphthol, and PND18 and 24-mo rats had similar, higher levels. The fastest clearance (shortest half-lives) was observed in 1- and 4-mo rats. Carbaryl levels were generally higher than 1-naphthol in brain and plasma, but in liver, 1-naphthol levels were similar to or greater than carbaryl. Brain ChE inhibition closely tracked brain carbaryl concentrations regardless of the time after dosing, but there was more variability in the relationship between RBC ChE and plasma carbaryl levels. Within-subject analyses suggested somewhat more brain ChE inhibition at lower carbaryl levels only in the PND18 rats. These findings may reflect maturation followed by decline in kinetic factors over the life span.

Time-course, dose-response, and age comparative sensitivity of N-methyl carbamates in rats.

N-Methyl carbamate insecticides are reversible inhibitors of central and peripheral acetylcholinesterase (ChE). Despite their widespread use, there are few studies of neurotoxicity in young animals. To study potential age-related differences, we evaluated seven carbamates (carbaryl, carbofuran, formetanate, methiocarb, methomyl, oxamyl, and propoxur) in preweanling (17 days old or postnatal day [PND] 17) male rats. Motor activity was monitored, and ChE inhibition was measured in brain and red blood cells (RBCs) using a radiometric assay that minimized reactivation of ChE. First, we conducted time-course studies in PND17 Long-Evans male rats, using a single oral dose of each carbamate. Almost all carbamates showed maximal ChE inhibition at a 45-min time point; only methomyl showed an earlier peak effect (15 min). At 24 h, most inhibition had recovered. Next, dose-response data were collected for each carbamate, using four doses and control, with motor activity testing beginning 15 min after dosing and tissue collection at 40-45 min. RBC ChE was generally inhibited to a greater degree than brain. Motor activity was not as sensitive a measure for some of the carbamates, with some differences across carbamates in the shapes of the dose-response curves. Additional studies documented age-related differences by comparing ChE inhibition in PND11, PND17, and adult rats following administration of carbaryl or carbofuran. Only the youngest (PND11) rats were more sensitive than adults to carbaryl, but both younger ages showed more effects than adults with carbofuran. Comparisons of the other carbamates to previous studies in adult rats suggest similar age-related sensitivity. Thus, these data show the time-course and dose-response characteristics for each carbamate and document greater sensitivity of the young for carbofuran and carbaryl.

Comparison of acute neurobehavioral and cholinesterase inhibitory effects of N-methylcarbamates in rat.

While the cholinesterase-inhibiting N-methyl carbamate pesticides have been widely used, there are few studies evaluating direct functional and biochemical consequences of exposure. In the present study of the acute toxicity of seven N-methyl carbamate pesticides, we evaluated the dose-response profiles of cholinesterase (ChE) inhibition in brain and erythrocytes (RBCs) as well as motor activity (both horizontally and vertically directed) and clinical signs of overt toxicity. The chemicals tested were carbaryl, carbofuran, formetanate, methiocarb, methomyl, oxamyl, and propoxur. All were administered orally, and rats were tested in 20-min activity sessions beginning 15 min after dosing; tissues were collected immediately after activity sessions. In general, motor activity was a sensitive measure of ChE inhibition for all these carbamate pesticides, and vertical activity showed the greatest magnitude of effect at the highest doses compared to either horizontal activity or ChE inhibition. Brain and RBC ChE activities were generally affected similarly. Pearson correlation coefficients of within-subject data showed good correlation between the behavioral and biochemical end points, with brain ChE inhibition and horizontal activity showing the highest correlation values. Determination of benchmark dose levels for 10% change in each end point also revealed that these two measures produced the lowest estimates. Thus, motor activity decreases are highly predictive of ChE inhibition for N-methyl carbamates, and vice versa.

Neurotoxicological evaluation of two disinfection by-products, bromodichloromethane and dibromoacetonitrile, in rats.

The Safe Drinking Water Act requires that the U.S. EPA consider noncancer endpoints for the assessment of adverse human health effects of disinfection by-products (DBPs). As an extension of our studies in which we demonstrated neurotoxicity at relatively low levels of dibromo- and dichloroacetic acids, we examined the potential neurotoxicity of other classes of DBPs. Bromodichloromethane (BDCM) and dibromoacetonitrile (DBAN) were administered to male and female F-344 rats via drinking water for 6 months. During exposure, rats were tested for neurobehavioral effects using a functional observational battery and motor activity, followed by perfusion fixation for neuropathological evaluation at the end of exposure. Calculating for chemical loss, fluid consumption, and body weight, average intakes were approximately: 9, 27, and 72mg/(kgday) BDCM, and 5, 12, and 29mg/(kgday) DBAN. Fluid consumption was decreased in most treatment groups, but body weight gain was altered only at the high concentrations. There were few neurobehavioral changes, and these were not considered toxicologically relevant. Of the general observations, there was only minimally decreased body tone in DBAN-treated high-dose males. Treatment-related neuropathological findings were not observed. Lowered fluid consumption was the most sensitive and consistent endpoint in the present studies. Thus, unlike the haloacetic acids, neurotoxicity may not be a concern for toxicity of halomethanes or haloacetonitriles.

Evaluation of developmental neurotoxicity of organotins via drinking water in rats: monomethyltin.

Organotins such as monomethyltin (MMT) are widely used as heat stabilizers in PVC and CPVC piping, which results in their presence in drinking water supplies. Concern for neurotoxicity produced by organotin exposure during development has been raised by published findings of a deficit on a runway learning task in rat pups perinatally exposed to MMT (Noland EA, Taylor DH, Bull RJ. Monomethyl and trimethyltin compounds induce learning deficiencies in young rats. Neurobehav Toxicol Teratol 1982;4:539-44). The objective of these studies was to replicate the earlier publication and further define the dose-response characteristics of MMT following perinatal exposure. In Experiment 1, female Sprague-Dawley rats were exposed via drinking water to MMT (0, 10, 50, 245 ppm) before mating and throughout gestation and lactation (until weaning at postnatal day [PND] 21). Behavioral assessments of the offspring included: a runway test (PND 11) in which the rat pups learned to negotiate a runway for dry suckling reward; motor activity habituation (PNDs 13, 17, and 21); learning in the Morris water maze (as adults). Other endpoints in the offspring included measures of apoptosis (DNA fragmentation) at PND 22 and as adults, as well as brain weights and neuropathological evaluation at PND 2, 12, 22, and as adults. There were no effects on any measure of growth, development, cognitive function, or apoptosis following MMT exposure. There was a trend towards decreased brain weight in the high dose group. In addition, there was vacuolation of the neuropil in a focal area of the cerebral cortex of the adult offspring in all MMT dose groups (1-3 rats per treatment group). In Experiment 2, pregnant rats were exposed from gestational day 6 until weaning to 500 ppm MMT in drinking water. The offspring behavioral assessments again included the runway task (PND 11), motor activity habituation (PND 17), and Morris water maze (as adults). In this second study, MMT-exposed females consumed significantly less water than the controls throughout both gestation and lactation, although neither dam nor pup weights were affected. As in Experiment 1, MMT-exposure did not alter pup runway performance, motor activity, or cognitive function. These results indicate that perinatal exposure to MMT, even at concentrations which decrease fluid intake, does not result in significant neurobehavioral or cognitive deficits. While mild neuropathological lesions were observed in the adult offspring, the biological significance of this restricted finding is unclear.

Differential profiles of cholinesterase inhibition and neurobehavioral effects in rats exposed to fenamiphos or profenofos.

The relationship between cholinesterase (ChE) inhibition and neurobehavioral changes was examined using two ChE-inhibiting organophosphorus (OP) pesticides, fenamiphos and profenofos. Both pesticides produce considerable blood ChE inhibition, but relatively little brain inhibition up to almost lethal doses. Interestingly, pronounced neurobehavioral signs were produced by fenamiphos but not profenofos. After a single oral dose, both pesticides greatly inhibited blood ChE (87-98% inhibition), yet whole brain ChE was only inhibited by 9-14% at the highest doses. Fenamiphos produced dose-dependent effects on many behavioral measures. Despite the similar ChE inhibition profile, profenofos produced no observable changes in behavior. Treatment with anticholinergic drugs was used to evaluate the contribution of peripheral versus central ChE inhibition. Scopolamine (SCO) and methylscopolamine (MSC) were used as central/peripheral and peripheral-only cholinergic receptor blockers, respectively, in combination with fenamiphos. Neither drug altered the effects of fenamiphos on ChE inhibition. Some behavioral effects of fenamiphos were blocked or attenuated only by SCO, whereas other effects were blocked by both drugs. These data indicate that some of the pronounced neurobehavioral changes observed following fenamiphos dosing may be centrally mediated (blocked by SCO only), despite the small amount of inhibition of brain ChE. Other behavioral changes may be mediated more peripherally (blocked by both MSC and SCO). To test the hypothesis that regionally specific ChE inhibition may be responsible for these effects, the same dose of fenamiphos used in the previous studies was given and one half of the brain was dissected into regions. There was significant ChE inhibition in the pons and medulla, cerebellum, striatum, hippocampus, and half-brain but not in the rest-of-brain and frontal cortex; however, the magnitude of inhibition was relatively small across the regions measured. Thus, the centrally mediated neurobehavioral effects of fenamiphos could not be explained based on differential regional brain ChE inhibition alone. Despite the low level of brain ChE inhibition, some behavioral effects of fenamiphos were centrally mediated, and there was little regional specificity of ChE inhibition that could account for the behavioral changes observed.