Relationship between brain and plasma carbaryl levels and cholinesterase inhibition.

Carbaryl is a N-methylcarbamate pesticide and, like others in this class, is a reversible inhibitor of cholinesterase (ChE) enzymes. Although studied for many years, there is a surprising lack of information relating tissue levels of carbaryl with ChE activity in the same animals. The present studies were undertaken to describe the dose-response relationship about 40 min (approximate time of maximal ChE inhibition) after oral treatment in adult, post-natal day (PND) 17, and PND11 rats. Additionally, the time-course of plasma ChE activity and carbaryl levels in adult rats was determined after a 30 mg/kg dosage of carbaryl. The time-course study found that carbaryl levels could be detected in plasma 1 h after dosing, but rapidly decreased below the level of quantitation by the 2 h time point. In the dose-response studies, treatment-related increases in plasma and brain carbaryl levels were observed 40 min after dosing. Plasma levels of carbaryl increased linearly, while brain levels appeared to asymptote after 75 mg/kg carbaryl. Plasma and brain levels of carbaryl appeared to be linearly related with a slope close to 1 after various dosages (range: 1-75 mg/kg) of carbaryl at the 40 min time point. Finally, the dose-related relationship between tissue levels of carbaryl and ChE activity was described using a first order exponential decay function with an asymptote. The parameters of this function did not appear to differ between adult, PND17, or PND11 rats. This indicates that age-related differences in brain ChE inhibition by carbaryl are unlikely to be the result of greater tissue levels of the pesticide in PND11 animals. These are the first studies to report the relationship between brain and plasma tissue levels of carbaryl and ChE activity on an individual animal basis. The results of these experiments will be useful to extend physiologically-based pharmacokinetic models for carbaryl and their application in risk assessment.

Single fiber electromyographic jitter and detection of acute changes in neuromuscular function in young and adult rats.

INTRODUCTION: Exposure to irreversible cholinesterase (ChE)-inhibiting compounds, such as organophosphates may produce neuromuscular dysfunction. However, less is known about changes in neuromuscular transmission after treatment with reversible ChE-inhibitors. These studies adapted single fiber electromyography (SFEMG) techniques to quantify neuromuscular jitter in adult and juvenile rats after treatment with agents that alter cholinergic neurotransmission. METHODS: Anesthetized adult and juvenile rats were tested using stimulation SFEMG, recorded in the gastrocnemius muscle, after stimulation in the sciatic notch region. The influence of electrode placement, treatment with decamethonium (to disrupt neuromuscular transmission) or physostigmine (reversible ChE inhibitor), and the impact of varied stimulation frequency were quantified. RESULTS: No significant effects of needle placement or criterion amplitude were observed when calculating the mean consecutive difference (MCD). Treatment with decamethonium did not alter MCD values in adult or juvenile rats. However, decamethonium produced an increased blocking of muscle action potentials (MAP) in juveniles. Also, when stimulated at 9.09 Hz, both adult and juvenile animals had a greater decrease in MAP amplitude between the first and fourth responses (T(1)-T(4) decrement) after treatment with decamethonium. Prior to treatment with decamethonium, the T(1)-T(4) decrement averaged about 3 fold greater in juveniles than adults, and was larger at 3.03 and 9.09 Hz than with 0.91 Hz stimulation. Treatment with physostigmine resulted in at least 50% inhibition of muscle ChE activity, but produced minimal changes in the MCD values in adults or juveniles. Combined over treatments and stimulation frequencies, the median MCD for juveniles (11.6 micros) was less than that for of adults (18.8 micros). In juveniles, the median MCD increased from 9.3 micros to 13.9 micros as the stimulation rate was increased from 0.91 to 9.09 Hz. This stimulus-dependent change was more evident in juveniles than in adults. DISCUSSION: A technique was developed to record stimulation SFEMG and neuromuscular jitter, in vivo, in adult and juvenile rats. The method was sufficiently sensitive to detect age-related differences, potentially allowing developmental processes to be examined. Based on the literature and the current data, the technique appears to be more sensitive to prolonged inhibition of ChE enzymes than the reversible inhibition produced by physostigmine.

Depression of the photic after discharge of flash evoked potentials by physostigmine, carbaryl and propoxur, and the relationship to inhibition of brain cholinesterase.

The effects of N-methyl carbamate pesticides on the photic after discharge (PhAD) of flash evoked potentials (FEPs) and the relationship between inhibition of brain cholinesterase (ChE) activity and the PhAD were evaluated. FEPs were recorded in Long Evans rats treated with physostigmine (s.c.) 0, 0.05, 0.1, 0.2 or 0.3mg/kg (free base), in an ascorbic acid/saline vehicle, carbaryl (p.o.) 0, 1, 3, 10, 30, 50 or 75 mg/kg, or propoxur (p.o.) 0, 0.3, 3, 10, 20, 30, or 40 mg/kg in a corn oil vehicle. Physostigmine served as positive control based on literature data. Early (e.g. peak N(36)) and late FEP components (peak N(166) and PhAD) are related to the initial retino-geniculate afferent volley and higher cortical processing of visual information, respectively. Compared to controls, the PhAD duration decreased following treatment with 0.1 and 0.3mg/kg physostigmine, 7 5 mg/kg carbaryl or 30 mg/kg propoxur. Lesser changes were noted in FEP amplitudes or peak latencies. Treatment with 0.2 or 0.3 mg/kg physostigmine increased peak N(36) latency. Peak N(166) latency increased only following exposure to 40 mg/kg propoxur. None of the compounds altered peak N(36) or N(166) amplitudes. Hypothermia was observed at doses greater than 0.05 mg/kg physostigmine, at 30 or 50 mg/kg carbaryl, and after treatment with 10, 20 or 40 mg/kg propoxur. Inhibition of brain ChE activity occurred at dosages greater than 0.05 mg/kg physostigmine, 1mg/kg carbaryl, and 0.3 mg/kg propoxur. Linear regression analysis indicated that the decrease in PhAD duration correlated with decrease in brain ChE activity. The results indicate that at 30 min after treatment, inhibition of brain ChE activity did not affect cortical processing of the input from the retino-geniculate volley (evidenced by unaltered peak N(36) amplitude). However, the data suggest that disruption of cortical processing of visual signals related to FEP late components, as indicated by depression of the PhAD, was related to inhibition of brain ChE activity.

Inhalational exposure to carbonyl sulfide produces altered brainstem auditory and somatosensory-evoked potentials in Fischer 344N rats.

Carbonyl sulfide (COS), a chemical listed by the original Clean Air Act, was tested for neurotoxicity by a National Institute of Environmental Health Sciences/National Toxicology Program and U.S. Environmental Protection Agency collaborative investigation. Previous studies demonstrated that COS produced cortical and brainstem lesions and altered auditory neurophysiological responses to click stimuli. This paper reports the results of expanded neurophysiological examinations that were an integral part of the previously published experiments (Morgan et al., 2004, Toxicol. Appl. Pharmacol. 200, 131-145; Sills et al., 2004, Toxicol. Pathol. 32, 1-10). Fisher 334N rats were exposed to 0, 200, 300, or 400 ppm COS for 6 h/day, 5 days/week for 12 weeks, or to 0, 300, or 400 ppm COS for 2 weeks using whole-body inhalation chambers. After treatment, the animals were studied using neurophysiological tests to examine: peripheral nerve function, somatosensory-evoked potentials (SEPs) (tail/hindlimb and facial cortical regions), brainstem auditory-evoked responses (BAERs), and visual flash-evoked potentials (2-week study). Additionally, the animals exposed for 2 weeks were examined using a functional observational battery (FOB) and response modification audiometry (RMA). Peripheral nerve function was not altered for any exposure scenario. Likewise, amplitudes of SEPs recorded from the cerebellum were not altered by treatment with COS. In contrast, amplitudes and latencies of SEPs recorded from cortical areas were altered after 12-week exposure to 400 ppm COS. The SEP waveforms were changed to a greater extent after forelimb stimulation than tail stimulation in the 2-week study. The most consistent findings were decreased amplitudes of BAER peaks associated with brainstem regions after exposure to 400 ppm COS. Additional BAER peaks were affected after 12 weeks, compared to 2 weeks of treatment, indicating that additional regions of the brainstem were damaged with longer exposures. The changes in BAERs were observed in the absence of altered auditory responsiveness in FOB or RMA. This series of experiments demonstrates that COS produces changes in brainstem auditory and cortical somatosensory neurophysiological responses that correlate with previously described histopathological damage.

Thermoregulatory response to an organophosphate and carbamate insecticide mixture: testing the assumption of dose-additivity.

Most toxicity data are based on studies using single compounds. This study assessed if there is an interaction between mixtures of the anticholinesterase insecticides chlorpyrifos (CHP) and carbaryl (CAR) using hypothermia and cholinesterase (ChE) inhibition as toxicological endpoints. Core temperature (T(c)) was continuously monitored by radiotelemetry in adult Long-Evans rats administered CHP at doses ranging from 0 to 50mg/kg and CAR doses of 0-150 mg/kg. The temperature index (TI), an integration of the change in T(c) over a 12h period, was quantified. Effects of mixtures of CHP and CAR in 2:1 and 1:1 ratios on the TI were examined and the data analyzed using a statistical model designed to assess significant departures from additivity for chemical mixtures. CHP and CAR elicited a marked hypothermia and dose-related decrease in the TI. The TI response to a 2:1 ratio of CHP:CAR was significantly less than that predicted by additivity. The TI response to a 1:1 ratio of CHP and CAR was not significantly different from the predicted additivity. Plasma and brain ChE activity were measured 4h after dosing with CHP, CAR, and mixtures in separate groups of rats. There was a dose-additive interaction for the inhibition of brain ChE for the 2:1 ratio, but an antagonistic effect for the 1:1 ratio. The 2:1 and 1:1 mixtures had an antagonistic interaction on plasma ChE. Overall, the departures from additivity for the physiological (i.e., temperature) and biochemical (i.e., ChE inhibition) endpoints for the 2:1 and 1:1 mixtures studies did not coincide as expected. An interaction between CHP and CAR appears to depend on the ratio of compounds in the mixture as well as the biological endpoint.

Evaluation of sensory evoked potentials in Long Evans rats gestationally exposed to mercury (Hg0) vapor.

Mercury is known to alter neuronal function and has been shown to cross the placental barrier. These experiments were undertaken to examine if gestational exposure to mercury vapor (Hg(0)) would result in alterations in sensory neuronal function in adult offspring. Dams were exposed to 0 or 4 mg/m(3) Hg(0) for 2 h/day from gestational days 6-15. This exposure paradigm has been shown to approximate a maximal tolerated dose of Hg(0) for the dams. Between postnatal days 140-168, male and female offspring (one of each gender/dam) were examined using a battery of sensory evoked potentials. Peripheral nerve action potentials, nerve conduction velocity, somatosensory evoked responses (cortical and cerebellar), brainstem auditory evoked responses, pattern evoked potentials, and flash evoked potentials were quantified. Gestational exposure to 4 mg/m(3) Hg(0) did not significantly alter any of the evoked responses, although there was a suggestion of a decrease in compound nerve action potential (CNAP) amplitudes in male animals for the 3 mA stimulus condition. However, this possible change in CNAP amplitudes was not replicated in a second experiment. All evoked potentials exhibited predictable changes as the stimulus was modified. This shows conclusively that the evoked responses were under stimulus control, and that the study had sufficient statistical power to detect changes of these magnitudes. These results indicate that gestational exposure to 4 mg/m(3) Hg(0) did not result in changes in responses evoked from peripheral nerves, or the somatosensory, auditory, or visual modalities.

Disassociation of carbon disulfide-induced depression of flash-evoked potential peak N166 amplitude and norepinephrine levels.

Exposure to organic solvents frequently causes functional impairment of the central nervous system (CNS). One method to examine the effects of solvent exposure on visual function is flash-evoked potentials (FEPs). Greater knowledge of the role of various neurotransmitters in generating FEP peaks would be beneficial for understanding the basis of neurotoxicant-induced changes. FEP peak N166 is influenced by the psychological construct of arousal, which in turn is believed to be influenced by the function of neurons containing norepinephrine (NE). Because of its known effects on both NE and FEPs, we utilized carbon disulfide (CS2) as a means to examine the possible role of NE in modulating the amplitude of FEP peaks N36 and N166. Our hypothesis was that CS2-induced alterations in cortical NE levels would be correlated with changes in FEP peak N36 and N166 amplitudes. Adult male Long-Evans rats were implanted with electrodes over their visual cortex and allowed to recover. To develop peak N166, FEPs were recorded for two days prior to dosing. On the third day, FEPs were recorded prior to dosing, and one group of animals was sacrificed to serve as pretreatment controls. The remaining animals were dosed ip with 0 (corn oil vehicle; 2 ml/kg), 100, 200, or 400 mg/kg CS2. The treated animals were retested at 1, 4, 8, or 24 h after dosing, immediately sacrificed, and samples of the cortex, cerebellum, striatum, and brain stem were frozen for high performance liquid chromatography (HPLC) analysis of monoamine levels. Treatment with CS2 decreased peak N166 amplitude at 1 h, and peak N36 amplitude was depressed at 4 h, relative to the subject's pretreatment values. Peak latencies were increased, and colonic temperature was decreased by treatment with CS2. Exposure to CS2 depressed NE levels in the cortex, brain stem, and cerebellum 4 h after treatment. Conversely, at 4 h, levels of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid were increased in the brain stem and cerebellum, and levels of the DA metabolite homovanillic acid were increased in the brain stem. Levels of serotonin were unaffected by CS2 treatment. There was a slight increase in striatal levels of the serotonin metabolite 5-hydroxyindole acetic acid at all times after treatment with CS2. There was no apparent association between the decreases in NE levels and the reductions in amplitudes for peaks N36 and N166. The neurochemical mechanism for CS2-induced reductions in FEP peak amplitudes remains to be determined.