Philip J. Bushnell

Neurotoxicity of environmental chemicals and their mechanism of action

Despite a ban on their manufacture in 1977, polychlorinated biphenyls (PCBs) are still found in significant quantities in the environment. Developmental exposure to PCBs and related compounds has been reported to be neurotoxic in human and animals. Research in our laboratory has focused on the possible site(s) and mechanism(s) of PCB-induced developmental neurotoxicity. Recent experiments with rats found that developmental exposure to Aroclor-1254 (ARC) affects the acquisition of a lever press response and produces long-term changes in calcium buffering and protein kinase C (PKC) activity in the brain. In vitro studies in our laboratory have found that ARC increases [3H]phorbol ester binding, an indirect measure of PKC translocation, and inhibits calcium buffering in microsomes and mitochondria. Other experiments indicate that PCB congeners with chlorine substitutions at ortho- or low lateral substitutions are active in vitro, while non-ortho-substituted congeners are less active or inactive. Other research suggests that the lack of coplanarity of the PCB molecule is related to in vitro activity of PCB congeners. These studies indicate that in vivo developmental exposure to PCBs alters behavior and second messenger systems during adulthood, while in vitro experiments indicate that nervous system activity is related to ortho-substituted congeners that tend to be non-coplanar in configuration. Our results are consistent with the hypothesis that developmental neurotoxicity of ARC is due, in part, to the presence of ortho-substituted PCB congeners.

Behavioral Toxicity of Cholinesterase Inhibitors

Publisher Summary Acute exposure to cholinesterase inhibitors produces a well-defined spectrum of behavioral effects in mammals that are generally well explained by overstimulation of cholinergic receptors in the central and peripheral nervous systems. Compensations for these neurochemical effects complicate the situation when exposure is prolonged or repeated. Acute poisoning can lead to persistent disorders in the cognitive, motor, sensory, and affective domains. This chapter reviews the acute behavioral effects of organophosphates (OP) and carbamate (CM) pesticides in humans and animals. The classic behavioral signs and symptoms experienced by humans poisoned with these compounds are described, and ways in which animal models have been used to characterize them are illustrated to understand the mechanisms by which they are produced. Studies on persistent effects of exposure to cholinesterase inhibitors are also reviewed. The human epidemiological literature is examined for evidence that persistent behavioral effects of exposure can be measured in populations with histories of acute poisoning and/or chronic ongoing exposure. Behavioral data from animal models designed to characterize the behavioral changes caused by experimental treatments with cholinesteraseinhibiting pesticides is discussed, and similarities with the literature on humans occupationally exposed to these compounds are identified. Age-related differences in sensitivity to cholinesterase inhibitors are also reviewed. Data comparing the sensitivity of young and adult animals to the neurochemical and behavioral effects of these compounds is presented, and the factors that appear to mediate differences in response during development is discussed.

Modeling the toxicokinetics of inhaled toluene in rats: influence of physical activity and feeding status.

Toluene is found in petroleum-based fuels and used as a solvent in consumer products and industrial applications. The critical effects following inhalation exposure involve the brain and nervous system in both humans and experimental animals, whether exposure duration is acute or chronic. The goals of this physiologically based pharmacokinetic (PBPK) model development effort were twofold: (1) to evaluate and explain the influence of feeding status and activity level on toluene pharmacokinetics utilizing our own data from toluene-exposed Long Evans (LE) rats, and (2) to evaluate the ability of the model to simulate data from the published literature and explain differing toluene kinetics. Compartments in the model were lung, slowly and rapidly perfused tissue groups, fat, liver, gut, and brain; tissue transport was blood-flow limited and metabolism occurred in the liver. Chemical-specific parameters and initial organ volumes and blood flow rates were obtained from the literature. Sensitivity analysis revealed that the single most influential parameter for our experimental conditions was alveolar ventilation; other moderately influential parameters (depending upon concentration) included cardiac output, rate of metabolism, and blood flow to fat. Based on both literature review and sensitivity analysis, other parameters (e.g., partition coefficients and metabolic rate parameters) were either well defined (multiple consistent experimental results with low variability) or relatively noninfluential (e.g. organ volumes). Rats that were weight-maintained compared to free-fed rats in our studies could be modeled with a single set of parameters because feeding status did not have a significant impact on toluene pharmacokinetics. Heart rate (HR) measurements in rats performing a lever-pressing task indicated that the HR increased in proportion to task intensity. For rats acclimated to eating in the lab during the day, both sedentary rats and rats performing the lever-pressing task required different alveolar ventilation rates to successfully predict the data. Model evaluation using data from diverse sources together with statistical evaluation of the resulting fits revealed that the model appropriately predicted blood and brain toluene concentrations with some minor exceptions. These results (1) emphasize the importance of experimental conditions and physiological status in explaining differing kinetic data, and (2) demonstrate the need to consider simulation conditions when estimating internal dose metrics for toxicity studies in which kinetic data were not collected.

Behavioral and electrophysiological estimates of visual thresholds in awake rats treated with 3,3', 4,4', 5-pentachlorobiphenyl (PCB 126)

Visual thresholds for luminance increments were obtained behaviorally and electrophysiologically from rats exposed to a polychlorinated biphenyl (PCB) during development. Male Long-Evans rats exposed to 0, 0.25, or 1.0 microg/kg/day of 3,3,4,4, 5-pentachlorobiphenyl (PCB 126) through gestation and weaning were trained as adults to perform a signal detection task. Estimates of threshold were derived from psychometric functions for each animal relating the proportion of hits to signal intensity. Thresholds derived under three luminance conditions did not differ significantly among the PCB-treated groups. After behavioral testing was completed, flash-evoked potentials were recorded from dark-adapted awake animals. Peak amplitudes increased linearly over approximately 3 log units of intensity. Extrapolations to 0 amplitude along the linear portion of the amplitude-log intensity functions produced estimates of absolute threshold of -5.44 to -5.53 log cd/m(2)-s. Waveforms recorded from awake animals had a large late negative component that was absent in previously reported anesthetized preparations. Developmental exposure to PCB 126 had no significant effect on absolute threshold or peak amplitudes and latencies.

Delay-dependent impairment of reversal learning in rats treated with trimethyltin

Recent theories of hippocampal function focus on its role in the formation of associations in the temporal domain. A reversal learning paradigm based on leverpress automaintenance was developed to vary the CS-US relationship along two independent dimensions, one temporal and one not: CS(+)-US delay and the probability of reinforcement [P(RFT)] following the CS+. Eight male hooded Long-Evans rats were trained to reverse these automaintained discriminations repeatedly, until stable performance was achieved. The neurotoxicant trimethyltin (TMT) was used to induce lesions in the CNS, including the CA3-4 region of Ammons Horn in dorsal hippocampus. Following iv injection of 7 mg/kg TMT to half the rats, reversal learning was assessed under varying conditions of delay and P(RFT). After recovery from the acute effects of TMT (1-2 weeks), treated rats reversed normally when no delay separated the CS+ and US; with delays of 2 to 4 s, they reversed less completely within a session than did controls. Changing P(RFT) did not affect reversal learning in either group, but reduced response rates similarly in both groups. Morphological damage was quantified by measuring the length of the remaining pyramidal cell line in sections of dorsal hippocampus. The degree of behavioral impairment correlated significantly with hippocampal damage only at nonzero CS(+)-US delays. These results indicate that TMT impaired ability of rats to integrate temporal relationships between stimulus events, and are consistent with theories of hippocampal mediation of temporal associations.

Characterization of the effects of inhaled perchloroethylene on sustained attention in rats performing a visual signal detection task

The aliphatic hydrocarbon perchloroethylene (PCE) has been associated with neurobehavioral dysfunction including reduced attention in humans. The current study sought to assess the effects of inhaled PCE on sustained attention in rats performing a visual signal detection task (SDT). Due to its similarities in physiological effect to toluene and trichloroethylene (TCE), two other commonly used volatile organic compounds (VOCs) known to reduce attention in rats, we hypothesized (1) that acute inhalation of PCE (0, 500, 1000, 1500 ppm) would disrupt performance of the SDT in rats; (2) that impaired accuracy would result from changes in attention to the visual signal; and (3) that these acute effects would diminish upon repetition of exposure. PCE impaired performance of the sustained attention task as evidenced by reduced accuracy [P(correct): 500 to 1500 ppm], elevated response time [RT: 1000 and 1500 ppm] and reduced number of trials completed [1500 ppm]. These effects were concentration-related and either increased (RT and trial completions) or remained constant [P(correct)] across the 60-min test session. The PCE-induced reduction in accuracy was primarily due to an increase in false alarms, a pattern consistent with reduced attention to the signal. A repeat of the exposures resulted in smaller effects on these performance measures. Thus, like toluene and TCE, inhaled PCE acutely impaired sustained attention in rats, and its potency weakened upon repetition of the exposure.

The Role of Physical Activity and Feeding Schedule on the Kinetics of Inhaled and Oral Toluene in Rats

Published studies of the kinetics of toluene in rats have shown that its concentration in the blood rises during inhalation and falls after exposure stops; a similar uptake profile and longer persistence in blood typify the kinetics after oral exposure. Because rats in these studies are typically inactive during exposure, and behavioral tests of the acute effects of toluene require physical activity and altered feeding schedules, this study examined the role of physical activity and feeding status on the uptake of toluene given by the two routes. Two groups of adult male Long-Evans rats were conditioned to eat in the lab during the day. A group of “conditioned-active” (C-A) rats performed a lever-pressing task (LPT) for 1 h, either while inhaling toluene vapor (2000 ppm) or after a gavage dose (800 mg/kg toluene in corn oil). Another group of “conditioned-sedentary” (C-S) rats was dosed similarly but did not perform the LPT. A third group of “home cage” (HC) rats was not conditioned to eat during the day, but was maintained under typical laboratory conditions (eating at night in the home cage) before receiving toluene by gavage. In the conditioned rats, physical activity during inhalation exposure increased the concentrations of toluene in blood (from 35.8 ± 2.5 to 45.2 ± 3.2 mg/L after 60 min) and brain (from 73.4 ± 5.3 to 103.0 ± 3.8 mg/L after 60 min), but did not affect those concentrations after oral toluene. The time course of the uptake of toluene into blood and brain of HC rats followed that of published data. In contrast, toluene concentrations in the blood and brain of orally dosed conditioned rats fell rapidly compared to HC rats and published data (at 60 min after dosing, blood concentrations were: C-S rats, 17.2 ± 1.7 mg/L; HC rats, 69.4 ± 9.6 mg/L; and brain concentrations were: C-S rats, 30.9 ± 5.0 mg/L; HC rats, 96.6 ± 18.5 mg/L). These studies demonstrate the importance of physical activity for the uptake of inhaled toluene, and the importance of feeding conditions for the elimination of oral toluene.

Behavioral effects of subchronic inhalation of toluene in adult rats

Whereas the acute neurobehavioral effects of toluene are robust and well characterized, evidence for persistent effects of repeated exposure to this industrial solvent is less compelling. The present experiment sought to determine whether subchronic inhalation of toluene caused persistent behavioral changes in rats. Adult male Long-Evans rats inhaled toluene vapor (0, 10, 100, or 1000 ppm) for 6h/day, 5 days/week for 13 weeks and were evaluated on a series of behavioral tests beginning 3 days after the end of exposure. Toluene delayed appetitively-motivated acquisition of a lever-press response, but did not affect motor activity, anxiety-related behavior in the elevated plus maze, trace fear conditioning, acquisition of an appetitively-motivated visual discrimination, or performance of a visual signal detection task. Challenges with acute inhalation of toluene vapor (1200-2400 ppm for 1 h) and injections of quinpirole (0.01-0.03 mg/kg) and raclopride (0.03-0.10 mg/kg) revealed no toluene-induced latent impairments in visual signal detection. These results are consistent with a pattern of subtle and inconsistent long-term effects of daily exposure to toluene vapor, in contrast to robust and reliable effects of acute inhalation of the solvent.

Extrapolating the Acute Behavioral Effects of Toluene from 1- to 24-h Exposures in Rats: Roles of Dose Metric and Metabolic and Behavioral Tolerance

Recent research on the acute effects of volatile organic compounds suggests that extrapolation from short (∼1 h) to long durations (up to 4 h) may be improved by using estimates of brain toluene concentration (Br[Tol]) instead of cumulative inhaled dose (C × t) as a metric of dose. This study compared predictions of these two dose metrics on the acute behavioral effects of inhaled toluene in rats during exposures up to 24 h in duration. We first evaluated estimates of Br[Tol] with a physiologically based toxicokinetic (PBTK) model for rats intermittently performing an operant task while inhaling toluene for up to 24 h. Exposure longer than 6 h induced P450-mediated metabolism of toluene. Adjusting the corresponding parameters of the PBTK model improved agreement between estimated and observed values of Br[Tol] in the 24-h exposure scenario. Rats were trained to perform a visual signal detection task and were then tested while inhaling toluene (0, 1125, and 1450 ppm for 24 h and 1660 ppm for 21 h). Tests occurred at times yielding equivalent C × t products but different estimates of Br[Tol], and also at 1 and 6 h afterexposure. Effects of toluene were better predicted by Br[Tol] than by C × t. However, even using Br[Tol] as the dose metric (after accounting for metabolic induction), acute dose-effect functions during 24-h exposures were shifted to the right relative to 1-h exposures, indicating that a dynamic behavioral tolerance also developed during prolonged exposure to toluene.

Acute inhalation of 2,2,4-trimethylpentane alters visual evoked potentials and signal detection behavior in rats☆

The volatile organic compound 2,2,4-trimethylpentane (TMP, isooctane) is a constituent of gasoline for which the current health effects data are insufficient to permit the US Environmental Protection Agency to conduct a risk assessment. The potential neurological impairment from acute inhalation exposure to TMP was evaluated in adult male Long-Evans rats using both electrophysiological and behavioral assessments. Visual evoked potentials (VEPs) were recorded from rats viewing modulated visual patterns (0.16 cycles per degree visual angle (cpd), 60% contrast, 4.55Hz appear/disappear). Rats (n=7-10/dose) were exposed to TMP vapors in concentrations of 0, 500, or 1000 ppm for 60-min. A VEP was recorded before exposure and at 10 min intervals during exposure and also for 60 min after exposure terminated. The spectral amplitude of the frequency-double component (F2) was significantly reduced after exposure to TMP. In behavioral assessments, rats (n=14) performed an appetitively motivated visual signal detection task while breathing 0, 500, 1500, 1000, 2000, or 2500 ppm TMP for 62 min. Slight reductions in accuracy of performance were observed at the 2500 ppm concentration. Concentrations of TMP in the brain were estimated using a physiologically based pharmacokinetic (PBPK) model to be less than 0.2mM after 62 min at 2500 ppm. Together these data demonstrate that TMP, like other volatile organic substances, impairs neurological function during acute inhalation exposure and that the small magnitude of the observed effects is consistent with the low concentrations of this hydrocarbon that were estimated to reach the CNS.