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People are often exposed to complex mixtures of environmental chemicals such as gasoline, tobacco smoke, water contaminants, or food additives. We developed an approach that applies chemical lumping methods to complex mixtures, in this case gasoline, based on biologically relevant parameters used in physiologically based pharmacokinetic (PBPK) modeling. Inhalation exposures were performed with rats to evaluate the performance of our PBPK model and chemical lumping method. There were 109 chemicals identified and quantified in the vapor in the chamber. The time-course toxicokinetic profiles of 10 target chemicals were also determined from blood samples collected during and following the in vivo experiments. A general PBPK model was used to compare the experimental data to the simulated values of blood concentration for 10 target chemicals with various numbers of lumps, iteratively increasing from 0 to 99. Large reductions in simulation error were gained by incorporating enzymatic chemical interactions, in comparison to simulating the individual chemicals separately. The error was further reduced by lumping the 99 nontarget chemicals. The same biologically based lumping approach can be used to simplify any complex mixture with tens, hundreds, or thousands of constituents.

Superior cognitive functions have allowed the human species to proliferate in a world of incredible biological diversity. Threats to these essential capacities cannot be ignored, and a strategy is needed to evaluate the hazard posed by exposure to chemical and other agents. Because people exposed to chemicals often complain about confusion and forgetfulness, it is commonly thought that cognitive functions should be sensitive indicators of adverse consequences of chemical exposure. For these reasons, complex tests of cognitive function have been developed and deployed in experimental animal laboratories for decades. However, the results of these tests are rarely used as points of departure for chemical risk assessments. Due to their high cost in time, animals, and equipment, the efficacy and utility of these tests need to be evaluated in relation to cheaper and faster whole-animal screening methods. This review examines evidence for the assertions that cognitive functions represent uniquely sensitive indicators of chemical exposure, and that animal models of these functions are necessary to detect and quantify the neurotoxicity of chemicals. Studies conducted since the early 1980s to compare these approaches to assess the neurotoxicity of chemicals are reviewed for both adult and perinatal exposures in experimental rodents. Forty-one studies of 35 chemicals were found that directly compared acute effects using complex tests (i.e., tests that require training animals) with acute effects using screening tests (i.e., tests that do not require training animals) in adult rodents. Complex tests detected effects of three substances (bitertanol, iso-amyl nitrite, and Pfiesteria toxin) that had no effect on screening tests; for an additional five chemicals (carbaryl, deltamethrin, methyl mercury, tetraethyl tin, and Isopar-C), complex tests identified effects at lower doses than did screening tests. Fewer comparable cases were found for developmental exposures: screening and complex tests were found to be equivalent for trimethyltin, n-propylthiouracil (PTU), and elemental mercury. Analysis of two studies yielded an inconclusive case for lead. Evidence for the insufficiency of screening tests was found for PCBs and inhaled ethanol, though it is not clear that the measured effects of these chemicals reflected cognitive deficits per se. Whether these benefits are worth the additional time and expense of conducting complex tests is a matter for discussion in the research and risk management communities.

The data generated by current high-throughput assays for chemical toxicity require information to link effects at molecular targets to adverse outcomes in whole animals. In addition, more efficient methods for testing volatile chemicals are needed. Here we begin to address these issues by determining the utility of measuring behavioral responses of Drosophila melanogaster to airborne volatile organic compounds (VOCs) as a potential model system for discovering adverse outcome pathways and as a method to test for toxicity. In these experiments, we measured motor activity in male and female flies to determine concentration-effect functions for three VOCs that differ in their mode of action: toluene, a narcotic; acrolein, an irritant; and vinyl chloride, a hepatocarcinogen. These experiments were conducted in Flyland, an outbred population of flies derived from 40 lines of the Drosophila Genetics Reference Panel (DGRP) (Mackay et al., 2012), in preparation for subsequent experiments with individual lines of the DGRP. Systematic, concentration-related changes in activity were observed with toluene, but not with acrolein; high concentrations of vinyl chloride reduced activity by a small amount. Despite higher activity levels in males than in females under control conditions, the sexes were equally sensitive to toluene. Transient increases in activity at the onset and offset of exposure to toluene and vinyl chloride suggested that the flies detected changes in air quality at concentrations that did not persistently suppress activity. The effects and potency of toluene are consistent with those observed in rodents. The lack of clear concentration-related changes in response to acrolein and vinyl chloride shows limitations of this method is for screening toxicity attributed to VOCs. This abstract does not reflect U.S. EPA policy.

Increased use of ethanol blends in gasoline suggests a need to assess the potential public health risks of exposure to these fuels. Ethanol consumed during pregnancy is a teratogen. However, little is known about the potential developmental neurotoxicity of ethanol delivered by inhalation, the most likely route of exposure from gasoline–ethanol fuel blends. We evaluated the potential cognitive consequences of ethanol inhalation by exposing pregnant Long Evans rats to clean air or ethanol vapor from gestational days 9–20, a critical period of neuronal development. Concentrations of inhaled ethanol (5000, 10,000, or 21,000 ppm for 6.5 h/day) produced modeled peak blood ethanol concentrations (BECs) in exposed dams of 2.3, 6.8, and 192 mg/dL, respectively. In offspring, no dose-related impairments were observed on spatial learning or working memory in the Morris water maze or in operant delayed match-to-position tests. Two measures showed significant effects in female offspring at all ethanol doses: 1) impaired cue learning after trace fear conditioning, and 2) an absence of bias for the correct quadrant after place training during a reference memory probe in the Morris water maze. In choice reaction time tests, male offspring (females were not tested) from the 5000 and 10,000 ppm groups showed a transient increase in decision times. Also, male offspring from the 21,000 ppm group made more anticipatory responses during a preparatory hold period, suggesting a deficit in response inhibition. The increase in anticipatory responding during the choice reaction time test shows that inhaled ethanol yielding a peak BEC of ~ 200 mg/dL can produce lasting effects in the offspring. The lack of a dose-related decrement in the effects observed in females on cue learning and a reference memory probe may reflect confounding influences in the exposed offspring possibly related to maternal care or altered anxiety levels in females. The surprising lack of more pervasive cognitive deficits, as reported by others at BECs in the 200 mg/dL range, may reflect route-dependent differences in the kinetics of ethanol. These data show that response inhibition was impaired in the offspring of pregnant rats that inhaled ethanol at concentrations at least 5 orders of magnitude higher than concentrations observed during normal automotive transport and fueling operations, which rarely exceed 100 ppb.

Interpretation and use of data from high-throughput assays for chemical toxicity require links between effects at molecular targets and adverse outcomes in whole animals. The well-characterized genome of Drosophila melanogaster provides a potential model system by which phenotypic responses to chemicals can be mapped to genes associated with those responses, which may in turn suggest adverse outcome pathways associated with those genes. To determine the utility of this approach, we used the Drosophila Genetics Reference Panel (DGRP), a collection of ∼200 homozygous lines of fruit flies whose genomes have been sequenced. We quantified toluene-induced suppression of motor activity in 123 lines of these flies during exposure to toluene, a volatile organic compound known to induce narcosis in mammals via its effects on neuronal ion channels. We then applied genome-wide association analyses on this effect of toluene using the DGRP web portal (http://dgrp2.gnets.ncsu.edu), which identified polymorphisms in candidate genes associated with the variation in response to toluene exposure. We tested ∼2 million variants and found 82 polymorphisms located in or near 66 candidate genes that were associated with phenotypic variation for sensitivity to toluene at P

Studies of humans chronically exposed to volatile organic solvents have reported impaired visual functions, including low contrast sensitivity and reduced color discrimination. These reports, however, lacked confirmation from controlled laboratory experiments. To address this question experimentally, we examined visual function by recording visual evoked potentials (VEP) and/or electroretinograms (ERG) from four sets of rats exposed repeatedly to toluene. In addition, eyes of the rats were examined with an ophthalmoscope and some of the retinal tissues were evaluated for rod and M-cone photoreceptor immunohistochemistry. The first study examined rats following exposure to 0, 10, 100 or 1000 ppm toluene by inhalation (6 hr/d, 5 d/wk) for 13 weeks. One week after the termination of exposure, the rats were implanted with chronically indwelling electrodes and the following week pattern-elicited VEPs were recorded. VEP amplitudes were not significantly changed by toluene exposure. Four to five weeks after completion of exposure, rats were dark-adapted overnight, anesthetized, and several sets of electroretinograms (ERG) were recorded. In dark-adapted ERGs recorded over a 5-log (cd-s/m 2 ) range of flash luminance, b-wave amplitudes were significantly reduced at high stimulus luminance values in rats previously exposed to 1000 ppm toluene. A second set of rats, exposed concurrently with the first set, was tested approximately one year after the termination of 13 weeks of exposure to toluene. Again, dark-adapted ERG b-wave amplitudes were reduced at high stimulus luminance values in rats previously exposed to 1000 ppm toluene. A third set of rats was exposed to the same concentrations of toluene for only 4 weeks, and a fourth set of rats exposed to 0 or 1000 ppm toluene for 4 weeks were tested approximately 1 year after the completion of exposure. No statistically significant reductions of ERG b-wave amplitude were observed in either set of rats exposed for 4 weeks. No significant changes were observed in ERG a-wave amplitude or latency, b-wave latency, UV- or green-flicker ERGs, or in photopic flash ERGs. There were no changes in the density of rod or M-cone photoreceptors. The ERG b-wave reflects the firing patterns of on-bipolar cells. The reductions of b-wave amplitude after 13 weeks of exposure and persisting for 1 year suggest that alterations may have occurred in the inner nuclear layer of the retina, where the bipolar cells reside, or the outer or inner plexiform layers where the bipolar cells make synaptic connections. These data provide experimental evidence that repeated exposure to toluene may lead to subtle persistent changes in visual function. The fact that toluene affected ERGs, but not VEPs, suggests that elements in the rat retina may be more sensitive to organic solvent exposure than the rat visual cortex.

A primary public health concern regarding environmental chemicals is the potential for persistent effects from long-term exposure, and approaches to estimate these effects from short-term exposures are needed. Toluene, a ubiquitous air pollutant, exerts well-documented acute and persistent CNS-mediated effects from a variety of exposure scenarios, and so provides a useful case for determining whether its persistent effects can be predicted from its acute effects on the CNS. We recently reported that acute inhalation of toluene produced transcriptional effects in rat brain 18 h following a single, acute 6-h exposure to toluene. The goal of the present study was to determine whether these acute effects are also evident after long-term (sub-chronic) exposure to toluene, and thereby provide a mechanistic basis for predicting its persistent effects from short-term exposures. Male Long–Evans rats were exposed to toluene via inhalation (0, 10, 100, 1000 ppm, n = 5/dose), 6 h/day for 64 days, excluding weekends. The day following the final exposure, total mRNA was extracted from the cerebral cortex and striatum, and gene expression evaluated using Affymetrix arrays. Principal component analysis using all samples showed a clear discrimination of tissues, with striatum having more within-group variance than cortex. Differentially-expressed genes (DEGs) whose expression was altered by toluene were identified in each tissue by ANOVA followed by mapping to pathways. Analysis of striatum revealed 22, 57, and 94 significant DEGs for the 10 ppm, 100 ppm, and 1000 ppm doses, respectively, far fewer than the 3352 DEGS previously observed after acute exposure. In addition, the direction of change in the 57 DEGs common to both exposures differed between acute and sub-chronic exposure scenarios. Thus, relative to acute toluene exposure, sub-chronic exposure yielded both quantitative and qualitative differences in transcriptional response. Based on the current data, long-term gene expression changes after toluene inhalation cannot be readily predicted by acute responses.

Biofuel blends of 10% ethanol (EtOH) and gasoline are common in the USA, and higher EtOH concentrations are being considered (15-85%). Currently, no physiologically-based pharmacokinetic (PBPK) models are available to describe the kinetics of EtOH-based biofuels. PBPK models were developed to describe life-stage differences in the kinetics of EtOH alone in adult, pregnant, and neonatal rats for inhalation, oral, and intravenous routes of exposure, using data available in the open literature. Whereas ample data exist from gavage and intravenous routes of exposure, kinetic data from inhalation exposures are limited, particularly at concentrations producing blood and target tissue concentrations associated with developmental neurotoxicity. Compared to available data, the three models reported in this paper accurately predicted the kinetics of EtOH, including the absorption, peak concentration, and clearance across multiple datasets. In general, model predictions for adult and pregnant animals matched inhalation and intravenous datasets better than gavage data. The adult model was initially better able to predict the time-course of blood concentrations than was the neonatal model. However, after accounting for age-related changes in gastric uptake using the calibrated neonate model, simulations consistently reproduced the early kinetic behavior in blood. This work provides comprehensive multi-route life-stage models of EtOH pharmacokinetics and represents a first step in development of models for use with gasoline-EtOH blends, with additional potential applicability in investigation of the pharmacokinetics of EtOH abuse, addiction, and toxicity.

Reports of behavioral effects of repeated inhalation of toluene in rats have yielded inconsistent findings. A recent study from this laboratory (Beasley et al., 2010) observed that after 13 weeks of inhaled toluene ("subchronic" exposure scenario), rats showed mild but persistent changes in behavior, primarily involving acquisition of an autoshaped lever-press response. The present experiment sought to systematically replicate these findings, using a 4-week "sub-acute" exposure scenario. Adult male Long-Evans rats inhaled toluene vapor (0, 10, 100, or 1000 ppm) for 6h/day, 5 days/week for 4 weeks. As in the subchronic study, toluene had no effect on motor activity, anxiety-related behavior in the elevated plus-maze, or acquisition of the visual discrimination. However, sub-acute toluene did not affect appetitively-motivated acquisition of the lever-press response, but did reduce accuracy of signal detection at the end of training. Analysis of the deficit in accuracy in the 1000 ppm group by means of manipulations of different task parameters suggested a greater influence of attentional impairment than visual or motor dysfunction as a source for the deficit. These results confirm a pattern of subtle and inconsistent long-term effects of repeated daily exposure to concentrations of toluene vapor of 1000 ppm and below, in contrast to robust and reliable effects of acute inhalation of the solvent at concentrations above 1000 ppm.

Attention can be readily measured in experimental animal models. Animal models of attention have been used to better understand the neural systems involved in attention, how attention is impaired, and how therapeutic treatments can ameliorate attentional deficits. This review focuses on the ways in which animal models are used to better understand the neuronal mechanism of attention and how to develop new therapeutic treatments for attentional impairment. Several behavioral test methods have been developed for experimental animal studies of attention, including a 5-choice serial reaction time task (5-CSRTT), a signal detection task (SDT), and a novel object recognition (NOR) test. These tasks can be used together with genetic, lesion, pharmacological and behavioral models of attentional impairment to test the efficacy of novel therapeutic treatments. The most prominent genetic model is the spontaneously hypertensive rat (SHR). Well-characterized lesion models include frontal cortical or hippocampal lesions. Pharmacological models include challenge with the NMDA glutamate antagonist dizocilpine (MK-801), the nicotinic cholinergic antagonist mecamylamine and the muscarinic cholinergic antagonist scopolamine. Behavioral models include distracting stimuli and attenuated target stimuli. Important validation of these behavioral tests and models of attentional impairments for developing effective treatments for attentional dysfunction is the fact that stimulant treatments effective for attention deficit hyperactivity disorder (ADHD), such as methylphenidate (Ritalin®), are effective in the experimental animal models. Newer lines of treatment including nicotinic agonists, α4β2 nicotinic receptor desensitizers, and histamine H₃ antagonists, have also been found to be effective in improving attention in these animal models. Good carryover has also been seen for the attentional improvement caused by nicotine in experimental animal models and in human populations. Animal models of attention can be effectively used for the development of new treatments of attentional impairment in ADHD and other syndromes in which have attentional impairments occur, such as Alzheimer's disease and schizophrenia.

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 solvent exposures may contribute to automobile accidents because they increase reaction time and decrease attention, in addition to impairing other behaviors. These effects resemble those of ethanol consumption, both with respect to behavioral effects and neurological mechanisms. These observations, along with the extensive data on the relationship between ethanol consumption and fatal automobile accidents, suggested a way to estimate the probability of fatal automobile accidents from solvent inhalation. The problem can be approached using the logic of the algebraic transitive postulate of equality: if A=B and B=C, then A=C. We first calculated a function describing the internal doses of solvent vapors that cause the same magnitude of behavioral impairment as ingestion of ethanol (A=B). Next, we fit a function to data from the literature describing the probability of fatal car crashes for a given internal dose of ethanol (B=C). Finally, we used these two functions to generate a third function to estimate the probability of a fatal car crash for any internal dose of organic solvent vapor (A=C). This latter function showed quantitatively (1) that the likelihood of a fatal car crash is increased by acute exposure to organic solvent vapors at concentrations less than 1.0 ppm, and (2) that this likelihood is similar in magnitude to the probability of developing leukemia from exposure to benzene. This approach could also be applied to other potentially adverse consequences of acute exposure to solvents (e.g., nonfatal car crashes, property damage, and workplace accidents), if appropriate data were available.

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 6 h/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.

The National Research Council (NRC) of the National Academies of Science recently published a report of its vision of toxicity testing in the 21st century. The report proposes that the current toxicity testing paradigm that depends upon whole-animal tests be replaced with a strategy based upon in vitro tests, in silico models and evaluations of toxicity at the human population level. These goals are intended to set in motion changes that will transform risk assessment into a process in which adverse effects on public health are predicted by quantitative structure–activity relationship (QSAR) models and data from suites of high-throughput in vitro tests. The potential roles for whole-animal testing in this futuristic vision are both various and undefined. A symposium was convened at the annual meeting of the Neurobehavioral Teratology Society in Rio Grande, Puerto Rico in June, 2009 to discuss the potential challenges and opportunities for behavioral scientists in developing and/or altering this strategy toward the ultimate goal of protecting public health from hazardous chemicals. R. Kavlock described the NRC vision, introduced the concept of the ‘toxicity pathway’ (a central guiding principle of the NRC vision), and described the current status of an initial implementation this approach with the EPA's ToxCast® program. K. Crofton described a pathway based upon disruption of thyroid hormone metabolism during development, including agents, targets, and outcomes linked by this mode of action. P. Bushnell proposed a pathway linking the neural targets and cellular to behavioral effects of acute exposure to organic solvents, whose predictive power is limited by our incomplete understanding of the complex CNS circuitry that mediates the behavioral responses to solvents. B. Weiss cautioned the audience regarding a pathway approach to toxicity testing, using the example of the developmental toxicity of phthalates, whose effects on mammalian sexual differentiation would be difficult to identify based on screening tests in vitro . Finally, D. Rice raised concerns regarding the use of data derived from toxicity screening tests to human health risk assessments. Discussion centered around opportunities and challenges for behavioral toxicologists regarding this impending paradigm shift. Opportunities include: identifying and characterizing toxicity pathways; informing the conditions and limits of extrapolation; addressing issues of susceptibility and variability; providing reality-checks on selected positives and negatives from screens; and performing targeted testing and dose-response assessments of chemicals flagged during screening. Challenges include: predicting behavior using models of complex neurobiological pathways; standardizing study designs and dependent variables to facilitate creation of databases; and managing the cost and efficiency of behavioral assessments. Thus, while progress is being made in approaching the vision of 21st century toxicology, we remain a long way from replacing whole-animal tests; indeed, some animal testing will be essential for the foreseeable future at least. Initial advances will likely provide better prioritization tools so that animal resources are used more efficiently and effectively.

We applied a neural network model of classical conditioning proposed by Schmajuk et al. (1996) to visual signal detection and discrimination tasks designed to assess sustained attention in rats (Bushnell, 1999). We used a sign-tracking description of signal detection behavior by assuming that rats approach the location of the lever that they expect will be rewarded. We also assumed that although the visual signals contribute to set the occasion for the approach response to be generated, they do not become strongly associated with reward. The model accurately described Bushnell's (1999) results showing an increased proportion of hits with increasing signal intensity, decreased proportion of hits with increasing trial rate, and lower accuracy in a discrimination task compared to a detection task. In addition, observation of the behavior of rats performing the task confirmed assumptions and predictions of the model: (a) rats learn to approach the location of the lever they expect to be rewarded; (b) during the pre-signal interval, rats approach the location of the blank lever because it matches the intensity of the light they experience during that interval; and (c) the rats' behavior is directed to the location of the levers and not towards the light, which acts only as an "occasion setter" for the lever to be selected and pressed.

The literature concerning the neurobehavioral and neurophysiological effects of long-term exposure to perchloroethylene (PERC) in humans was meta-analyzed to provide a quantitative review and synthesis in the form of dose-effect curves. The useable database from this literature comprised studies reporting effects of long-term exposure to PERC, effects that included slowed reaction times, cognitive deficits, impaired color vision, and reduced visual contrast sensitivity. For the meta-analyses, dose was defined as the product of the concentration inhaled PERC and the duration of exposure, expressed in unites of ppm-h/1000 (for numerical convenience). Dose-related results were highly variable across studies. Reports involving low exposure concentrations characteristic of nonoccupational exposures consistently produced effects of a magnitude that were comparable to those reported for higher concentration occupational studies. If this finding is reliable and general, studies of occupationally exposed persons may underestimate the magnitude of effects of PERC and other chemicals in the total population. Given the limited scope of the available data for PERC and its methodological and reporting problems (small sample sizes, testers were not blind to the subjects' exposure conditions, and the timing and location of testing were insufficiently documented), it seems important to test this conclusion with a well-documented study of two groups (occupational and nonoccupational exposure) in which subjects are evaluated in randomized order, using the same procedures and with the testers kept blind to the status of the subjects.

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.

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.

Acute exposure to toluene was assessed in two experiments to determine the relationship between brain toluene concentration and changes in neurophysiological function. The concentration of toluene in brain tissue at the time of assessment was estimated using a physiologically based pharmacokinetic model. Brain neurophysiological function was measured using pattern-elicited visual evoked potentials (VEP) recorded from electrodes located over visual cortex of adult male Long-Evans rats. In the first experiment, VEPs were recorded before and during exposure to control air or toluene at 1000 ppm for 4 h, 2000 ppm for 2 h, 3000 ppm for 1.3 h, or 4000 ppm for 1 h. In the second experiment, VEPs were recorded during and after exposure to clean air or 3000 or 4000 ppm toluene. In both experiments, the response amplitude of the major spectral component of the VEP (F2 at twice the stimulus rate in steady-state responses) was reduced by toluene. A logistic function was fit to baseline-adjusted F2 amplitudes from the first experiment that described a significant relationship between brain toluene concentration and VEP amplitude deficits. In the second experiment, 3000 ppm caused equivalent VEP deficits during or after exposure as a function of estimated brain concentration, but 4000 ppm showed a rapid partial adaptation to the acute effects of toluene after exposure. In general, however, the neurophysiological deficits caused by acute toluene exposure could be described by estimates of the momentary concentration of toluene in the brain at the time of VEP evaluation.

Knowledge of the appropriate metric of dose for a toxic chemical facilitates quantitative extrapolation of toxicity observed in the laboratory to the risk of adverse effects in the human population. Here, we utilize a physiologically based toxicokinetic (PBTK) model for toluene, a common volatile organic compound (VOC), to illustrate that its acute behavioral effects in rats can be quantitatively predicted on the basis of its concentration in the brain. Rats previously trained to perform a visual signal detection task for food reward performed the task while inhaling toluene (0, 1200, 1600, 2000, and 2400 ppm in different test sessions). Accuracy and speed of responding were both decreased by toluene; the magnitude of these effects increased with increasing concentration of the vapor and with increasing duration of exposure. Converting the exposure conditions to brain toluene concentration using the PBTK model yielded a family of overlapping curves for each end point, illustrating that the effects of toluene can be described quantitatively by its internal dose at the time of behavioral assessment. No other dose metric, including inhaled toluene concentration, duration of exposure, the area under the curve of either exposure (ppm h), or modeled brain toluene concentration (mg-h/kg), provided unambiguous predictions of effect. Thus, the acute behavioral effects of toluene (and of other VOCs with a similar mode of action) can be predicted for complex exposure scenarios by simulations that estimate the concentration of the VOC in the brain from the exposure scenario.

Synthesizing information about the acute neurotoxicity of organic solvents into predictive relationships between exposure and effect in humans is difficult because (1) data are usually derived from experimental animals whose sensitivity to the chemical relative to humans is unknown; (2) the specific endpoints measured in laboratory animals seldom translate into effects of concern in humans; and (3) the mode of action of the chemical is rarely understood. We sought to develop approaches to estimate the hazard and cost of exposure to organic solvents, focusing on the acute behavioral effects of toluene in rats and humans. Available published data include studies of shock avoidance behavior in rats and choice reaction time in humans. A meta-analysis of these data suggested that a 10% change in rat avoidance behavior occurs at a blood concentration of toluene 25 times higher than the concentration at which a 10% change in human choice reaction time occurs. In contrast, our in vitro studies of nicotinic acetylcholine receptors indicated that human and rat receptors do not differ in sensitivity to toluene. Analysis of other dose-response relationships for visual and cognitive functions in rats suggests that the apparent difference between rats and humans may be driven by the specific endpoints measured in the two species rather than by inherent differences in sensitivity to toluene. We also explored the hypothesis that dose-equivalence relationships may be used to compare the societal costs of two chemicals. For example, ethanol-induced changes in choice reaction time, for which societal costs are estimatable, may be used as a benchmark effect for estimating the monetary benefits of controlling exposure to organic solvents. This dose-equivalence method is applicable for solvents because this set of data fulfills three important assumptions about equivalence relationships based on a single effect: (1) a common dose metric (concentration of the chemical in the brain); (2) a common effect to provide a linking variable (choice reaction time); and (3) a common mode of action (interference with neuronal ion channel function).

Previous work showed that trichloroethylene (TCE) impairs sustained attention as evidenced by a reduction in accuracy and elevation of response latencies in rats trained to perform a visual signal detection task (SDT). This work also showed that these effects abate during repeated exposures if rats inhale TCE while performing the SDT. The present experiment sought to determine whether toluene, another commonly-used solvent, would induce tolerance similarly if inhaled repeatedly during SDT testing. Sixteen male, Long-Evans rats were trained to perform the SDT. Upon completion of training, rats were divided into 2 groups. In Phase I, concentration-effect functions were determined for toluene (0, 1200, 1600, 2000, 2400 ppm) in both groups. Toluene reduced the proportion of correct responses [P(correct)], and increased response time (RT) and response failures. In Phase II, Group-Tol inhaled 1600 ppm toluene while Group-Air inhaled clean air during 11 daily SDT sessions. In Group-Tol the effect of toluene on P(correct) abated after 3 days, while RT remained elevated for the duration of the repeated exposures. In Phase III, toluene concentration-effect functions were re-determined for both groups. Group-Air remained impaired on all test measures, whereas for Group-Tol, toluene did not reduce P(correct), but continued to increase RT. These data confirm our previous hypothesis that animals can develop tolerance to chemical exposures that impair appetitively-motivated behaviors if that impairment leads to loss of reinforcement.

The effects of exposure to volatile organic compounds (VOCs), which are of concern to the EPA, are poorly understood, in part because of insufficient characterization of how human exposure duration impacts VOC effects. Two inhalation studies with multiple endpoints, one acute and one subchronic, were conducted to seek effects of the VOC, toluene, in rats and to compare the effects between acute and subchronic exposures. Adult male Long-Evans rats were exposed to toluene vapor (n=6 per group) at a concentration of 0 or 1019 ± 14 ppm for 6h in the acute study and at 0 ± 0, 10 ± 1.4, 97 ± 7, or 995 ± 43 ppm for 6h/d, 5d/week for 13 weeks in the subchronic study. For the acute study, brains were dissected on ice within 30 min of the end of exposure, while for the subchronic study, brains were dissected 18 h after the last exposure. Frontal cortex, hippocampus, cerebellum, and striatum were assayed for a variety of oxidative stress (OS) parameters including total aconitase (TA), protein carbonyls, glutathione peroxidase (GPX), glutathione reductase (GRD), glutathione transferase (GST), γ-glutamylcysteine synthetase (GCS), superoxide dismutase (SOD), total antioxidants (TAS), NADPH quinone oxidoreductase-1 (NQO1), and NADH ubiquinone reductase (UBIQ-RD) activities using commercially available kits. Following acute exposure, UBIQ-RD, GCS and GRD were increased significantly only in the cerebellum, while TAS was increased in frontal cortex. On the other hand, subchronic exposure affected several OS markers including increases in NQO1 and UBIQ-RD. The effect of subchronic toluene exposure on SOD and TAS was greater in the striatum than in the other brain regions. TA activity (involved in maintaining iron homeostasis and an indicator of DNA damage) was inhibited in striatum and cerebellum, increased in hippocampus, and unchanged in frontal cortex. Protein carbonyls increased significantly in both the frontal cortex and cerebellum. In general, the results showed that acute exposure to toluene affected OS parameters to a lesser extent than did subchronic exposure. These results suggest that toluene exposure induces OS in the brain and this may be a component of an adverse outcome pathway for some of the neurotoxic effects reported following toluene exposure.

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.

To provide useful alternatives to in vivo animal studies, in vitro assays for dose-response assessments of xenobiotic chemicals must use concentrations in media and target tissues that are within biologically-plausible limits. Determining these concentrations is a complex matter, which can be facilitated by applying physiologically-based pharmacokinetic (PBPK) models in an in vitro to in vivo extrapolation (IVIVE) paradigm. We used ethanol (EtOH), a ubiquitous chemical with defined metrics for in vivo and in vitro embryotoxicity, as a model chemical to evaluate this paradigm. A published series of life-stage PBPK models for rats was extended to mice, yielding simulations that adequately predicted in vivo blood EtOH concentrations (BECs) from oral, intraperitoneal, and intravenous routes in nonpregnant and pregnant adult mice. The models were then extrapolated to nonpregnant and pregnant humans, replicating BEC data within a factor of two. The rodent models were then used to conduct IVIVEs for rodent and whole-embryo culture embryotoxicity data (neural tube closure defects, morphological changes). A second IVIVE was conducted for exposure scenarios in pregnant women during critical windows of susceptibility for developmental toxicity, such as the first 6-to-8 weeks (prerecognition period) or mid-to-late pregnancy period, when EtOH consumption is associated with fetal alcohol spectrum disorders. Incorporation of data from human embryonic stem cell studies led to a model-supported linkage of in vitro concentrations with plausible exposure ranges for pregnant women. This effort demonstrates benefits and challenges associated with use of multispecies PBPK models to estimate in vivo tissue concentrations associated with in vitro embryotoxicity studies.

The mechanisms underlying the acute neurophysiological and behavioral effects of volatile organic compounds (VOCs) remain to be elucidated. However, the function of neuronal ion channels is perturbed by VOCs. The present study examined effects of toluene (TOL), trichloroethylene (TCE), and perchloroethylene (PERC) on whole-cell calcium current (ICa) in nerve growth factor-differentiated pheochromocytoma (PC12) cells. All three VOCs affected ICa in a reversible, concentration-dependent manner. At +10-mV test potentials, VOCs inhibited ICa, whereas at test potentials of -20 and -10 mV, they potentiated it. The order of potency for inhibition (IC50) was PERC (270 microM) > TOL (720 microM) > TCE (1525 microM). VOCs also changed ICa inactivation kinetics from a single- to double-exponential function. Voltage-ramp experiments suggested that VOCs shifted ICa activation in a hyperpolarizing direction; this was confirmed by calculating the half-maximal voltage of activation (V1/2, act) in the absence and presence of VOCs using the Boltzman equation. V(1/2, act) was shifted from approximately -2 mV in control to -11, -12, and -16 mV by TOL, TCE, and PERC, respectively. Similarly, VOCs shifted the half-maximal voltage of steady-state inactivation (V1/2, inact) from approximately -16 mV in control to -32, -35, and -20 mV in the presence of TOL, TCE, and PERC, respectively. Inhibition of ICa by TOL was confirmed in primary cultures of cortical neurons, where 827 microM TOL inhibited current by 61%. These data demonstrate that VOCs perturb voltage-sensitive Ca2+ channel function in neurons, an effect that could contribute to the acute neurotoxicity of these compounds.

Risk assessment procedures can be improved through better understanding and use of tissue dose information and linking tissue dose level to adverse outcomes. For volatile organic compounds, such as toluene and trichloroethylene (TCE), blood and brain concentrations can be estimated with physiologically based pharmacokinetic (PBPK) models. Acute changes in the function of the nervous system can be linked to the concentration of test compounds in the blood or brain at the time of neurological assessment. This set of information enables application to a number of risk assessment situations. For example, we have used this approach to recommend duration adjustments for acute exposure guideline levels (AEGLs) for TCE such that the exposure limits for each exposure duration yield identical tissue concentrations at the end of the exposure period. We have also used information on tissue concentration at the time of assessment to compare sensitivity across species, adjusting for species-specific pharmacokinetic differences. Finally this approach has enabled us to compare the relative sensitivity of different compounds on a tissue dose basis, leading to expression of acute solvent effects as ethanol-dose equivalents for purposes of estimating cost–benefit relationships of various environmental control options.

We are developing an exposure–dose–response (EDR) model for volatile organic compounds (VOCs) to predict acute effects of VOCs on nervous system function from exposure data (concentration and duration of inhalation). This model contains both toxicokinetic and toxicodynamic components. One advantage of the EDR model will be its ability to relate in vitro effects of solvents on cellular ion channels (putative targets) to in vivo effects, using a combination of physiologically-based toxicokinetic (PBTK) modeling (to estimate VOC concentrations in the blood and brain) and in vitro studies to clarify the mode of action of the VOCs. Recent work in vitro has focused on quantifying the inhibitory effects of toluene, trichloroethylene (TCE) and perchloroethylene (PERC) on ion channel currents. All three VOCs inhibit current through voltage-sensitive calcium channels (VSCCs) in pheochromocytoma cells; PERC blocked calcium currents and altered the current–voltage relationship at lower concentrations than did toluene or TCE. Recombinant nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes, were also inhibited by PERC and toluene in a concentration-dependent manner. PERC inhibited α7 receptors more than α4β2 receptors in recombinant human and rat nAChRs. However, human and rat α7 receptors were equally sensitive to PERC and TOL. These in vitro studies will be used to identify an appropriate neuronal receptor system to serve as an index of acute effects of VOCs in vivo. The PBTK model incorporates physiological input parameters derived from radiotelemetered heart rate data from rats performing operant tests of cognitive and motor functions. These studies should improve predictions of target organ concentrations of inhaled VOCs in subjects actively performing behavioral tests over a range of physical activity levels.

The relative sensitivity of rats and humans to volatile organic compounds (VOCs) such as toluene (TOL) and perchloroethylene (PERC) is unknown and adds to uncertainty in assessing risks for human exposures to VOCs. Recent studies have suggested that ion channels, including nicotinic acetylcholine receptors (nAChRs), are targets of TOL effects. However, studies comparing TOL effects on human and rat ligand-gated ion channels have not been conducted. To examine potential toxicodynamic differences between these species, the sensitivity of human and rat nAChRs to TOL was assessed. Since PERC has similar effects, in vivo, to TOL, effects of PERC on nAChR function were also examined. Two-electrode voltage-clamp techniques were utilized to measure acetylcholine-induced currents in neuronal nAChRs (alpha4beta2, alpha3beta2, and alpha7) expressed in Xenopus oocytes. PERC (0.065 mM) inhibited alpha7 nAChR currents by 60.1 +/- 4.0% (human, n = 7) and 40 +/- 3.5% (rat, n = 5), and inhibited alpha4beta2 nAChR currents by 42.0 +/- 5.2% (human, n = 6) and 52.2 +/- 5.5% (rat, n = 8). Likewise, alpha3beta2 nAChRs were significantly inhibited by 62.2 +/- 3.8% (human, n = 7) and 62.4 +/- 4.3% (rat, n = 8) in the presence of 0.065 mM PERC. TOL also inhibited both rat and human alpha7, alpha4beta2, and alpha3beta2 nAChRs. Statistical analysis indicated that although there was not a species (human vs. rat) difference with PERC (0.0015-0.065 mM) or TOL (0.03-0.9 mM) inhibition of alpha7, alpha4beta2, or alpha3beta2 nAChRs, all receptor types were more sensitive to PERC than TOL. These results demonstrate that human and rat nACh receptors represent a sensitive target for VOCs. This toxicodynamic information will help decrease the uncertainty associated with animal to human extrapolations in the risk assessment of VOCs.

Trichloroethylene (TCE) is an organic solvent with robust acute effects on the nervous system, but poorly documented long-term effects. This study employed a signal detection task (SDT) to assess the persistence of effects of repeated daily inhalation of TCE on sustained attention in rats. Adult male Long-Evans rats inhaled TCE at 0, 1600, or 2400 ppm, 6 h/day for 20 days (n=8/group) and began learning the SDT 3 weeks later. Rats earned food by pressing one retractable response lever in a signal trial and a second lever in a blank (no signal) trial. TCE did not affect acquisition of the response rule or performance of the SDT after the intertrial interval (ITI) was changed from a constant value to a variable one. Increasing the trial presentation rate reduced accuracy equivalently in all groups. Injections of ethanol (0, 0.5, 1.0, 1.5 g/kg ip) and d-amphetamine (0, 0.1, 0.3, 1.0 mg/kg sc) systematically impaired performance as functions of drug dose. d-Amphetamine (1.0 mg/kg) reduced P(hit) more in the 2400-ppm TCE group than in the other groups. All rats required remedial training to learn a reversal of the response contingencies, which TCE did not interfere with. Thus, a history of exposure to TCE did not significantly alter learning or sustained attention in the absence of drugs. Although ethanol did not differentially affect the TCE groups, the effect of d-amphetamine is consistent with solvent-induced changes in dopaminergic functions in the CNS. Calculations indicated power values of 0.5 to 0.8 to detect main effects of TCE for the three primary endpoints.

Risk assessments often must consider exposures that vary over time or for which the exposure duration of concern differs from the available data, and a variety of extrapolation procedures have been devised accordingly. The present experiments explore the relationship(s) between exposure concentration (C) and time (t) to investigate procedures for assessing the risks of short-term solvent exposures. The first hypothesis tested was that the product of C x t would produce a constant health effect (Haber's rule). The second hypothesis tested was that exposure conditions produce effects in proportion to the tissue concentrations created. Awake, adult, male Long-Evans (LE) rats were exposed to trichloroethylene (TCE) vapor in a head-only exposure chamber while pattern onset/offset visual evoked potentials (VEPs) were recorded. Exposure conditions were designed to provide C x t products of 0 ppm/h (0 ppm for 4 h) or 4000 ppm/h created through four exposure scenarios: 1000 ppm for 4 h; 2000 ppm for 2 h; 3000 ppm for 1.3 h; or 4000 ppm for 1h (n = 9-10/concentration). The amplitude of the VEP frequency double component (F2) was decreased significantly by exposure; this decrease was related to C but not to t or to the C x t product, indicating that Haber's rule did not hold. The mean amplitude (+/- SEM in muV) of the F2 component in the control and treatment groups measured 4.4 +/- 0.5 (0 ppm/4 h), 3.1 +/- 0.5 (1000 ppm/4 h), 3.1 +/- 0.4 (2000 ppm/2 h), 2.3 +/- 0.3 (3000 ppm/1.3 h), and 1.9 +/- 0.4 (4000 ppm/1 h). A physiologically based pharmacokinetic (PBPK) model was used to estimate the concentrations of TCE in the brain achieved during each exposure condition. The F2 amplitude of the VEP decreased monotonically as a function of the estimated peak brain concentration but was not related to the area under the curve (AUC) of the brain TCE concentration. In comparison to estimates from the PBPK model, extrapolations based on Haber's rule yielded approximately a 6-fold error in estimated exposure duration when extrapolating across only a 4-fold change in exposure concentration. These results indicate that the use of a linear form of Haber's rule will not predict accurately the risks of acute exposure to TCE, nor will an estimate of AUC of brain TCE. However, an estimate of the brain TCE concentration at the time of VEP testing predicted the effects of TCE across exposure concentrations and durations.

The practice of cross-species extrapolation (CSE) in toxicology is a special case of comparative physiology in which experimental data collected in nonhumans are used to estimate or predict effects of toxicants in humans. Conceptually, data from nonhumans are adjusted to account for physiological differences between species and applied to humans. The components of the process are as follows: (a) adjust for differences in internal doses that result from differences in physiology due to body size and pharmacokinetic differences, (b) account for differences in the sensitivity of species due to physiological processes, and (c) make the adjustments in numerical terms with confidence limits. Steps (a) and (b) are usually done by applications of mathematical/mechanistic physiological models to the extent possible. Step (c) is sometimes done using mechanistic models and sometimes empirically. The state of practice and limitations are discussed. The practice is in early stages of development. Keywords: species extrapolation; comparative physiology/biology; quantitative extrapolation; mechanistic methods; empirical methods

Ethanol (EtOH) exposure induces a variety of concentration-dependent neurological and developmental effects in the rat. Physiologically-based pharmacokinetic (PBPK) models have been used to predict the inhalation exposure concentrations necessary to produce blood EtOH concentrations (BEC) in the range associated with these effects. Previous laboratory reports often lacked sufficient detail to adequately simulate reported exposure scenarios associated with BECs in this range, or lacked data on the time-course of EtOH in target tissues (e.g. brain, liver, eye, fetus). To address these data gaps, inhalation studies were performed at 5000, 10 000, and 21 000 ppm (6 h/d) in non-pregnant female Long-Evans (LE) rats and at 21 000 ppm (6.33 h/d) for 12 d of gestation in pregnant LE rats to evaluate our previously published PBPK models at toxicologically-relevant blood and tissue concentrations. Additionally, nose-only and whole-body plethysmography studies were conducted to refine model descriptions of respiration and uptake within the respiratory tract. The resulting time-course and plethysmography data from these in vivo studies were compared to simulations from our previously published models, after which the models were recalibrated to improve descriptions of tissue dosimetry by accounting for dose-dependencies in pharmacokinetic behavior. Simulations using the recalibrated models reproduced these data from non-pregnant, pregnant, and fetal rats to within a factor of 2 or better across datasets, resulting in a suite of model structures suitable for simulation of a broad range of EtOH exposure scenarios.

Pfiesteria piscicida is a toxic dinoflagellate that has caused massive fish kills in estuaries along the East Coast of the United States, and exposure of humans to toxic Pfiesteria has been associated with cognitive impairment. A visual signal detection task was used to determine the possible importance of attentional and visual processes in Pfiesteria effects on cognitive function. Adult female rats were trained to perform the signal detection task. After training, the rats were injected subcutaneously with fish culture water containing toxic Pfiesteria (35,600 or 106,800 cells of Pfiesteria/kg of rat body weight) or with (control) fish culture water containing no Pfiesteria. Effects of toxic Pfiesteria on maintenance of signal detection behavior were assessed for 2 weeks after treatment. Then, the signal-response contingencies were reversed. After the discrimination was reestablished on the reversed levers, the rats received a second dose of toxic Pfiesteria. The rats were again tested for 2 weeks, after which a second reversal was imposed. Pfiesteria did not affect behavior in the signal detection task during 2 weeks of prereversal testing after either exposure. However, a significant Pfiesteria-induced deficit emerged when the signal-response contingencies were reversed. These findings suggest that Pfiesteria-induced deficits emerge during periods of behavioral transition and not during performance of previously learned tasks.

Previous work showed that rats develop tolerance to the acute behavioral effects of trichloroethylene (TCE) on signal detection if they inhale TCE while performing the task and that this tolerance depends more upon learning than upon changes in metabolism of TCE. The present study sought to characterize this tolerance by assessing signal detection in rats during three phases of TCE exposures. Tolerance was induced in Phase 1 (daily 1-h test sessions concurrent with TCE exposure), extinguished in Phase 2 (daily tests in air with intermittent probe tests in TCE), and reinduced in Phase 3. Original induction in Phase 1 required 2 weeks, whereas reinduction in Phase 3 required less than 1 week. Tolerance persisted for 2 (accuracy) or 8 weeks [response time] in Phase 2 and was resistant to changes in test conditions in Phase 3. The slow induction, gradual extinction, savings during reinduction and lack of disruption from altered test conditions suggest mediation by instrumental learning processes. These data and most other evidence for behavioral tolerance to solvents can be explained by solvent-induced loss of reinforcement.

Interpretation and use of data from high-throughput assays for chemical toxicity require links between effects at molecular targets and adverse outcomes in whole animals. The well-characterized genome of Drosophila melanogaster provides a potential model system by which phenotypic responses to chemicals can be mapped to genes associated with those responses, which may in turn suggest adverse outcome pathways associated with those genes. To determine the utility of this approach, we used the Drosophila Genetics Reference Panel (DGRP), a collection of ∼200 homozygous lines of fruit flies whose genomes have been sequenced. We quantified toluene-induced suppression of motor activity in 123 lines of these flies during exposure to toluene, a volatile organic compound known to induce narcosis in mammals via its effects on neuronal ion channels. We then applied genome-wide association analyses on this effect of toluene using the DGRP web portal (http://dgrp2.gnets.ncsu.edu), which identified polymorphisms in candidate genes associated with the variation in response to toluene exposure. We tested ∼2 million variants and found 82 polymorphisms located in or near 66 candidate genes that were associated with phenotypic variation for sensitivity to toluene at P

Developmental exposure to polychlorinated biphenyls (PCBs) has been associated with behavioral and cognitive deficits in humans and animal models. Perinatal exposure to PCBs has also been associated with sensory deficits in animal models. These effects were hypothesized to be mediated in part by ortho-substituted PCBs, which do not or weakly bind to the aryl hydrocarbon (Ah) receptor. The present studies were designed to determine whether perinatal exposure to Aroclor 1254, a commercial mixture of > 99% ortho-substituted PCBs, would affect cognitive and sensory function in Long-Evans rats. Adult male and female offspring of female rats fed Aroclor 1254 (Lot #124-191; doses of 0, 1, or 6 mg/kg/day; gestational day 6 through postnatal day 21; n = eight/group) were trained to perform a signal detection task capable of assessing sensory thresholds. Training included autoshaping and operant conditioning. Thresholds for detecting a 1-s light stimulus were determined under background illuminations ranging from 2 lux to complete darkness. Female rats exposed to Aroclor 1254 autoshaped more rapidly than control females, at a rate akin to control males. Control females had lower thresholds than control males at all levels of background illumination. These differences were abolished by Aroclor 1254, which reduced thresholds in males and increased thresholds in females. These data extend previous findings of gender-specific effects of PCBs on neurobehavioral development to measures of acquisition and sensory function.

It is often assumed that cognitive function is more sensitive to neurotoxic chemicals than are the unconditioned behaviors employed in neurobehavioral screens; however, direct comparisons of the sensitivity of these test methods are lacking. The present studies were conducted to compare the effects of the widely used cholinesterase-inhibiting insecticide, chlorpyrifos (O,O'-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothionate, CPF), on a visual signal detection task (SDT) with its effects on a neurobehavioral test battery. Adult male Long-Evans rats were trained to perform the SDT, dosed with CPF, and then assessed with both test instruments. Oral CPF (50 mg/kg) impaired signal detection for 8 days, and subcutaneous CPF (250 mg/kg) did so for 4 weeks. CPF (30 and 50 mg/kg po and 250 mg/kg sc) also lowered activity in the test battery for up to 18 days. Thus, CPF impaired attention and altered behavior in the test battery in the same dose ranges under two very different dosing scenarios.

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 μg/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/m2-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.

A symposium was convened to discuss recent developments in the assessment of attention and the effects of drugs and toxic chemicals on attention at the 17th annual meeting of the Behavioral Toxicology Society on May 1, 1999, in Research Triangle Park, NC. Speakers addressed issues including the methodology of assessing cognitive function, the neurobiology of specific aspects of attention, the dual roles of attention as a target of intoxication and as a mediating variable in the development of addiction to psychoactive drugs, the changes in attention that accompany neuropsychological disorders of schizophrenia, senile dementia of the Alzheimer type and attention deficit hyperactivity disorder, and potential therapies for these disorders. This article provides an overview of the objectives of the symposium, followed by summaries of each of the talks given.

Strategies are needed for assessing the risks of exposures to airborne toxicants that vary over concentrations and durations. The goal of this project was to describe the relationship between the concentration and duration of exposure to inhaled trichloroethylene (TCE), a representative volatile organic chemical, tissue dose as predicted by a physiologically based pharmacokinetic model, and neurotoxicity. Three measures of neurotoxicity were studied: hearing loss, signal detection behavior, and visual function. The null hypothesis was that exposure scenarios having an equivalent product of concentration and duration would produce equal toxic effects, according to the classic linear form of Haber's Rule ((italic)C(/italic) times t = k), where C represents the concentration, t, the time (duration) of exposure, and k, a constant toxic effect. All experiments used adult male, Long-Evans rats. Acute and repeated exposure to TCE increased hearing thresholds, and acute exposure to TCE impaired signal detection behavior and visual function. Examination of all three measures of neurotoxicity showed that if Haber's Rule were used to predict outcomes across exposure durations, the risk would be overestimated when extrapolating from shorter to longer duration exposures, and underestimated when extrapolating from longer to shorter duration exposures. For the acute effects of TCE on behavior and visual function, the estimated concentration of TCE in blood at the time of testing correlated well with outcomes, whereas cumulative exposure, measured as the area under the blood TCE concentration curve, did not. We conclude that models incorporating dosimetry can account for differing exposure scenarios and will therefore improve risk assessments over models considering only parameters of external exposure.

The possibility that the acute neurotoxic effects of organic solvents change with repeated exposure will affect risk assessment of these pollutants. We observed previously that rats inhaling trichloroethylene (TCE) showed a progressive attenuation of impairment of signal detection behavior across several weeks of intermittent exposure, suggesting the development of tolerance. Here, we explored the development of tolerance to TCE during two weeks of daily exposures, and the degree to which learned behavioral modifications (“behavioral tolerance”) could account for the effect. Adult Long-Evans rats were trained to perform a visual signal detection task (SDT) in which a press on one lever yielded food if a visual stimulus (a “signal”) had occurred on that trial, and a press on a second lever produced food if no signal had been presented. In two experiments, with 2000 and 2400 ppm of TCE respectively, trained rats were divided into two groups ( n = 8/group) with equivalent accuracy and then exposed to TCE in two-phase studies. In Phase 1, one group of rats received daily SDT tests paired with 70-min TCE exposures, followed by 70-min exposures to clean air after testing. The other group received daily SDT tests in clean air, followed by 70-min exposures to TCE (unpaired exposure and testing). All rats thus received the same number and daily sequence of exposures to TCE that differed only in the pairing with SDT testing. Both concentrations of TCE disrupted performance of the paired groups and this disruption abated over the 9 days of exposure. In Phase 2, the pairing of exposure and test conditions were reversed for the two groups. The groups that were shifted from unpaired to paired exposures (Unpaired–Paired groups) showed qualitatively similar patterns of deficit and recovery as did the rats whose tests were initially paired with TCE (Paired–Unpaired groups), indicating that task-specific learning was involved in the development of tolerance. Quantitative differences in the magnitude and duration of the effects of TCE in the two groups indicated that other factors, not specific to the SDT, also contributed to the development tolerance to TCE. Published by Elsevier Science Inc.

Evidence from humans suggests that cognitive dysfunction may result from perinatal exposure to polychlorinated biphenyls (PCBs), and the results of some animal research with PCBs have been interpreted in terms of possible impairment of attention. Long-Evans rats were fed 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), a coplanar congener, at doses of 0.25 or 1 mg/kg/day throughout gestation and nursing. Male offspring of these rats were trained as adults to perform 2 tests of attention for food reward. First, a cued target-detection task, modeled after Posner's covert orienting method for humans, was used to assess visuospatial attention. In this task, a visual target stimulus was presented in 1 visual hemifield on each trial, preceded either by a valid cue, an invalid cue, or no cue. A valid cue appeared in the same hemifield as the target, and an invalid cue appeared in the opposite hemifield. As expected, valid cues increased accuracy and speed of target detection and invalid cues decreased accuracy and speed; moreover, these effects were systematically related to changes in cue intensity and target duration. However, perinatal exposure to PCB 126 did not affect acquisition or performance of this task. The second task assessed sustained attention by means of a signal detection method in which a brief, spatially-constant but temporally unpredictable, visual signal indicated which of 2 responses would yield food. Varying the intensity of the signal greatly affected the probability of correctly reporting the signal. Perinatal exposure to PCB 126 did not affect acquisition of the response rule or performance of the task. Finally, all rats were challenged with chlordiazepoxide (CDP) at doses of 0, 3, 5, 8, or 12 mg/kg SC, 20 min before testing in the sustained attention task. In control rats, low doses (3, 5, and 8 mg/kg) of CDP reduced accuracy at low signal intensities only, suggesting an increase in visual threshold. The high dose of CDP reduced accuracy at all signal intensities and increased the false-alarm rate as well, suggesting an impairment of attention. The rats exposed perinatally to PCB 126 at 0.25 mg/kg were unaffected by CDP, and those exposed to PCB 126 at 1 mg/kg showed a smaller decrement in performance after CDP than did the controls. Taken together, these data provide little support for the possibility that perinatal exposure to PCB 126 causes deficits in attention, but suggest that PCB 126 may alter GABA-mediated pathways in the CNS during development. Published by Elsevier Science Inc.

Animal models of human cognitive processes are being developed for investigating the neurobiological mechanisms of these processes and for identifying potential therapies for intoxication and neurodegenerative diseases. One promising method involves assessment of sustained attention in rats by use of a discrete-trial signal detection task. Several psychophysical and procedural factors have been identified in the literature that affect sustained attention in normal human subjects. Three key parameters that affect the level of performance, and whether that performance level deteriorates over time, include the quality of the signal, the event rate, and the type of task employed (simultaneous or successive discrimination). These three parameters were manipulated in this study to assess the degree of similarity in the behavior patterns engendered in rats by this signal detection task, in relation to the behavior observed in humans performing a variety of sustained attention tasks. Signal quality was manipulated by varying the increment in the intensity of a lamp (duration=300 ms), and event rate was varied among values of 4, 7, and 10 trials/min. The ‘standard’ detection task was used as a simultaneous discrimination and a successive discrimination task was designed in which a dim light flash was defined as a non-signal event and any of three brighter flashes were signal events. Accuracy of signal detection was quantified by the proportion of correct detections of the signal [P(hit)] and the proportion of false alarms [P(fa), i.e., incorrect responses on non-signal trials]. P(hit) fell with decreasing signal intensity, increasing event rate, and was lower in the discrimination task compared to the detection task. P(fa) increased with increasing event rate, but only in the detection task. A decrement in P(hit) across trial blocks was observed in the discrimination task primarily, and was most evident with a high event rate and dim signals. These data confirm that these tasks assess process(es) in rats that are very similar to those considered as sustained attention in humans.

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 paradigms applicable for use in both human and nonhuman subjects for investigating aspects of working/short-term memory are presented with a view towards exploring their strengths, weaknesses, and utility in a variety of experimental situations. Such procedures can be useful in teasing out specific aspects of mnemonic processes including discrimination, encoding, and retention. Delayed matching-to-position, delayed matching-to-sample (DMTS), and titrating matching-to-sample procedures are highlighted. Additionally, the application of DMTS tasks in preclinical and clinical settings is presented: drug effects on memory processes can be explored preclinically in animal models; normative data have been developed in human populations where they have been used in adults to explore the relationships between mnemonic processes and specific clinical entities such as Parkinsonism, senile dementia of the Alzheimer's type, schizophrenia, and depression. Studies in children indicate that encoding and retention processes improve rapidly in the early years, plateauing prior to puberty. Noninvasive imaging techniques such as positron emission tomography (PET) indicate that activity in specific brain areas is associated with DMTS task performance and may serve to confirm roles for such structures in mnemonic processes.