Application of Biologically Based Lumping To Investigate the Toxicokinetic Interactions of a Complex Gasoline Mixture.

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.

Use of novel inhalation kinetic studies to refine physiologically-based pharmacokinetic models for ethanol in non-pregnant and pregnant rats.

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.

Development of multi-route physiologically-based pharmacokinetic models for ethanol in the adult, pregnant, and neonatal rat.

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.

Estimated rate of fatal automobile accidents attributable to acute solvent exposure at low inhaled concentrations.

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.

Long-term perchloroethylene exposure: a meta-analysis of neurobehavioral deficits in occupationally and residentially exposed groups.

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.

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.

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.

Quantitative comparisons of the acute neurotoxicity of toluene in rats and humans.

The behavioral and neurophysiological effects of acute exposure to toluene are the most thoroughly explored of all the hydrocarbon solvents. Behavioral effects have been experimentally studied in humans and other species, for example, rats. The existence of both rat and human dosimetric data offers the opportunity to quantitatively compare the relative sensitivity to acute toluene exposure. The purpose of this study was to fit dose-effect curves to existing data and to estimate the dose-equivalence equation (DEE) between rats and humans. The DEE gives the doses that produce the same magnitude of effect in the two species. Doses were brain concentrations of toluene estimated from physiologically based pharmacokinetic models. Human experiments measuring toluene effects on choice reaction time (CRT) were meta-analyzed. Rat studies employed various dependent variables: amplitude of visual-evoked potentials (VEPs), signal detection (SIGDET) accuracy (ACCU) and reaction time (RT), and escape-avoidance (ES-AV) behaviors. Comparison of dose-effect functions showed that human and rat sensitivity was practically the same for those two task regimens that exerted the least control over the behaviors being measured (VEP in rats and CRT in humans) and the sensitivity was progressively lower for SIGDET RT, SIGDET ACCU, and ES-AV behaviors in rats. These results suggested that the sensitivity to impairment by toluene depends on the strength of control over the measured behavior rather than on the species being tested. This interpretation suggests that (1) sensitivity to toluene would be equivalent in humans and rats if both species performed behaviors that were controlled to the same extent, (2) the most sensitive tests of neurobehavioral effects would be those in which least control is exerted on the behavior being measured, and (3) effects of toluene in humans may be estimated using the DEEs from rat studies despite differences in the amount of control exerted by the experimental regimen or differences in the behaviors under investigation.

A dosimetric analysis of the acute behavioral effects of inhaled toluene in rats.

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.

Acute toluene exposure and rat visual function in proportion to momentary brain concentration.

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.

Evaluating the NMDA-glutamate receptor as a site of action for toluene, in vivo.

Acute exposure to toluene and other volatile organic solvents results in neurotoxicity characterized by nervous system depression, cognitive and motor impairment, and alterations in visual function. In vitro, toluene disrupts the function of N-methyl-D-aspartate (NMDA)-glutamate receptors, indicating that effects on NMDA receptor function may contribute to toluene neurotoxicity. NMDA-glutamate receptors are widely present in the visual system and contribute to pattern-elicited visual-evoked potentials (VEPs) in rodents, a measure that is altered by toluene exposure. The present study tested the hypothesis that effects on NMDA receptors contribute to toluene-induced alterations in pattern-elicited VEPs. Prior to examining the effects of NMDA receptor agonists and antagonists on toluene-exposed animals, a dose-range study was conducted to determine the optimal dose for NMDA (agonist) and MK801 (antagonist). Dose levels of 2.5 mg/kg NMDA and 0.1 mg/kg MK801 were selected from these initial studies. In the second study, Long-Evans rats were exposed to toluene by inhalation, and VEPs were measured during toluene exposure in the presence or absence of NMDA or MK801. Pattern-elicited VEPs were collected by exposing rats to a sinusoidal pattern modulated at a temporal frequency of 4.55 Hz. Following collection of baseline VEPs, rats were injected with either saline, NMDA (2.5 mg/kg, ip), or MK801 (0.1 mg/kg, ip) and 10 min later were exposed to air or toluene (2000 ppm). VEP amplitudes were calculated for 1x (F1) and 2x stimulus frequency (F2). The F2 amplitude was reduced by approximately 60, 60, and 50% in the toluene-exposed groups (TOL): SALINE/TOL (n = 11), NMDA/TOL (2.5 mg/kg; n = 13), and NMDA/TOL (10 mg/kg, n = 11), respectively. Thus, NMDA (2.5 and 10 mg/kg) did not significantly affect toluene-mediated F2 amplitude effects. Administration of 0.1 mg/kg MK801 prior to toluene exposure blocked the F2 amplitude decreases caused by toluene (n = 9). However, when 0.1 mg/kg MK801 was administered 20 min after the onset of toluene exposure, toluene-mediated F2 amplitude decreases persisted despite the challenge by MK801. These data support the hypothesis that acute actions of toluene on pattern-elicited VEPs involve NMDA receptors.

Approaches to extrapolating animal toxicity data on organic solvents to public health.

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).

Repeated inhalation of toluene by rats performing a signal detection task leads to behavioral tolerance on some performance measures.

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.

Cardiovascular effects of oral toluene exposure in the rat monitored by radiotelemetry.

Toluene is a hazardous air pollutant that can be toxic to the nervous and cardiovascular systems. The cardiotoxicity data for toluene come from acute studies in anesthetized animals and from clinical observations made on toluene abusers and there is little known on the response of the cardiovascular and other autonomic processes to graded doses of toluene. This study assessed the effects of toluene (0.4, 0.8, and 1.2 g/kg; gavage) on heart rate (HR), blood pressure, core temperature (Tc), and motor activity (MA) in unrestrained, male Long-Evans rats monitored by telemetry. Toluene doses of 0.8 and 1.2 g/kg elicited significant elevations in HR, characterized by a transient 100 beats/min increase in HR lasting 1 h followed with a steady state tachycardia lasting >6 h. Overall, HR increased by 25 and 50 beats/min in the 0.8 and 1.2 g/kg groups, respectively. MA increased markedly in the 0.8 and 1.2 g/kg groups but the tachycardia persisted in spite of recovery of MA in the 0.8 g/kg group. There was a small (<0.5 degrees C) increase in Tc above controls in rats dosed with 0.8 g/kg toluene, whereas 1.2 g/kg toluene elicited a transient reduction in Tc followed by a small elevation lasting several hours. In a second study, rats were implanted with transmitters to monitor blood pressure (BP), and were administered with toluene as in the first study. HR, Tc, and MA were also monitored. The tachycardic effects of toluene at 0.8 and 1.2 g/kg were associated with a rise in blood pressure. Doses of 0.8 and 1.2 g/kg elicited a mean BP elevation of 6 and 16 mm Hg, respectively, for 7-hour post-dosing. The biphasic tachycardia to toluene suggests multiple sites for eliciting the cardiotoxic effects of this toxicant.

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.

Integrating epidemiology and toxicology in neurotoxicity risk assessment.

Neurotoxicity risk assessments depend on the best available scientific information, including data from animal toxicity studies, human experimental studies and human epidemiology studies. There are several factors to consider when evaluating the comparability of data from studies. Regarding the epidemiology literature, issues include choice of study design, use of appropriate controls, methods of exposure assessment, subjective or objective evaluation of neurological status, and assessment and statistical control of potential confounding factors, including co-exposure to other agents. Animal experiments must be evaluated regarding factors such as dose level and duration, procedures used to assess neurological or behavioural status, and appropriateness of inference from the animal model to human neurotoxicity. Major factors that may explain apparent differences between animal and human studies include: animal neurological status may be evaluated with different procedures than those used in humans; animal studies may involve shorter exposure durations and higher dose levels; and most animal studies evaluate a single substance whereas humans typically are exposed to multiple agents. The comparability of measured outcomes in animals and humans may be improved by considering functional domains rather than individual test measures. The application of predictive models, weight of evidence considerations and meta-analysis can help evaluate the consistency of outcomes across studies. An appropriate blend of scientific information from toxicology and epidemiology studies is necessary to evaluate potential human risks of exposure to neurotoxic substances.

Editor's Highlight: Genetic Targets of Acute Toluene Inhalation in Drosophila melanogaster.

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 < 5 × 10-5, and human orthologs for 52 of these candidate Drosophila genes. None of these orthologs are known to be involved in canonical pathways for mammalian neuronal ion channels, including GABA, glutamate, dopamine, glycine, serotonin, and voltage sensitive calcium channels. Thus this analysis did not reveal a genetic signature consistent with processes previously shown to be involved in toluene-induced narcosis in mammals. The list of the human orthologs included Gene Ontology terms associated with signaling, nervous system development and embryonic morphogenesis; these orthologs may provide insight into potential new pathways that could mediate the narcotic effects of toluene.

Toluene inhalation exposure for 13 weeks causes persistent changes in electroretinograms of Long-Evans rats.

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 1000ppm toluene by inhalation (6hr/d, 5d/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 1000ppm 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 1000ppm 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 1000ppm toluene for 4 weeks were tested approximately 1year 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 1year 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.

Human neurobehavioral effects of long-term exposure to styrene: a meta-analysis.

Many reports in the literature suggest that long-term exposure to styrene may exert a variety of effects on the nervous system, including increased choice reaction time and decreased performance of color discrimination and color arrangement tasks. Sufficient information exists to perform a meta-analysis of these observations quantifying the relationships between exposure (estimated from biomarkers) and effects on two measures of central nervous system function: reaction time and color vision. To perform the meta-analysis, we pooled data into a single database for each end point. End-point data were transformed to a common metric of effect magnitude (percentage of baseline). We estimated styrene concentration from biomarkers of exposure and fitted linear least-squares equations to the pooled data to produce dose-effect relationships. Statistically significant relationships were demonstrated between cumulative styrene exposure and increased choice reaction time as well as increased color confusion index. Eight work-years of exposure to 20 ppm styrene was estimated to produce a 6.5% increase in choice reaction time, which has been shown to significantly increase the probability of automobile accidents. The same exposure history was predicted to increase the color confusion index as much as 1.7 additional years of age in men.

Effects of chronic dietary and repeated acute exposure to chlorpyrifos on learning and sustained attention in rats.

Cognitive and motor impairment often follow acute poisoning with an organophosphorous (OP) pesticide. However, the persistence of these effects and the conditions necessary for their appearance are not clear: two specific concerns are whether symptomatic poisoning is necessary for persistent effects, and whether inhibition of cholinesterase (ChE) activity is a protective metric of OP exposure. This study examined the effects of chronic dietary and repeated high-level acute exposure to the pesticide chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate, CPF) on learning and attention. Beginning at 3 months of age, male Long-Evans rats received dietary CPF at a daily dose of 0, 1, or 5 mg/kg for 1 year. Half of each dietary group also received an acute oral dose of CPF (initial dose at 60 mg/kg, 5 doses at 45 mg/kg) every 2 months. Beginning 2 weeks before the fourth acute dose, behavioral assessments were conducted on the eight rats in each of the six exposure groups (0-Oil, 0-CPF, 1-Oil, 1-CPF, 5-Oil, and 5-CPF). Using an auto-shaping procedure, the groups learned to press a lever for food in the following order: 5-Oil, 5-CPF, 1-Oil, and 0-Oil. The 0-CPF and 1-CPF groups did not learn the response in three 50-trial sessions. Chronic CPF did not affect acquisition of other behaviors required by a signal detection task (SDT) designed to assess sustained attention. The sixth acute CPF dose significantly disrupted the SDT in all dosed groups. Two months after the end of dosing, performance of the SDT was impaired in the 5-CPF group. These data suggest that learning the contingency between an action and reward may be accelerated by chronic exposure to CPF and inhibited by previous symptomatic exposure to CPF, and that persistent cognitive impairment may follow if CPF exposure inhibits brain ChE activity and is accompanied by acute doses sufficient to induce signs of toxicity.