Issues in assessing the health risks of n-butanol.

A literature review and health effects evaluation were conducted for n-butanol, a chemical that occurs naturally in some foods, which is an intermediate in the production of butyl esters and can be used as a gasoline additive or blend. Studies evaluating n-butyl acetate were included in the review as n-butyl acetate is rapidly converted to n-butanol following multiple routes of exposure. The primary n-butanol health effects identified were developmental and nervous system endpoints. In conducting the literature review and evaluating study findings, the following observations were made: (1) developmental findings were consistently identified; (2) neurodevelopmental findings were inconsistent; (3) evidence for nervous system effects was weak; (4) comparing internal doses from oral and inhalation exposures using physiologically based pharmacokinetic models introduces uncertainties; and (5) a lack of mechanistic information for n-butanol resulted in the reliance on mechanistic data for ethanol, which may or may not be applicable to n-butanol. This paper presents findings from a literature review on the health effects of n-butanol and proposes research to help reduce uncertainty that exists due to database limitations.

An overview of butanol-induced developmental neurotoxicity and the potential mechanisms related to these observed effects.

The purpose of this article is to briefly review the published literature on the developmental neurotoxic effects, including potential mechanisms, of four butanols: n-butanol, sec-butanol, tert-butanol, isobutanol, and identify data gaps and research needs for evaluation of human health risks in this area. Exposure potential to these four butanols is considerable given the high production volume (>1 billion lb) of n- and tert-butanol and moderate production volumes (100-500 million lb) of sec- and isobutanol. With the impetus to derive cleaner gasoline blends, butanols are being considered for use as fuel oxygenates. Notable signs of neurotoxicity and developmental neurotoxicity have been observed in some studies where laboratory animals (rodents) were gestationally exposed to n- or tert-butanol. Mechanistic data relevant to the observed developmental neurotoxicity endpoints were also reviewed to hypothesize potential mechanisms associated with the developmental neurotoxicity outcome. Data from the related and highly characterized alcohol, ethanol, were included to examine consistencies between this compound and the four butanols. It is widely known that alcohols, including butanols, interact with several ion channels and modulate the function of these targets following both acute and chronic exposures. In addition, n- and sec-butanol have been demonstrated to inhibit fetal rat brain astroglial cell proliferation. Further, rat pups exposed to n-butanol in utero were also reported to have significant increases in brain levels of dopamine and serotonin, but decreases in serotonin levels were noted with gestational exposure to tert-butanol. tert-Butanol was reported to inhibit muscarinic receptor-stimulated phosphoinositide metabolism which has been hypothesized to be a possible target for the neurotoxic effects of ethanol during brain development. The mechanistic data for the butanols support developmental neurotoxicity that has been observed in some of the rodent studies. However, careful studies evaluating the neurobehavior of developing pups in sensitive strains, as well as characterizing the plausible mechanisms involved, need to be conducted in order to further elucidate the neurodevelopmental effects of butanols for risk evaluation.

Human health effects of dichloromethane: key findings and scientific issues.

BACKGROUND: The U.S. EPA's Integrated Risk Information System (IRIS) completed an updated toxicological review of dichloromethane in November 2011. OBJECTIVES: In this commentary we summarize key results and issues of this review, including exposure sources, identification of potential health effects, and updated physiologically based pharmacokinetic (PBPK) modeling. METHODS: We performed a comprehensive review of primary research studies and evaluation of PBPK models. DISCUSSION: Hepatotoxicity was observed in oral and inhalation exposure studies in several studies in animals; neurological effects were also identified as a potential area of concern. Dichloromethane was classified as likely to be carcinogenic in humans based primarily on evidence of carcinogenicity at two sites (liver and lung) in male and female B6C3F1 mice (inhalation exposure) and at one site (liver) in male B6C3F1 mice (drinking-water exposure). Recent epidemiologic studies of dichloromethane (seven studies of hematopoietic cancers published since 2000) provide additional data raising concerns about associations with non-Hodgkin lymphoma and multiple myeloma. Although there are gaps in the database for dichloromethane genotoxicity (i.e., DNA adduct formation and gene mutations in target tissues in vivo), the positive DNA damage assays correlated with tissue and/or species availability of functional glutathione S-transferase (GST) metabolic activity, the key activation pathway for dichloromethane-induced cancer. Innovations in the IRIS assessment include estimation of cancer risk specifically for a presumed sensitive genotype (GST-theta-1+/+), and PBPK modeling accounting for human physiological distributions based on the expected distribution for all individuals 6 months to 80 years of age. CONCLUSION: The 2011 IRIS assessment of dichloromethane provides insights into the toxicity of a commonly used solvent.

Human health effects of tetrachloroethylene: key findings and scientific issues.

BACKGROUND: The U.S. Environmental Protection Agency (EPA) completed a toxicological review of tetrachloroethylene (perchloroethylene, PCE) in February 2012 in support of the Integrated Risk Information System (IRIS). OBJECTIVES: We reviewed key findings and scientific issues regarding the human health effects of PCE described in the U.S. EPA's Toxicological Review of Tetrachloroethylene (Perchloroethylene). METHODS: The updated assessment of PCE synthesized and characterized a substantial database of epidemiological, experimental animal, and mechanistic studies. Key scientific issues were addressed through modeling of PCE toxicokinetics, synthesis of evidence from neurological studies, and analyses of toxicokinetic, mechanistic, and other factors (tumor latency, severity, and background rate) in interpreting experimental animal cancer findings. Considerations in evaluating epidemiological studies included the quality (e.g., specificity) of the exposure assessment methods and other essential design features, and the potential for alternative explanations for observed associations (e.g., bias or confounding). DISCUSSION: Toxicokinetic modeling aided in characterizing the complex metabolism and multiple metabolites that contribute to PCE toxicity. The exposure assessment approach-a key evaluation factor for epidemiological studies of bladder cancer, non-Hodgkin lymphoma, and multiple myeloma-provided suggestive evidence of carcinogenicity. Bioassay data provided conclusive evidence of carcinogenicity in experimental animals. Neurotoxicity was identified as a sensitive noncancer health effect, occurring at low exposures: a conclusion supported by multiple studies. Evidence was integrated from human, experimental animal, and mechanistic data sets in assessing adverse health effects of PCE. CONCLUSIONS: PCE is likely to be carcinogenic to humans. Neurotoxicity is a sensitive adverse health effect of PCE.

Correlating in vitro data to in vivo findings for risk assessment.

A special session at the Toxicology and Risk Assessment Conference in Cincinnati, OH, USA in May, 2012 presented approaches expanding upon current uses of in vitro toxicity data for risk assessment. Evaluation of xenobiotics through use of in vitro study methods is increasing exponentially and these methodologies offer a relatively fast and considerably cheaper way to determine toxicities in comparison to traditional approaches. One of the challenges with in vitro data is to effectively use this information for risk assessment purposes. Currently, in vitro studies are used as supportive for hazard characterization and identifying mechanisms associated with toxicity. Being able to effectively correlate in vitro effects to in vivo observations represents a major challenge for risk assessors. The presentations in this special session provided innovative approaches toward effectively using in vitro data for the human health risk assessment process.

Developmental neurotoxicity of engineered nanomaterials: identifying research needs to support human health risk assessment.

Increasing use of engineered nanomaterials (ENM) in consumer products and commercial applications has helped drive a rise in research related to the environmental health and safety (EHS) of these materials. Within the cacophony of information on ENM EHS to date are data indicating that these materials may be neurotoxic in adult animals. Evidence of elevated inflammatory responses, increased oxidative stress levels, alterations in neuronal function, and changes in cell morphology in adult animals suggests that ENM exposure during development could elicit developmental neurotoxicity (DNT), especially considering the greater vulnerability of the developing brain to some toxic insults. In this review, we examine current findings related to developmental neurotoxic effects of ENM in the context of identifying research gaps for future risk assessments. The basic risk assessment paradigm is presented, with an emphasis on problem formulation and assessments of exposure, hazard, and dose response for DNT. Limited evidence suggests that in utero and postpartum exposures are possible, while fewer than 10 animal studies have evaluated DNT, with results indicating changes in synaptic plasticity, gene expression, and neurobehavior. Based on the available information, we use current testing guidelines to highlight research gaps that may inform ENM research efforts to develop data for higher throughput methods and future risk assessments for DNT. Although the available evidence is not strong enough to reach conclusions about DNT risk from ENM exposure, the data indicate that consideration of ENM developmental neurotoxic potential is warranted.

Human health effects of trichloroethylene: key findings and scientific issues.

BACKGROUND: In support of the Integrated Risk Information System (IRIS), the U.S. Environmental Protection Agency (EPA) completed a toxicological review of trichloroethylene (TCE) in September 2011, which was the result of an effort spanning > 20 years. OBJECTIVES: We summarized the key findings and scientific issues regarding the human health effects of TCE in the U.S. EPA's toxicological review. METHODS: In this assessment we synthesized and characterized thousands of epidemiologic, experimental animal, and mechanistic studies, and addressed several key scientific issues through modeling of TCE toxicokinetics, meta-analyses of epidemiologic studies, and analyses of mechanistic data. DISCUSSION: Toxicokinetic modeling aided in characterizing the toxicological role of the complex metabolism and multiple metabolites of TCE. Meta-analyses of the epidemiologic data strongly supported the conclusions that TCE causes kidney cancer in humans and that TCE may also cause liver cancer and non-Hodgkin lymphoma. Mechanistic analyses support a key role for mutagenicity in TCE-induced kidney carcinogenicity. Recent evidence from studies in both humans and experimental animals point to the involvement of TCE exposure in autoimmune disease and hypersensitivity. Recent avian and in vitro mechanistic studies provided biological plausibility that TCE plays a role in developmental cardiac toxicity, the subject of substantial debate due to mixed results from epidemiologic and rodent studies. CONCLUSIONS: TCE is carcinogenic to humans by all routes of exposure and poses a potential human health hazard for noncancer toxicity to the central nervous system, kidney, liver, immune system, male reproductive system, and the developing embryo/fetus.

Translating neurobehavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts.

The developing nervous system is particularly vulnerable to chemical insults. Exposure to chemicals can result in neurobehavioural alterations, and these have been used as sensitive readouts to assess neurotoxicity in animals and man. Deconstructing neurobehaviour into relevant cellular and molecular components may allow for detection of specific neurotoxic effects in cell-based systems, which in turn may allow an easier examination of neurotoxic pathways and modes of actions and eventually inform the regulatory assessment of chemicals with potential developmental neurotoxicity. Here, current developments towards these goals are reviewed. Imaging genetics (CB) provides new insights into the neurobiological correlates of cognitive function that are being used to delineate neurotoxic mechanisms. The gaps between in vivo neurobehaviour and real-time in vitro measurements of neuronal function are being bridged by ex vivo measurements of synaptic plasticity (RW). An example of solvent neurotoxicity demonstrates how an in vivo neurological defect can be linked via the N-methyl-d-aspartate (NMDA)-glutamate receptor as a common target to in vitro readouts (AB). Axonal and dendritic morphology in vitro proved to be good correlates of neuronal connectivity and neurobehaviour in animals exposed to polychlorinated biphenyls and organophosphorus pesticides (PJL). Similarly, chemically induced changes in neuronal morphology affected the formation of neuronal networks on structured surfaces. Such network formation may become an important readout for developmental neurotoxicity in vitro (CvT), especially when combined with human neurons derived from embryonic stem cells (ML). We envision that future in vitro test systems for developmental neurotoxicity will combine the above approaches with exposure information, and we suggest a strategy for test system development and cell-based risk assessment.

Insights from epidemiology into dichloromethane and cancer risk.

Dichloromethane (methylene chloride) is a widely used chlorinated solvent. We review the available epidemiology studies (five cohort studies, 13 case-control studies, including seven of hematopoietic cancers), focusing on specific cancer sites. There was little indication of an increased risk of lung cancer in the cohort studies (standardized mortality ratios ranging from 0.46 to 1.21). These cohorts are relatively small, and variable effects (e.g., point estimates ranging from 0.5 to 2.0) were seen for the rarer forms of cancers such as brain cancer and specific hematopoietic cancers. Three large population-based case-control studies of incident non-Hodgkin lymphoma in Europe and the United States observed odds ratios between 1.5 and 2.2 with dichloromethane exposure (ever exposed or highest category of exposure), with higher risk seen in specific subsets of disease. More limited indications of associations with brain cancer, breast cancer, and liver and biliary cancer were also seen in this collection of studies. Existing cohort studies, given their size and uneven exposure information, are unlikely to resolve questions of cancer risks and dichloromethane exposure. More promising approaches are population-based case-control studies of incident disease, and the combination of data from such studies, with robust exposure assessments that include detailed occupational information and exposure assignment based on industry-wide surveys or direct exposure measurements.

A review of potential neurotoxic mechanisms among three chlorinated organic solvents.

The potential for central nervous system depressant effects from three widely used chlorinated solvents, trichloroethylene (TCE), perchloroethylene (PERC), and dichloromethane (DCM), has been shown in human and animal studies. Commonalities of neurobehavioral and neurophysiological changes for the chlorinated solvents in in vivo studies suggest that there is a common mechanism(s) of action in producing resultant neurotoxicological consequences. The purpose of this review is to examine the mechanistic studies conducted with these chlorinated solvents and to propose potential mechanisms of action for the different neurological effects observed. Mechanistic studies indicate that this solvent class has several molecular targets in the brain. Additionally, there are several pieces of evidence from animal studies indicating this solvent class alters neurochemical functions in the brain. Although earlier evidence indicated that these three chlorinated solvents perturb the lipid bilayer, more recent data suggest an interaction between several specific neuronal receptors produces the resultant neurobehavioral effects. Collectively, TCE, PERC, and DCM have been reported to interact directly with several different classes of neuronal receptors by generally inhibiting excitatory receptors/channels and potentiating the function of inhibitory receptors/channels. Given this mechanistic information and available studies for TCE, DCM, and PERC, we provide hypotheses on primary targets (e.g. ion channel targets) that appear to be most influential in producing the resultant neurological effects.

A proposal to facilitate weight-of-evidence assessments: Harmonization of Neurodevelopmental Environmental Epidemiology Studies (HONEES).

The ability to conduct weight-of-evidence assessments to inform the evaluation of potential environmental neurotoxicants is limited by lack of comparability of study methods, data analysis, and reporting. There is a need to establish consensus guidelines for conducting, analyzing, and reporting neurodevelopmental environmental epidemiologic studies, while recognizing that consistency is likewise needed for epidemiology studies examining other health outcomes. This paper proposes a set of considerations to be used by the scientific community at-large as a tool for systematically evaluating the quality of proposed and/or published studies in terms of their value for weight-of-evidence assessments. Particular emphasis is placed on evaluating factors influencing the risk of incorrect conclusions at the level of study findings. The proposed considerations are the first step in what must be a larger consensus-based process and can serve to catalyze such a discussion. Achieving consensus in these types of endeavors is difficult; however, opportunities exist for further interdisciplinary discussion, collaboration, and research that will help realize this goal. Broad acceptance and application of such an approach can facilitate the expanded use of environmental epidemiology studies of potential neurodevelopmental toxicants in the protection of public health, and specifically children's health.

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).

Volatile organic compounds inhibit human and rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

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.

Role of NMDA, nicotinic, and GABA receptors in the steady-state visual-evoked potential in rats.

Agonists and antagonists at the NMDA, GABA, and nicotinic acetylcholine receptors were administered to adult male rats to evaluate the contribution of these pathways to the visual-evoked potential (VEP). Rats were presented with an onset/offset pattern at a temporal frequency (4.55 Hz) resulting in a steady-state VEP. Averaged VEPs were Fourier transformed and VEP amplitudes were calculated at 1x stimulus frequency (F1) and 2x stimulus frequency (F2). About 30 min after administration, NMDA (10 mg/kg, i.p.; n = 9) increased F1 amplitude by 350% and decreased F2 amplitude by 48%. Memantine (4.5 mg/kg, i.p.; n = 10) increased F1 amplitude by 50%, 10 min post-injection. Similarly, nicotine (0.1 mg/kg, s.c.; n = 9) increased F1 amplitude by 55%, 20 min after drug administration. Muscimol (1 mg/kg, i.p.; n = 10) increased F1 amplitude significantly from 20 to 45 min post-injection. Mecamylamine (6 mg/kg, i.p.; n = 10) decreased F2 amplitude by 70% during the 60-min testing session. Bicuculline (0-0.5 mg/kg, i.p.; n = 8-10 rats/dose) did not significantly alter either F1 or F2 amplitudes. Results indicate important roles for glutamate and nicotinic acetylcholine receptors in both F1 and F2, while GABA receptors contribute to F1.

Developing an exposure-dose-response model for the acute neurotoxicity of organic solvents: overview and progress on in vitro models and dosimetry.

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 α4ß2 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.

Evaluation of toluene dependence and cross-sensitization to diazepam.

Acute effects of the abused inhalant toluene resemble those of CNS depressant drugs. Since abuse of toluene involves repeated use, the purpose of the present study was to evaluate the effects of repeated or continuous exposure to toluene and to compare these effects to those of other inhalants and depressants. In experiment 1, ICR mice exposed continuously to 250 ppm toluene via inhalation for four days developed mild dependence upon termination that was characterized by an increase in severity of handling-induced convulsions. However, administration of the convulsants, N-methyl-D-aspartate (NMDA) or pentylenetetrazole (PTZ), did not differentially affect toluene- vs. air-exposed mice. In experiment 2, CFW mice (but not ICR mice) developed cross-sensitization to the initial locomotor stimulatory effects of toluene following four days of injections with 10 mg/kg/day diazepam. Previous findings have shown that 1,1,1-trichloroethane (TCE) produced robust dependence and cross-sensitization to diazepam's locomotor effects when tested under similar conditions. The present results suggest that the dependence and cross-sensitization with diazepam produced by toluene are milder than those induced by TCE. Further, these studies add to increasing evidence that abused inhalants do not have identical pharmacological effects.

Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene.

1 Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA) and glycine receptors at concentrations that do not affect non-NMDA receptors. 2 We utilized two-electrode voltage-clamp and whole cell patch-clamp techniques to assess the effects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 micro M to 10 mM) produced a reversible, concentration-dependent inhibition of acetylcholine-induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The alpha4beta2 and alpha3beta2 subunit combinations were significantly more sensitive to toluene inhibition than the alpha4beta4, alpha3beta4 and alpha7 receptors. 3 Receptors composed of alpha4 and beta2(V253F) subunits showed alpha4beta4-like toluene sensitivity while those containing alpha4 and beta4(F255V) subunits showed alpha4beta2-like sensitivity. 4 In hippocampal neurons, toluene (50 micro M to 10 mM) dose-dependently inhibited ACh-mediated responses with an IC(50) of 1.1 mM. 5 Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit-dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural effects of toluene.