Ambuja S. Bale

Human Health Effects of Dichloromethane: 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. Citation: Guyton KZ, Hogan KA, Scott CS, Cooper GS, Bale AS, Kopylev L, Barone S Jr, Makris SL, Glenn B, Subramaniam RP, Gwinn MR, Dzubow RC, Chiu WA. 2014. Human health effects of tetrachloroethylene: key findings and scientific issues. Environ Health Perspect 122:325–334; http://dx.doi.org/10.1289/ehp.1307359

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.

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.

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 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.

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.

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 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 childrens health.

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.

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.

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.