Applying comprehensive environmental assessment to research planning for multiwalled carbon nanotubes: Refinements to inform future stakeholder engagement.

Risk assessments and risk management efforts to protect human health and the environment can benefit from early, coordinated research planning by researchers, risk assessors, and risk managers. However, approaches for engaging these and other stakeholders in research planning have not received much attention in the environmental scientific literature. The Comprehensive Environmental Assessment (CEA) approach under development by the United States Environmental Protection Agency (USEPA) is a means to manage complex information and input from diverse stakeholder perspectives on research planning that will ultimately support environmental and human health decision making. The objectives of this article are to 1) describe the outcomes of applying lessons learned from previous CEA applications to planning research on engineered nanomaterial, multiwalled carbon nanotubes (MWCNTs) and 2) discuss new insights and refinements for future efforts to engage stakeholders in research planning for risk assessment and risk management of environmental issues. Although framed in terms of MWCNTs, this discussion is intended to enhance research planning to support assessments for other environmental issues as well. Key insights for research planning include the potential benefits of 1) ensuring that participants have research, risk assessment, and risk management expertise in addition to diverse disciplinary backgrounds; 2) including an early scoping step before rounds of formal ratings; 3) using a familiar numeric scale (e.g., US dollars) versus ordinal rating scales of "importance"; 4) applying virtual communication tools to supplement face-to-face interaction between participants; and 5) refining criteria to guide development of specific, actionable research questions.

A web-based tool to engage stakeholders in informing research planning for future decisions on emerging materials.

Prioritizing and assessing risks associated with chemicals, industrial materials, or emerging technologies is a complex problem that benefits from the involvement of multiple stakeholder groups. For example, in the case of engineered nanomaterials (ENMs), scientific uncertainties exist that hamper environmental, health, and safety (EHS) assessments. Therefore, alternative approaches to standard EHS assessment methods have gained increased attention. The objective of this paper is to describe the application of a web-based, interactive decision support tool developed by the U.S. Environmental Protection Agency (U.S. EPA) in a pilot study on ENMs. The piloted tool implements U.S. EPA's comprehensive environmental assessment (CEA) approach to prioritize research gaps. When pursued, such research priorities can result in data that subsequently improve the scientific robustness of risk assessments and inform future risk management decisions. Pilot results suggest that the tool was useful in facilitating multi-stakeholder prioritization of research gaps. Results also provide potential improvements for subsequent applications. The outcomes of future CEAWeb applications with larger stakeholder groups may inform the development of funding opportunities for emerging materials across the scientific community (e.g., National Science Foundation Science to Achieve Results [STAR] grants, National Institutes of Health Requests for Proposals).

Developmentally-regulated sodium channel subunits are differentially sensitive to alpha-cyano containing pyrethroids.

Juvenile rats have been reported to be more sensitive to the acute neurotoxic effects of the pyrethroid deltamethrin than adults. While toxicokinetic differences between juveniles and adults are documented, toxicodynamic differences have not been examined. Voltage-gated sodium channels, the primary targets of pyrethroids, are comprised of alpha and beta subunits, each of which have multiple isoforms that are expressed in a developmentally-regulated manner. To begin to test whether toxicodynamic differences could contribute to age-dependent deltamethrin toxicity, deltamethrin effects were examined on sodium currents in Xenopus laevis oocytes injected with different combinations of rat alpha (Na(v)1.2 or Na(v)1.3) and beta (beta(1) or beta(3)) subunits. Deltamethrin induced tail currents in all isoform combinations and increased the percent of modified channels in a concentration-dependent manner. Effects of deltamethrin were dependent on subunit combination; Na(v)1.3-containing channels were modified to a greater extent than were Na(v)1.2-containing channels. In the presence of a beta subunit, deltamethrin effects were significantly greater, an effect most pronounced for Na(v)1.3 channels; Na(v)1.3/beta(3) channels were more sensitive to deltamethrin than Na(v)1.2/beta(1) channels. Na(v)1.3/beta(3) channels are expressed embryonically, while the Na(v)1.2 and beta(1) subunits predominate in adults, supporting the hypothesis for age-dependent toxicodynamic differences. Structure-activity relationships for sensitivity of these subunit combinations were examined for other pyrethroids. Permethrin and tetramethrin did not modify currents mediated by either subunit combination. Cypermethrin, beta-cyfluthrin, esfenvalerate and fenpropathrin all modified sodium channel function; effects were significantly greater on Na(v)1.3/beta(3) than on Na(v)1.2/beta(1) channels. These data demonstrate a greater sensitivity of Na(v)1.3 vs Na(v)1.2 channels to deltamethrin and other cyano-containing pyrethroids, particularly in the presence of a beta subunit.

Concentration-dependent accumulation of [3H]-deltamethrin in sodium channel Nav1.2/beta1 expressing Xenopus laevis oocytes.

Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown. Therefore, accumulation of [(3)H]-DLT in non-transfected, sham (water)-transfected and VSSC (Na(v)1.2+beta(1))-transfected oocytes after a 1h exposure was measured using liquid scintillation counting. Successful transfection of Na(v)1.2+beta(1) VSSCs in X. laevis oocytes was confirmed by two-electrode voltage-clamp; inward, tetrodotoxin (TTX)-sensitive currents were obtained in 98% of all oocytes examined (n=60 in nine experiments). DLT (1.0 microM) induced tail currents in all VSSC-transfected oocytes; TTX also blocked these DLT-induced tail currents. In 0.1 microM DLT solution, non-transfected oocytes accumulated 0.098+/-0.01 ppm [(3)H]-DLT, sham-transfected oocytes accumulated 0.06+/-0.01 ppm DLT, and VSSC-transfected oocytes accumulated 0.050+/-0.009 ppm DLT. In 1.0 microM DLT solution, non-transfected oocytes accumulated 0.62+/-0.08 ppm DLT, sham-transfected oocytes accumulated 0.60+/-0.09 ppm DLT, and VSSC-transfected oocytes accumulated 0.51+/-0.07 ppm DLT. There was a significant difference in DLT accumulation between VSSC-transfected oocytes and non-transfected controls, where the transfected oocytes consistently had less accumulation.

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.

Time and concentration dependent accumulation of [3H]-deltamethrin in Xenopus laevis oocytes.

A primary target of pyrethroid insecticides are the voltage-sensitive sodium channels (VSSCs). Expression of VSSCs in oocytes from Xenopus laevis is an experimental model used to study the effects of pyrethroids. A common assumption when utilizing this model is that media concentration is an accurate substitute for tissue dose. This assumption may not hold true for lipophilic chemicals. [3H]-deltamethrin (DLT) was used to test the hypothesis that media concentration is a good surrogate for tissue concentration. Accumulation of DLT (0.001-10 microM) in non-transfected oocytes exposed for 20 min was determined using liquid scintillation counting. The time course (1.0-180 min) of tissue accumulation of DLT (approximately 1.0 microM (0.50 ppm) in media) was also determined. Results demonstrate that tissue dose increases as a function of time with media concentration underestimating tissue dose at long incubation times (approximately 2.0-fold at 180 min) and overestimating tissue dose short incubation times (approximately 8.6-fold at 5 min). Tissue dose increases as a function of media concentration, with overestimation of tissue dose ranging from 1.5-fold at 0.0005 ppm to 4.1-fold at 5.0 ppm. These data suggest that media concentration does not accurately predict tissue dose at all times for a broad range of deltamethrin concentrations in X. laevis oocytes.

An embryonic chick pancreas organ culture model: characterization and neural control of exocrine release.

An embryonic chick (Gallus domesticus) whole-organ pancreas culture system was developed for use as an in vitro model to study cholinergic regulation of exocrine pancreatic function. The culture system was examined for characteristic exocrine function and viability by measuring enzyme release, and noting histological, morphological, and anti-amylase immuno-fluorescence staining changes over a series of incubation times. This embryonic culture system exhibits loss of viability and morphological degeneration after 12 h of incubation time. Characterization and development of this exocrine model system was an important aspect of this study. Assessment of the 18-day-old embryonic chick pancreas model clearly indicated biochemical and cholinergic functionality, and morphological integrity, of the tissue after 4-h incubation. This embryonic age and incubation period were utilized for all subsequent cholinergic studies. The in vitro model was used to study parasympathetic regulation of exocrine function via the muscarinc receptors present in the embryonic chick pancreas. The effects of synthetic muscarinic agonists (bethanechol and carbachol) and subtype-specific antagonists affected amylase release to varying degrees suggesting heterogeneity of receptors. The effects of the muscarinic receptor antagonists atropine (non-specific), pirenzepine (M(1)-selective) and 4-DAMP [4-diphenylacetoxy-N-methyl-piperidine methiodide] (M(3)-selective) on bethanechol-stimulated amylase release were examined. Atropine and 4-DAMP at concentrations of 2 microM and higher significantly inhibited (p<0.05) agonist-stimulated amylase release, while pirenzipine did not at 2 microM, but did at 200 microM. The M(3) subtype selective antagonist 4-DAMP (2 pM-2 mM) significantly inhibited (p<0.05) 5 mM bethanechol-stimulated amylase release at concentrations of 2 microM and greater (amylase activity decreased from 100.61 to 49.41 U/l/mg). The data suggest the existence of a muscarinic receptor subtype for the embryonic chick pancreas exocrine cells characteristic to the mammalian M(3) glandular subtype.

Ontogeny of voltage-sensitive calcium channel alpha(1A) and alpha(1E) subunit expression and synaptic function in rat central nervous system.

Immunohistochemical expression in the neocortex, hippocampus and cerebellum of the alpha(1A) or alpha(1E) subunit of the voltage-sensitive Ca(2+) channel was examined in Long-Evans hooded rats on gestational day 18 and postnatal days 1, 4, 7, 10, 14, 21, 90, 360 and 720. On gestational day 18 and postnatal day 1, alpha(1A) immunoreactivity was more dense in the neocortex and hippocampus than the cerebellum. By postnatal day 7, levels of alpha(1A) immunoreactivity increased dramatically in the cerebellum, while in neocortex, alpha(1A) immunoreactivity became more sparse, which approached the more diffuse pattern of cellular staining in the mature brain. Expression of alpha(1E) in the neocortex, hippocampus and cerebellum was much less dense than alpha(1A) between gestational day 18 and postnatal day 4. There was also significant alpha(1E) immunoreactivity in the mossy fibers of the hippocampus and in dendrites of Purkinje cells of the cerebellum. Depolarization-dependent 45Ca(2+) influx was examined in rat brain synaptosomes on postnatal days 4, 7, 10, 14, 21 and >60. In neocortical and hippocampal synaptosomes, 45Ca(2+) influx increased steadily with age and reached adult levels by postnatal day 10. In cerebellar synaptosomes, 45Ca(2+) influx was constant across all ages, except for a spike in activity which was observed on postnatal day 21. In neocortical and hippocampal synaptosomes, 100 nM omega-conotoxin MVIIC significantly inhibited 45Ca(2+) influx on postnatal day 10 and 14, respectively, or after. In cerebellar synaptosomes, influx was inhibited by omega-conotoxin MVIIC only on postnatal day 10 or prior. On postnatal day 7, 45Ca(2+) influx was not inhibited in neocortical and hippocampal synaptosomes by a combination of 10 microM nifedipine, 1 microM omega-conotoxin GVIA and 1 microM omega-conotoxin MVIIC, suggesting that an 'insensitive' flux predominates at this age. Overall, the results suggest that expression of voltage-sensitive Ca(2+) channels during development is dynamic and is important in central nervous system development.

Effects of prolonged exposure to nanomolar concentrations of methylmercury on voltage-sensitive sodium and calcium currents in PC12 cells.

The neurotoxicant methylmercury (CH(3)Hg(+)) inhibits voltage-sensitive Na(+) and Ca(2+) currents in neuronal preparations following acute, in vitro, exposure. In the present study, effects on voltage-sensitive Na(+) (I(Na)) and Ca(2+) (I(Ca)) currents in pheochromocytoma (PC12) cells were examined following prolonged exposure to CH(3)Hg(+). When PC12 cells cultured in the presence of nerve growth factor (NGF) for 7 days ('primed') were replated in the presence of NGF and 30 nM CH(3)Hg(+), I(Ca), but not I(Na), amplitude was reduced (29%) significantly approximately 24 h later. Quantitative assessment of morphology indicated that this approximately 24 h exposure to CH(3)Hg(+) significantly reduced neurite length. The N-type voltage-sensitive Ca(2+) channel (VSCC) antagonist omega-conotoxin GVIA (500 pM) was without significant effect on current amplitude or morphology in this exposure protocol. When undifferentiated cells were cultured in the presence of NGF and 10 nM CH(3)Hg(+) for 6 days, I(Ca) and I(Na) amplitude were reduced by 36 and 52%, respectively. I(Ca) at the end of a 150 ms test pulse was also reduced by 40% in CH(3)Hg(+)-treated cells. Thus, both inactivating and non-inactivating I(Ca) were reduced equally. There was no change in [(3)H]saxitoxin or omega-[(125)I]conotoxin GVIA binding, nor were there any morphological alterations in cells treated with CH(3)Hg(+) for 6 days. Omega-conotoxin GVIA (500 pM, 6 days), reduced significantly I(Ca), but not I(Na), but was without effect on morphology. These results demonstrate that prolonged exposure to low concentrations of CH(3)Hg(+) reduces cationic currents in differentiating PC12 cells, but that current reduction is not always associated with morphological alteration.