Updating the Standards of Professional Competence in the Field of Toxicology: The Second Generation of Best Practice.

The American Board of Toxicology (ABT), in consultation with ACT Credentialing & Career Services (ACT), performed a practice analysis study of general toxicology in 2020-21. This work follows up on an initial practice analysis commissioned by the ABT and conducted in 2014-2015, results of which were published in 2016. The purpose of the current, second-generation study was to update and validate the existing process-based delineation of practice of general toxicologists, including major domains of responsibility and tasks performed in practice. In addition, the study included the review, update, and validation of the knowledge areas required by toxicologists developed by subject-matter experts (SMEs) that have been used for ABT examination development initiatives. Consistent with best practices in the field of credentialing, ABT also contracted with ACT to conduct 2 follow-on activities: a study to evaluate the reliability of a reduced-length ABT examination and a standard setting study to establish a valid passing score for the updated examination. In addition to informing ongoing ABT certification examination and question writing activities, it is anticipated that the results of this practice analysis will be of value to those responsible for developing graduate and undergraduate toxicology curricula, creating continuing education content, and authoring textbooks covering the contemporary practice of toxicology.

Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M(3) muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M(3) muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study, we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25-100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert-butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

Astrocytes play an important role in neuronal development through the release of soluble factors that affect neuronal maturation. Shotgun proteomics followed by gene ontology analysis was used in this study to identify proteins present in the conditioned medium of primary rat astrocytes. One hundred and thirty three secreted proteins were identified, the majority of which were never before reported to be produced by astrocytes. Extracellular proteins were classified based on their biological and molecular functions; most of the identified proteins were involved in neuronal development. Semi-quantitative proteomic analysis was carried out to identify changes in the levels of proteins released by astrocytes after stimulation with the cholinergic agonist carbachol, as we have previously reported that carbachol-treated astrocytes elicit neuritogenesis in hippocampal neurons through the release of soluble factors. Carbachol up-regulated secretion of 15 proteins and down-regulated the release of 17 proteins. Changes in the levels of four proteins involved in neuronal differentiation (thrombospondin-1, fibronectin, plasminogen activator inhibitor-1, and plasminogen activator urokinase) were verified by western blot or ELISA. In conclusion, this study identified a large number of proteins involved in neuronal development in the astrocyte secretome and implicated extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

Biological fate of low-calorie sweeteners.

With continued efforts to find solutions to rising rates of obesity and diabetes, there is increased interest in the potential health benefits of the use of low- and no-calorie sweeteners (LNCSs). Concerns about safety often deter the use of LNCSs as a tool in helping control caloric intake, even though the safety of LNCS use has been affirmed by regulatory agencies worldwide. In many cases, an understanding of the biological fate of the different LNSCs can help health professionals to address safety concerns. The objectives of this review are to compare the similarities and differences in the chemistry, regulatory status, and biological fate (including absorption, distribution, metabolism, and excretion) of the commonly used LNCSs: acesulfame potassium, aspartame, saccharin, stevia leaf extract (steviol glycoside), and sucralose. Understanding the biological fate of the different LNCSs is helpful in evaluating whether reports of biological effects in animal studies or in humans are indicative of possible safety concerns. Illustrations of the usefulness of this information to address questions about LNCSs include discussion of systemic exposure to LNCSs, the use of sweetener combinations, and the potential for effects of LNCSs on the gut microflora.

Muscarinic receptor-activated signal transduction pathways involved in the neuritogenic effect of astrocytes in hippocampal neurons.

Astrocytes have been shown to release factors that affect various aspects of neuronal development. We have previously shown that the acetylcholine analog carbachol, by activating muscarinic M(3) receptors in rat astrocytes, increases their ability to promote neuritogenesis in hippocampal neurons. This effect was mediated by an increased expression and release by astrocytes of several permissive factors, a most relevant of which was fibronectin. In the present study we investigated the signal transduction pathways involved in these effects of carbachol in astrocytes. Results show that multiple pathways are involved in the effects of carbachol on astrocyte-mediated increases in fibronectin expression and neuritogenesis. These include the phospholipase D pathway, leading to sequential activation of protein kinase C (PKC) ζ, p70S6 kinase and nuclear factor-κB; the phosphoinositide-3 kinase pathway; and the PKC ε pathway leading to activation of mitogen activated protein kinase. These pathways were shown to mediate the effect of carbachol on neurite outgrowth as well as the increased expression of fibronectin, further substantiating the important role of the latter in astrocyte-mediated neuritogenesis. Interference with these signaling pathways would be expected to impair astrocyte-neurons communication leading to impaired neuronal development.

Modulation of neuritogenesis by astrocyte muscarinic receptors.

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.