Citation:

Johnstone, A., C. Mack, M. Valdez, T. Shafer, D. Herr, P. Kodavanti, AND R. Lo Pachin. Acute In Vitro Effects on Embryonic Rat Dorsal Root Ganglion (DRG) Cultures by In Silico Predicted Neurotoxic Chemicals: Evaluations on Cytotoxicity, Neurite Length, and Neurophysiology. TOXICOLOGY IN VITRO. Elsevier Science Ltd, New York, NY, 69(104989):1, (2020). https://doi.org/10.1016/j.tiv.2020.104989

Impact/Purpose:

Many chemicals are proposed to produce toxic responses through non-receptor mediated actions of the parent compound (or its metabolites) with endogenous proteins and cellular macromolecules (Thomas et al., 2013). For reproductive and developmental toxicants, predictions based on structure activity modeling have been proposed (Blackburn et al., 2011). A method based on chemical structure, known as the Hard and Soft Acids and Bases (HSAB) hypothesis, has recently been proposed to predict peripheral neurotoxicity (LoPachin et al., 2012). Peripheral neurotoxicity (i.e. paresthesia, hypesthesia, dysesthesia, anaphia, etc) continues to be a human health problem that is not easily predicted in advance. In spite of recent advances in chemical screening (e.g., EPA’s ToxCast) such programs do not have exhaustive coverage of molecular initiating events (MIE’s) for all forms of toxicity(e.g. neurotoxicity and reproductive toxicity) and are based on “acute exposures” in vitro that may not account for the effects of metabolism on chemicals. To address these data gaps in EPA’s existing screening programs, we propose to develop an AOP for certain types of toxicities based on HSAB predictions from chemical structures . The HSAB hypothesis was developed for neurotoxicants, and will be expanded and tested using additional chemicals. Neuropathies will be evaluated by the evaluation of protein adduct formation in functionally crucial neuronal macromolecules. We will use the existing findings to “follow the data”, provide proof of principle, and expand the number of chemicals (chemical space) tested using the HSAB theory, thereby helping to establish its predictive validity. Linking the predictions from the HSAB theory with traditional adverse outcomes is important for increasing its use in the risk assessment process. Successful predictions will allow the development of Adverse Outcome Pathways (AOPs) for peripheral neurotoxicity. If successful in predicting neurotoxicants, the theory can be further expanded into other types of toxicity. This research project will increase the predictive capability of EPA’s screening process for multiple types of toxicity.

Description:

The Hard-Soft Acid and Base hypothesis can be used to predict the potential bio-reactivity (electrophilicity) of a chemical with intracellular proteins, resulting in neurotoxicity. Twelve chemicals predicted to be neurotoxic were evaluated in vitro in rat dorsal root ganglia (DRG) for effects on cytotoxicity (%LDH), neuronal structure (total neurite length/neuron, NLPN), and neurophysiology (mean firing rate, MFR). DRGs were treated acutely on days in vitro (DIV) 7 (1–100 μM) with test chemical; %LDH and NLPN were measured after 48 h. 4-cyclohexylhexanone (4-C) increased %LDH release at 50 (29%) and 100 μM (56%), citronellal (Cit) and 1-bromopropane increased %LDH at 100 μM (22% and 26%). 4-C, Cit, 2,5 Hexanedione (2,5Hex), phenylacetylaldehyde (PAA) and 2-ethylhexanal decreased mean NLPN at 48 h; 50 and 100 μM for 4-C (28% and 60%), 100 μM Cit (52%), 100 μM 2,5- Hex (37%) 100 μM PAA (41%) and 100 μM for 2-ethylhexanal (23%). Separate DRG cultures were treated on DIV 14 and changes in MFR measured. Four compounds decreased MFR at 50 or 100 μM: Acrylamide (−83%), 3,4-dichloro-1-butene (−93%), 4-C (−89%) and hexane (−79%, 50 μM). Changes in MFR and NLPN occurred in absence of cytotoxicity. While the current study showed little cytotoxicity, it gave insight to initial changes in MFR. Results provide insight for future chronic exposure experiments to evaluate neurotoxicity.

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