An Evaluation of Neurotoxic Chemicals: ‘Omics Approach to Identify Neurotoxic Adverse Outcome Pathways

Humans are exceedingly exposed to various chemicals over a lifetime, of which more than 100,0000 require testing for potential adverse effects. The National Academy of Science has suggested utilizing advances in molecular biology to identifying adverse outcome pathways (AOPs) for the development of new approach methods (NAMs) to prioritize chemicals that require testing for neurotoxicity including developmental exposure.  Current methods in neurotoxicity testing that rely on in vivo neurobehavior and gross pathology may not be sensitive enough to detect changes caused by realistic low levels chemical exposures. We hypothesize that exposure leads to several initiating cascades culminating in an adverse effect.  Our focus will be on the structure as well as function of the nervous system in addition to looking at the neurobehavior. With our in vitro model using primary neuronal cultures, we will perform broad range proteomics (LC-MS) and transcriptomics (RNA-Seq) to develop proteomic and genomic signatures for each test chemical. Our first set of chemicals included positive controls chlorpromazine (CAS# 69-09-0) and deltamethrin (CAS# 52918-63-5) along with five test chemicals: fipronil (CAS# 120068-37-3), kainic acid (CAS# 52918-63-5), triethyltin bromide (CAS# 2767-54-6), bis(tributyltin) oxide (CAS# 56-35-9), and lindane (CAS# 58-89-9). Lactate dehydrogenase (LDH) assays for cytotoxicity were conducted for each chemical with a range of concentrations and time points over 48 hours. Dose-response profiles were established for each compound for use in future transcriptomics/proteomics studies. For each chemical, high and low doses have been determined that do not exceed 20% released LDH for two time points: 3 hours (short exposure) and 24 hours (long exposure). Proteomic and genomic information will be correlated with AOPs representing neuronal structure and function in in vitro and in vivo.  By identifying commonalities between chemical signatures, we can develop an empirical model and identify a set of genes/proteins which can be used for screening untested chemicals for their neurotoxicity or developmental neurotoxicity. The overall goal is to move from expensive and time-consuming in vivo testing batteries to faster and less expensive batteries that allow true screening and prioritization of large numbers of chemicals. (This abstract does not necessarily reflect USEPA policy).

Impact/Purpose

Identifying adverse outcome pathways (AOPs) for prioritization of chemical testing is greatly lacking for brain health.  This study is an attempt to identify several AOPs responsible for the effects on the nervous system and to further develop an in vitro cell culture model that ultimately can be used to design a computational model that includes pharmacokinetic, SAR, and AOP information to increase the predictability of untested chemicals.  Proteomic signatures (LC-MS) along with genomic signatures (RNA-Seq) will not only provide AOP data but link toxicity initiating events with their adverse outcomes. Since this model includes SAR, guidance will be provided to develop safer products for a sustainable environment with minimal to no impact on human health. In this study we began with a test set that includes known neurotoxicants with known negative controls. The overall goal is to move from expensive and time-consuming in vivo testing batteries to faster and less expensive paradigms that allow accurate screening and prioritization of large numbers of chemicals.

Citation

Valdez, M., B. Langenbach, C. Mack, D. Freeborn, T. Shafer, D. Herr, AND Prasada Rao Kodavanti. An Evaluation of Neurotoxic Chemicals: ‘Omics Approach to Identify Neurotoxic Adverse Outcome Pathways. Society of Toxicology (SOT 2022) - 61st Annual Meeting and ToxExpo, San Diego, California, March 27 - 31, 2022.