Friday, May 30, 2025
5/30/2025
Novel behavioral screening tool for therapeutics against organophosphorus agents - Objectives. Terrorist acts or accidental poisoning involving acute exposure to organophosphorus (OP) agents, such as nerve gases and OP pesticides (OPPs), pose a serious threat to induce mass casualties. Acute OP toxicity results from inhibition of acetylcholinesterase (AChE), causing acetylcholine accumulation and cholinergic crisis. Symptoms include increased secretions, respiratory distress, loss of consciousness, and status epilepticus, causing permanent brain damage and death if untreated. Acute OP toxicity is currently treated with atropine and pralidoxime (2-PAM) to mitigate cholinergic hyperstimulation, and midazolam to stop epileptic seizures. This treatment reduces mortality, but only if administered within minutes of exposure and is thus not realistic for treating the civilian population during a mass casualty. Moreover, adverse health effects may remain after treatment. Thus, there is an urgent need for inexpensive therapeutics that can reduce mortality and alleviate adverse effects when administered later. The rigor of the prior research is hampered because current mammalian screening methods are slow and expensive, restricting the number of candidates that can be tested. High- throughput screening (HTS) platforms that can rapidly and cheaply screen possible candidates promise to accelerate the development of new therapeutics. The overarching goal of this research is to develop a cost- effective HTS organismal platform to streamline and accelerate first-tier screening of possible therapeutics using the planarian D. japonica. The specific objective of this proposal is to test the hypothesis that planarian HTS is “fit for purpose” as a screening tool for therapeutic candidates that can alleviate the symptoms of acute OPP exposure. Planarians are small flatworms with tractable, evolutionarily conserved neuronal circuits and a wide repertoire of complex behaviors that are amenable to HTS. As invertebrates, they are considered a non-animal organism. Unique to the planarian system, developing organisms are metabolically competent and can be screened from exposure onset into adulthood, allowing for the assessment of toxicants in mixed populations. Experimental approach. In Aim 1 will study the acute toxicity of 2 OPPs of concern, diisopropyl fluorophosphate and parathion. Using HTS, we will characterize their toxicity profiles and evaluate lethality, morphology and behavior in adult planarians at 30 minutes and 24 hours of exposure. In aim 2, we will verify that OPP-induced seizures in planarians are true seizures by developing quantitative metrics for comparison with verified planarian seizures and blocking of OPP-induced seizures with midazolam. In aim 3, we will demonstrate that acute OPP toxicity in planarians is responsive to therapeutics and that OPP-induced symptoms can be alleviated using combinatory treatment with atropine, 2-PAM, and midazolam, to establish a baseline to compare to novel drugs. Expected results. This proposal will demonstrate the value of planarian HTS for testing therapeutic candidates to treat OPP acute toxicity. The development of this rapid organismal screening method could be easily adopted to other test paradigms, including different life stages, lengths of exposure, and chemical domains.
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