Sub-Chronic Chlorpyrifos Exposure Leads to Epigenetic and Sex-Specific Behavioral Changes in Adult Mice

Alyssa R. Daniel; Noah Gernander; Sara Dodge; Cessily Hayes; Emma Simpson-Wade; Emese H. C. Kovács; Gibson Dowd; Jared M. McLendon; Benjamin Hing; Marie E. Gaine

doi:10.64898/2026.06.08.730429

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Sub-Chronic Chlorpyrifos Exposure Leads to Epigenetic and Sex-Specific Behavioral Changes in Adult Mice

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Sub-Chronic Chlorpyrifos Exposure Leads to Epigenetic and Sex-Specific Behavioral Changes in Adult Mice

Alyssa R. Daniel, Noah Gernander, Sara Dodge, Cessily Hayes, Emma Simpson-Wade, Emese H. C. Kovács, Gibson Dowd, Jared M. McLendon, Benjamin Hing and Marie E. Gaine

bioRxiv

Cold Spring Harbor Laboratory Preprints

06/11/2026

DOI: 10.64898/2026.06.08.730429

PMCID: PMC13277971

PMID: 42327306

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https://doi.org/10.64898/2026.06.08.730429View

Preprint (Author's original) This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

Chlorpyrifos is a widely used organophosphate pesticide that exerts its primary toxic effect through inhibition of acetylcholinesterase (AChE). Although the acute neurotoxicity of chlorpyrifos is well characterized, the lasting biochemical, behavioral, and epigenetic consequences of sub-chronic exposure remain poorly understood, particularly when considering sex-specific differences. Therefore, we exposed male and female C57BL/6J mice to either peanut oil (n=19), low chlorpyrifos exposure (1 mg/kg/day; n=19), or high chlorpyrifos exposure (10 mg/kg/day n=10) repeatedly for 21 days via subcutaneous injection. Blood AChE activity, behavior, and hippocampal DNA methylation were measured across groups. During exposure, AChE activity decreased in both males and females but only returned to baseline after behavioral testing in females exposed to low chlorpyrifos levels. Behavioral tests also revealed a sex-specific phenotype, with females in the low exposure group exhibiting reduced forced swim test immobility and a significant time by exposure interaction in open field habituation. No significant behavioral effects were observed in males. Significant DNA methylation changes were observed at 3,538 CpG sites in male and female mice after high exposure. Sex-specific analyses revealed two female-specific differentially methylated CpGs after high exposure. Pathways enriched for differentially methylated genes included several related to synaptic remodeling, cholinergic synapse, and various endocrine systems. These findings demonstrate that repeated high chlorpyrifos exposure leads to persistent cholinergic disruption and DNA methylation changes. However, the female-specific behavioral changes seen are independent of AChE activity and widespread DNA methylation changes, suggesting additional mechanisms, present only in females, may underlie behavioral sensitivity to chlorpyrifos.

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Title: Subtitle: Sub-Chronic Chlorpyrifos Exposure Leads to Epigenetic and Sex-Specific Behavioral Changes in Adult Mice
Creators: Alyssa R. Daniel - University of Iowa
Noah Gernander - University of Iowa
Sara Dodge - University of Iowa
Cessily Hayes - University of Iowa
Emma Simpson-Wade - University of Iowa
Emese H. C. Kovács - University of Iowa
Gibson Dowd - University of Iowa
Jared M. McLendon - University of Iowa
Benjamin Hing - University of Iowa
Marie E. Gaine - University of Iowa
Resource Type: Preprint
Publication Details: bioRxiv
DOI: 10.64898/2026.06.08.730429
PMID: 42327306
PMCID: PMC13277971
eISSN: 2692-8205
Publisher: Cold Spring Harbor Laboratory Preprints
Language: English
Date posted: 06/11/2026
Academic Unit: Molecular Physiology and Biophysics; Psychiatry; Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Internal Medicine
Record Identifier: 9985175374002771

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