In vitro characterization of thymol-induced effects in IPEC-J2 intestinal cells

Background: Natural compounds are gaining interest in veterinary medicine as alternatives to conventional pharmaceuticals. Thymol (THY) is a natural volatile monoterpenoid phenol, widely known for its antibacterial, antiviral, antioxidant, and anti-inflammatory properties, also including antiparasitic activity against gastrointestinal parasites. However, a thorough understanding of its selectivity and safety for the animal host remains limited. Hypothesis: Despite its use as a feed additive, THY has limited selectivity as an antiparasitic compound because of its intestinal toxicity. Methods: To calculate THY selectivity index (SI), efficacy (EC50) against Cryptosporidium parvum and Giardia duodenalis was assessed using previously established methods, while toxicity (TC50) on 21-day differentiated IPEC-J2 cells was evaluated using Alamar Blue (AB), WST-1, and CellTiter-Glo® (GLO) assays. To assess THY transport and its effects on cell barrier integrity and transcriptome, polarized IPEC-J2 cells were incubated for 48 h with two subcytotoxic concentrations within the EC50 range (32 and 48 μg/mL). Transepithelial Electrical Resistance (TEER) was monitored; media and cell monolayer samples were subjected to LC-MS/MS and RNA-seq analyses. Results: THY showed poor selectivity (1.6 < SI < 2.3), with TC50 of 64, 80, and 95 μg/mL (AB, WST-1, and GLO, respectively), and EC50 of 23–58 and 10–50 μg/mL against C. parvum and G. duodenalis, respectively. Further analyses on the transwell supports demonstrated THY transport across the epithelial barrier. At 48 μg/mL, THY slightly decreased barrier integrity, consistent with transcriptional downregulation of cytoskeletal and adhesion-related genes. Moreover, it modulated genes involved in cell-cycle regulation and chromosome organization, beyond immune signaling, lipid transport, and aldehyde metabolism. Conclusion: THY's poor selectivity and intestinal toxicity are likely attributable to a non-specific mechanism of action. Our findings emphasize the need for a comprehensive understanding of essential oils' mode of action, toxicity, and precise composition before their use in vivo. Acknowledgements: This study received funding from La Veterinaria srl; EU Horizon Europe Project 101136346 EUPAHW – SOA19.