An Insectified Caco-2 Cell-Based Assay to Monitor Pesticide Transport Processes and Pharmacokinetics

In vitro intestinal models such as Caco-2 cells are integral to pharmacokinetic research. However, insect intestinal cells have proven less tractable for pharmacokinetic studies limiting our knowledge of pesticide pharmacokinetics in these species. To overcome this, we have developed a Caco-2 cell line that has been genetically modified to express insect P-glycoprotein (Pgp), providing a robust ‘insectified’ screening platform to monitor xenobiotic transport. This platform consists of wild-type (Pgpwild type) cells, a Pgp knockout (PgpKO) line which eliminates endogenous background interference, and species-specific Pgp rescue lines. These rescue lines allow for the stable expression of human (Hs Pgp), cotton bollworm (Ha Pgp), and malaria mosquito (Ag Pgp) homologs, enabling direct comparisons of efflux kinetics across different pesticides and species. Functional validation using the substrate Digoxin confirmed high Pgp dependency within the platform. Methyl parathion, an organophosphate insecticide, was recognized and transported by both insect and human Pgps consistent with the low mammalian selectivity of this compound. In contrast, Triflumezopyrim exhibited a high efflux ratio that remained remarkably stable even in the absence of MDR1, indicating that the uptake and pharmacokinetics of this compound are not Pgp-dependent. By successfully differentiating between transporter-specific and independent pathways, these cell lines can serve as a high-fidelity screening tool for predicting novel pesticide selectivity and possibly validating the potential role of transporters in resistance. Future work can extend this system to other transporters.