In vivo verification of fish keratocyte explant culture for use as innate immunotoxicity screening assay: 2,4-Dicholorphenoxyacetic acid effects in fathead minnow (Pimephales promelas) keratocyte cell sheet migration

2,4-Dichlorphenoxyacetic acid (2,4-D) is the active ingredient in selective herbicides commonly used worldwide to control non-native aquatic plants. Limited research on the impacts of 2,4-D exposure on teleost innate immunity, such as barrier defense provided by epidermis, has been conducted. Fish keratocyte explant culture serves as a rapid, low-cost, cutaneous wound healing in vitro model of cell differentiation and migration. To test the hypothesis that this assay can serve as an innate immunotoxicity screening tool, scales containing keratocyte sheets were non-lethally extracted from adult male fathead minnows (Pimephales promelas), incubated in culture medium with increasing concentrations of 2,4-D (0–2,560 ppm), and cell sheet migration was measured. At 24h post scale pull, keratocyte cell sheet farthest migration distance significantly increased upon exposure to 0.04 ppm and significantly decreased upon exposure to ≥160 ppm 2,4-D as compared to controls. A punch biopsy on adult male fathead minnows was performed at the start of a 24h exposure to environmental application concentrations of 2,4-D (0, 0.04, 0.4, 4 ppm), followed by a dye penetration assay to observe keratocyte cell sheet migration in vivo. At 6h post biopsy, keratocytes migrated significantly farther across the wound bed in fish exposed to 0.04 and 0.4 ppm 2,4-D as compared to controls. Results suggest that keratocyte explant culture can model environmentally relevant in vivo scenarios and that 0.04–4 ppm 2,4-D does not negatively impact keratocyte migration rate. Future studies using other environmental toxicants or fish species may continue to explore the value of this assay as a practical screening tool for toxicological effects on cutaneous wound healing.