Measurement of multixenobiotic resistance activity in enchytraeids as a tool in soil ecotoxicology

Abstract

The multixenobiotic resistance (MXR) mechanism is the first defense line against xenobiotics. Enchytraeids, a model organism in soil ecotoxicology, are often exposed to various xenobiotics, some of which may influence MXR activity. Since MXR activity has not been studied in these organisms, the aim of this paper was to establish a methodology for the implementation of the dye assay in enchytraeids. Enchytraeus albidus and Enchytraeus crypticus were exposed to model chemosensitizers: cyclosporine A (CA), dexamethasone (DEX), ivermectin (IVM), rifampicin (RIF), verapamil (VER), and fungicide propiconazole (PCZ). Thereafter, a dye assay with specific fluorescent dyes rhodamine B and rhodamine 123 was performed. Changes in MXR activity caused by variations in dye accumulation were measured fluorometrically. CA, IVM, and VER were found to inhibit the MXR system and increase the fluorescence 2.2-fold, while DEX and RIF induced the MXR system and decreased the fluorescence. CA was the strongest inhibitor in both E. albidus (IC50 5.48 ± 1.25 μM) and E. crypticus (IC50 5.20 ± 3.10 μM). In the validation experiment, PCZ was found to inhibit the MXR system. The IC₅₀ varied between species and exposure substrates: water (E. albidus - IC₅₀ 0.74 ± 0.24 mg/L; E. crypticus - 1.31 ± 0.24 mg/L) or soil (E. albidus - 1.79 ± 0.42 mg/kg; E. crypticus - 1.79 ± 0.17 mg/kg). In conclusion, the tested compounds changed the MXR activity, which confirms the applicability of this method as a valuable complementary biomarker in soil ecotoxicology.

Keywords: Chemosensitizers; Efflux pump; Enchytraeus; MXR protein.