Dimethyl Sulfoxide as a Biocompatible Extractant for Enzymatic Bioluminescent Toxicity Assays: Experimental...

Diesel fuel is among the most persistent petroleum-derived pollutants in soils, posing long-term ecological and toxicological risks, especially in cold-climate regions where natural degradation is limited. Reliable assessment of diesel-contaminated soils remains difficult because conventional solvent-based analyses are incompatible with bioassays, while aqueous extracts underestimate hydrocarbon toxicity. This study evaluated dimethyl sulfoxide (DMSO) as a biocompatible extractant for enzymatic bioluminescent toxicity assays employing the coupled NAD(P)H:FMN-oxidoreductase and bacterial luciferase (BLuc-Red) system. Soil samples artificially contaminated with diesel fuel were analyzed using DMSO extracts in combination with molecular dynamics (MD) simulations to examine enzyme stability in solvent environments. Moderate DMSO concentrations (4-6% v/v) maintained enzymatic activity, whereas higher levels caused partial inhibition. Diesel hydrocarbons dissolved in DMSO strongly suppressed luminescence, and soil extracts exhibited a clear dose-response relationship between contamination level and enzymatic inhibition. MD simulations confirmed that neither DMSO nor diesel induced large-scale unfolding of luciferase or reductase, though localized flexibility changes and partial dehydration of active site residues was observed, which may account for the detected inhibition of luminescence at higher DMSO concentrations. These results demonstrate that DMSO provides an effective and biocompatible extraction medium for enzymatic bioluminescent assays, enabling accurate toxicity evaluation of petroleum-contaminated soils and offering a promising tool for ecotoxicological risk assessment in oil-impacted environments.