Differential dihydroethidium fluorescence spectra in cell-free and cellular superoxide models: roles of riboflavin, FMN, FAD, and ions.

Dihydroethidium (DHE) is widely used for superoxide detection, yet reported excitation and emission values vary across studies. To address this, we employed full-spectrum scanning to compare DHE fluorescence between a xanthine oxidase (XO)-based cell-free system and a rotenone-treated cellular model, and to assess factors contributing to spectral shifts. In the XO system, the excitation peak was ∼480 nm, whereas in cells it shifted to ∼520 nm. Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) accounted for this shift, while calcium and bicarbonate ions modulated both peak position and fluorescence intensity. Riboflavin depletion reduced intracellular flavin levels but did not restore the peak to 480 nm, indicating additional roles for FMN and FAD. Among scavengers, only tiron directly inhibited DHE fluorescence in the cell-free system, with enhanced activity in the presence of Ca²⁺ and Mg²⁺. In contrast, responses in cells varied by type and rotenone concentration, suggesting indirect modulation through endogenous antioxidant defenses. Addition of FMN, FAD, or cell lysates to the cell-free system attenuated scavenger efficacy, supporting intracellular interference. These findings demonstrate that riboflavin metabolism and ionic microenvironments critically shape DHE spectral behavior. Accurate interpretation of DHE-based superoxide detection therefore requires prior spectral evaluation to distinguish genuine superoxide signals from cofactor- or ion-dependent effects.