Evaluation of Oxidative Stress and Antioxidant Effects of Methylxanthines in Adult Zebrafish Exposed to Zinc Oxide Nanoparticles (ZnO-NPs)

<i>Background and Objectives:</i> Oxidative stress plays a central role in numerous pathological and toxicological processes, and in vivo investigations are essential for understanding integrated systemic responses. Methylxanthines have been reported to modulate redox homeostasis through multiple mechanisms, but their effects in aquatic vertebrate models under metal nanoparticle-induced oxidative stress remain poorly characterized. <i>Materials and Methods:</i> In the present study, adult zebrafish were exposed for 15 days to ZnO nanoparticles (0.69 mg/L) as a pro-oxidant model, and to methylxanthines (caffeine, theobromine, theophylline; 50 mg/L). Oxidative stress biomarkers were assessed by measuring the levels of glutathione peroxidase 1 (GPx1), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) in whole-body homogenates using ELISA. Complementary molecular docking was performed to investigate methylxanthine-enzyme interactions. <i>Results:</i> The most substantial change was observed for SOD level, which significant increased compared to the control group (from 0.122 to 1.090 ng/g; <i>p</i> = 0.001), followed by CAT, which rose from 38.3 pg/g to 100.8 pg/g; <i>p</i> = 0.001), and GPX1 which increased from 84.3 pg/g to 142.2 pg/g; <i>p</i> = 0.011). In parallel, GSH levels decreased by 58.7% (<i>p</i> = 0.001). Co-exposure to methylxanthines significantly modulated the ZnO-NPs exposure response, by mitigating the increase in antioxidant enzyme levels and restoring glutathione. Among the tested compounds, theobromine exerted the strongest protective effect on GPx1 and GSH and caffeine primarily influenced CAT and SOD, whereas theophylline showed overall weaker responses. The molecular docking investigation indicated that all tested methylxanthines can attach to different cavities of the antioxidant enzymes. Theophylline and theobromine established hydrogen bonds and π-stacking interactions with the interfacing amino acids, potentially contributing to the modulation of enzymes stabilization and function under physiological conditions. <i>Conclusions:</i> ZnO-NPs trigger a robust systemic response in zebrafish, whereas methylxanthines display distinct compound-specific modulating effects.