"From shadows to shores"-quantitative analysis of CuO nanoparticle-induced apoptosis and DNA damage in fish erythrocytes: A multimodal approach combining experimental, image-based quantification, docking and molecular dynamics

The usage of copper (II) oxide nanoparticles (CuO NPs) has significantly expanded across industries and biomedical fields. However, the potential toxic effects on non-target organisms and humans lack comprehensive understanding due to limited research on molecular mechanisms. With this study, by combining the 96 h in vivo exposure of crucian carp fish, Carassius carassius, to sub-lethal CuO NPs doses (0.5 and 1 mg/dL) with imagebased quantification, and docking and molecular dynamics approaches, we aimed to understand the mechanism of CuO NPs-induced cyto-genotoxicity in the fish erythrocytes. The results revealed that both doses of copper NPs used were toxic to erythrocytes causing oxidative stress response and serious red blood cell morphological abnormalities, and genotoxicity. Docking and 10-ns molecular dynamics confirmed favorable interactions (Delta G = -2.07 kcal mol-1) and structural stability of Band3-CuO NP complex, mainly through formation of H-bonds, implying the potential of CuO NPs to induce mitotic nuclear abnormalities in C. carassius erythrocytes via Band3 inhibition. Moreover, conventional and multiple ligand simultaneous docking with DNA revealed that single, double and triple CuO NPs bind preferentially to AT-rich regions consistently in the minor grooves of DNA. Of note, the DNA-binding strength subtantially increased (Delta G = -2.13 kcal mol- 1, Delta G = -4.08 kcal mol- 1, and Delta G = -6.03 kcal mol- 1, respectively) with an increasing number of docked CuO NPs, suggesting that direct structural perturbation on DNA could also count for the molecular basis of in-vivo induced DNA damage in C. carassius erythrocytes. This study introduces the novel term "erythrotope" to describe comprehensive red blood cell morphological abnormalities. It proves to be a reliable and cost-effective biomarker for evaluating allostatic erythrocyte load in response to metallic nanoparticle exposure, serving as a distinctive fingerprint to assess fish erythrocyte health and physiological fitness.