Abstract
The previously unexplored physiological implications of Hesperetin (Hes) on Drosophila melanogaster's DNA (dmDNA), in terms of either genotoxicity or antigenotoxicity, were systematically investigated for the first time against the ethyl methanesulfonate (EMS), an alkylating agent and mutagen, that initiates replication/transcription leading to unnatural G = T and T = G pairing, mutagenicity, toxicity, and carcinogenicity. Either genotoxic or antigenotoxic features of Hes were revealed using an alkaline comet assay against, and compared to Genistein (Gen), Doxorubicin (Dox), Dexrazoxane (ICRF-187), and Resveratrol (Res) (and various mixtures), as internal pharmacological standards in three different concentrations: 0.5, 1, and 2 mM. Adopting a machine-learning-inspired analytical strategy, the study unveiled previously unrecognized synergistic, antagonistic, and additive dynamics among the evaluated agents. Individual compounds and mixtures were likewise assayed for either relaxation or decatenation of dmDNA while interfering with dmTopoisomerase IIα, as well as for listed processes energy deprivation (i.e., ATP hydrolysis denial) while interacting with FRET-labeled dmATPase. The optimal pharmacological profile of Hes, in terms of negligible genotoxicity and pronounced antigenotoxic activity across all tested conditions, was observed at 1 mM: against EMS, it exerted a distinguished antigenotoxic potential while in mixtures, Hes attenuated the genotoxic potential of other agents. Mechanistically, Hes acts through a previously unrecognized dual-mode pharmacology: (1) it functions as a catalytic inhibitor of dmTopIIα, preventing dmDNA relaxation and decatenation and thus limiting EMS-induced damage; and (2) it serves as a mixed-type competitive inhibitor of dmATPase, blocking its dimerization and consequently disrupting the ATP-dependent catalytic cycle of dmTopIIα. This dual-mode pharmacological profile positions Hes as a potential adjuvant in chemotherapy, capable of counteracting the mutagenic and carcinogenic consequences associated with alkylating agents.
