Evaluation of DNA/Chromosome Integrity and Cell Death in Human Metabolically Noncompetent and Competent Cells Exposed to <i>N</i> ′‐(3,5‐Difluorobenzylidene)Pyridine‐4‐Carbohydrazide

The N-acylhydrazone scaffold is recognized as a privileged structure for the design of bioactive substances with increasing applications in medicinal chemistry research. Ensuring the safety of newly developed molecules is a critical step for both human health and environmental protection. Accordingly, this study aimed to evaluate the cytotoxic and genotoxic properties of N'-(3,5-difluorobenzylidene)pyridine-4-carbohydrazide in two cellular models: nonmetabolizing leukocytes and metabolically active hepatic cells (HepG2/C3A). The resazurin-based cytotoxicity analysis, performed with concentrations between 1 and 600 μg/mL, indicated that only the uppermost concentration caused a marked decrease in viability of both cell populations after 48 h of incubation. Regarding genotoxicity at 50, 100, and 200 μg/mL concentrations, no DNA damage was detected in the comet assay, but in the micronucleus test, a significant increase in chromosome alterations in leukocytes at 200 μg/mL concentration was detected, with a decrease in cell proliferation in both cell types. The data indicate that, at the concentrations where the biological effects of acylhydrazone were previously observed, the substance appeared to be safe, but at higher concentrations and/or during chronic exposure, caution and further studies are needed.