Multi-endpoint toxicological assessment of nifuroxazide through experimental and molecular docking analyses

In this study, the potential genetic and biochemical toxicities of the antibiotic active substance nifuroxazide on Allium cepa L., a non-target model organism, were evaluated in detail. Mitotic index (MI), micronucleus (MN) frequency, chromosomal abnormality (CAs) rate, malondialdehyde (MDA) level, superoxide dismutase (SOD) and catalase (CAT) enzyme activities, and chlorophyll a and b contents were used as key indicators for determining toxic effects. The effect of nifuroxazide on DNA integrity was investigated using the Comet test, while its interaction with DNA, proteins, and enzymes was studied using the molecular docking method. In the experimental setup, the A. cepa bulbs were randomly divided into four experimental sets. One was an untreated control group, and the other three were separate treatment groups. Onions in the control group were germinated in tap water, while onions in the treatment groups were exposed to nifuroxazide solutions prepared at concentrations of 0.0179, 0.0357, and 0.0714 mg/L throughout the germination period. After the germination period, the root and leaf tissues obtained were evaluated for biochemical and genetic analyses. The findings revealed that MI, DNA content, and chlorophyll (a and b) levels were highest in the control group (Group I). In contrast, MN frequency, CAs, MDA level, and SOD and CAT activities increased markedly in the nifuroxazide-treated groups. Parallel to the increasing nifuroxazide doses, statistically significant decreases ( p < 0.05) were observed in MI, DNA, and chlorophyll contents, while significant increases were detected in MN formation, CAs, and oxidative stress parameters. Compared with the control group, the highest nifuroxazide dose (Group IV) resulted in an approximately 22% reduction in MI, a 55.59% decrease in Head DNA (%), a 1.9-fold reduction in chlorophyll a, and a 3.3-fold reduction in chlorophyll b, while the MDA level, SOD activity, and CAT activity increased by 2.6-, 1.6-, and 2.0-fold, respectively. Nifuroxazide exposure induced various CAs such as vagrant chromosome, sticky chromosome, fragment, bridge, unequal chromatin distribution, binucleated cells, and spindle-associated orientation disorders. The Comet analysis showed a decrease in head DNA percentage and an increase in tail DNA percentage with increasing doses, confirming the deterioration of DNA integrity. Molecular docking analyses suggested that nifuroxazide may interact with DNA, tubulin proteins, topoisomerase enzymes, and chlorophyll biosynthesis-related enzymes, providing supportive mechanistic evidence for the experimentally observed genetic and biochemical alterations. Overall, the results indicate that environmental antibiotic residues may cause significant stress and damage to ecosystem components. The uncontrolled release of antibiotics such as nifuroxazide into the environment may disrupt critical metabolic and genetic processes in non-target plants; therefore, limiting the environmental dissemination of these substances and regularly monitoring residue levels are of great importance from an eco-toxicological perspective.