2,4-Dichlorophenoxyacetic acid (2,4-D) is used as a selective herbicide and associated with a variety of toxicities in mammals. In contrast, melatonin is an antioxidant that promotes the elimination of free radicals. In the present study, the protective effects of melatonin (10 mg/kg body weight) against 2,4- D (low, mid, and high dose-16.5, 33.0, and 66.0 mg/kg body weight) induced testicular steroidogenesis alteration were examined using in vivo and in silico models. Doses of 2,4-D and melatonin were administered orally for 28 days. The evaluated parameters were body weight, total protein, markers for male reproductive function, and steroidogenesis i.e. testis weight, total lipid, cholesterol, testosterone, 3 beta-hydroxysteroid dehydrogenase, 17 betahydroxysteroid dehydrogenase, total sperm count, sperm motility, and sperm viability along with the histopathology of the testis. The statistical significant value was considered at p<0.05. Molecular docking study was performed for interaction of 2,4-D and melatonin with steroid binding proteins. In vivo results revealed that 2,4-D treatment showed a significant dose-dependent alteration in above all studied parameters. No significant auto-recovery was observed in the withdrawal study, on the contrarily, the altered parameters were normalized and comparable to control when melatonin was given alone and in combination with 2,4-D. In silico results also demonstrated that the binding affinity of melatonin with steroid binding proteins is higher than 2,4-D. Collectively, these in vivo and in silico findings indicated that 2,4-D induced testicular toxicity accompanied by steroidogenesis upset and can be reduced by melatonin significantly by interacting directly and strongly with studied molecular markers.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a systemic phenoxy herbicide that induces oxidative stress. In contrast, melatonin is a secretory product of the pineal gland with antioxidant properties. In the present study, the ameliorative potential of melatonin (10 mg/kg body weight) was investigated against 2,4-D (low, mid, and high dose-16.5, 33.0, and 66.0 mg/kg body weight) induced altered haematological variables using in vivo and in silico models. Doses of 2,4-D and melatonin were administered orally for 28 days. The evaluated haematological indices in the present study were Haemoglobin (Hb), Red Blood Corpuscles (RBC), Haematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), White Blood Corpuscles (WBC), Lymphocytes, Monocytes, Granulocytes, Platelet Count (PT), Mean Platelet Volume (MPV), Plateletcrit (PCT), and Erythrocyte Sedimentation Rate (ESR). The statistical significant value was considered at p<0.05. Molecular docking study was performed for interaction of 2,4-D and melatonin with haemoglobin. In vivo results revealed that 2,4-D treatment showed a significant dose-dependent alteration in above all studied haematological indices. No significant auto reversal effects were observed in the withdrawal study, on the contrarily, the altered haematological indices were normalized and comparable to control when melatonin was given alone and in combination with 2,4-D. In silico results also demonstrated that 2,4-D and melatonin showed competitive bindings with haemoglobin. In nutshell, these in vivo and in silico findings depicted those haematological indices were altered by 2,4-D toxicity and can be abridged by melatonin attributed to its ameliorative potential as also evidenced by molecular docking.