Hepatoprotective and antioxidant activity of novel hydrazone derivatives in experimental models of hepatotoxicity in vitro

The safety profile of two newly synthesized series of hydrazone derivatives (7 a–e and 8 a–e ) was studied in different liver-derived cell and subcellular culture models, i.e., isolated rat liver hepatocytes and microsomes, and the HepG2 cell line. The hydrazones, which have previously been reported to possess neuroprotective and antioxidant activity, were further assessed for possible hepatoprotective and antioxidant properties in several in vitro models of hepatotoxicity: non-enzymatic lipid peroxidation in isolated rat liver microsomes; metabolic bioactivation induced by carbon tetrachloride (CCl 4 , 86 µM) in rat hepatocytes; and oxidative stress models induced by tert-butyl hydroperoxide (t-BuOOH, 75 µM) in rat hepatocytes and hydrogen peroxide (H 2 O 2 , 0.1 mM) in HepG2 cells. Compounds 7d and 8d demonstrated the lowest hepatotoxic potential in isolated rat liver microsomes and hepatocytes. In HepG2 cells, all tested hydrazones exhibited low or negligible in vitro toxicity, with IC₅₀ values ranging from 64.28 to 205.10 µM. Compound 7c showed the lowest cytotoxicity, followed by 7a and 8d. All hydrazones exhibited significant antioxidant activity under conditions of non-enzymatic lipid peroxidation, with 7d and 8d displaying the most pronounced effects. In the CCl₄-induced hepatotoxicity model, all compounds demonstrated statistically significant hepatoprotective and antioxidant activity, as evidenced by improved hepatocyte viability, reduced lactate dehydrogenase (LDH) leakage, preserved intracellular glutathione (GSH) levels, and decreased malondialdehyde (MDA) production. Similar protective trends were observed in the model of t-BuOOH-induced oxidative stress, again with 7d and 8d showing superior effects. In the H 2 O 2 -induced oxidative stress model in HepG2 cells, compounds 7c, 7d, 7e, 8d, and 8e exhibited protective activity, with 7e and 8d providing the strongest protection. Overall, compounds 7d and 8d emerged as the most promising candidates, combining low hepatotoxicity with pronounced hepatoprotective and antioxidant effects, supporting their potential for further pharmacological development.