Diurnal and Circannual Variations Influence the Severity of Artesunate–Gentamicin–Induced Liver Injury

Objective: This study aims to determine whether time-of-day (12:00 h vs. 00:00 h) and season (rainy vs. dry) modify liver injury induced by gentamicin and artesunate in male Wistar rats. Methods: A total of 128 male Wistar rats were randomized to 16 groups (n=7–8/group) and treated once daily intraperitoneal for 7 days with normal saline, gentamicin (120 mg/kg), artesunate (100 mg/kg), or the combination during the rainy (August 2023) and dry (February 2024) seasons. Primary outcomes were serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP); secondary outcomes included superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and liver histology. Normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene tests. Two-way analysis of variance (ANOVA; season×dosing time) with Bonferroni correction was used to evaluate effect sizes (partial η<sup>2</sup>) and 95% confidence intervals (CIs). Results: Two-way ANOVA revealed significant season×dosing-time interactions for ALT, AST, and ALP (all p<0.01; partial η<sup>2</sup>≈0.10–0.25). Overall, dry-season exposure produced larger enzyme elevations and greater reductions in antioxidant activities than rainy-season exposure. Nighttime dosing (00:00 h) in the dry season produced the largest ALT increases compared with rainy-season daytime saline (p<0.001). Histology corroborated biochemical findings, showing more pronounced necrosis and inflammatory infiltration with dry-season night dosing. Artesunate attenuated some gentamicin-induced changes in the rainy season but not in the dry season. Conclusion: Season and dosing time jointly modulate artesunate–gentamicin hepatotoxicity in rats; dry-season nighttime dosing markedly increases injury. These results support chronotherapeutic consideration in settings where seasonal environmental stressors vary.