Effects of cyclophosphamide on the metabolism of primary mouse hepatocytes

Cyclophosphamide (CTX) is limited by hepatotoxicity, whose underlying mechanism remains obscure. Since the liver is the central organ for metabolism, we thereby aimed to investigate the effect of CTX on the metabolism of the primary mouse hepatocytes. The cells were divided into the normal and CTX groups, following the cell counting kit-8 (CCK8), Annexin V and enzyme-linked immunosorbent assay (ELISA) were employed. Our results indicated that the level of 4-hydroxycyclophosphamide, the key active metabolite of CTX, elevated with prolonged incubation. This accumulation was accompanied by marked cytotoxic effects on primary mouse hepatocytes. CTX treatment significantly inhibited cell viability. Subsequent to 4 hours of CTX treatment, cell viability was significantly decreased by 27.6% compared to the normal group (P 6 vs 1.38 × 106, P P P P P P P P P P P (PPP) activation was evidenced by increased 6-phosphogluconate dehydrogenase (G6PD) (9.813 ± 0.70 vs 7.148 ± 0.950, P < 0.05), and mitochondrial impairment was evidenced by elevated malic acid (5.41 ± 1.81 vs 1.47 ± 0.4, P < 0.05). Collectively, these findings demonstrate that CTX drives hepatocytes into a "high consumption, low storage" stress-adapted metabolic phenotype by inducing mitochondrial dysfunction and oxidative stress, disrupting the integrated metabolic network of lipids, amino acids and glucose. The accumulation of 4-hydroxycyclophosphamide might serve as the upstream driver of this metabolic disruption.