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 elevated with prolonged incubation. This accumulation was accompanied by marked cytotoxic effects on primary mouse hepatocytes. CTX treatment significantly inhibited cell viability, impaired the morphology, reduced the numbers and accelerated the early apoptotic rate of the primary mouse hepatocytes. For lipid metabolism, CTX significantly increased glycerol kinase (GK) and decreased triglycerides (TG) levels; Acetyl-CoA was markedly elevated. For amino acid metabolism, aspartate aminotransferase (AST) was significantly reduced. In glucose metabolism, glycolysis was enhanced by CTX treatment, as evidenced by significantly elevated lactic acid (LA) levels and markedly increased pyruvic acid (PA) levels. Glycogen phosphorylase (GP) was increased, and glycogen (GN) was decreased. Although glucokinase (GCK) was significantly upregulated by CTX treatment, intracellular glucose (Glu) levels were significantly reduced. G6PC levels were significantly increased alongside a paradoxical decrease in fructose-1,6-diphosphate (FDP). 6-phosphogluconate dehydrogenase (G6PD) was increased, and malic acid was elevated. 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.