Effects of cyclophosphamide on the metabolism of primary mouse hepatocytes

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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 < 0.01), and this inhibitory effect was sustained for up to 8 hours. Microscopy examination indicated that primary hepatocytes treated with CTX for 6 hours, compared to cells in normal group, exhibited marked morphological changes, characterized by wrinkled contours, coarse cellular membranes, and a grainy cytoplasm. Furthermore, CTX exposure led to a substantial decrease in cell count, with a 50% reduction observed (2.56 × 106 vs 1.38 × 106, P < 0.001). Flow cytometry demonstrated a significant increase in early apoptosis following CTX treatment compared to controls (37.9% ± 0.3% vs 0.6% ± 0.6%, P < 0.0001). For lipid metabolism, CTX significantly increased glycerol kinase (GK) (12.36 ± 1.191U vs 4.88 ± 2.053, P < 0.01) and decreased triglycerides (TG) levels (0.465 ± 0.14 vs 2.295 ± 0.365, P < 0.01), indicating that decomposition was increased and compensatory lipid synthesis was insufficient. Acetyl-CoA was markedly elevated (7.68 ± 0.66 vs 1.641 ± 0.721, P < 0.01), reflecting active β-oxidation but impaired tricarboxylic acid (TCA) cycle flux. For amino acid metabolism, aspartate aminotransferase (AST) was significantly reduced (1174 ± 267.4 vs 2061 ± 241.4, P < 0.05), suggesting specific mitochondrial isoform depletion. In glucose metabolism, glycolysis was enhanced by CTX treatment, as evidenced by significantly elevated lactic acid (LA) levels (2.26 ± 0.34 vs 1.05 ± 0.15, P < 0.01) and markedly increased pyruvic acid (PA) levels (3.86 ± 0.31 vs 1.07 ± 0.25, P < 0.001). Glycogen mobilization was confirmed by increased glycogen phosphorylase (GP) (1.687 ± 0.073 vs 0.824 ± 0.067, P < 0.001),and decreased glycogen (GN) (2.777 ± 0.742 vs 5.096 ± 1.768, P < 0.05). Although GK was significantly upregulated by CTX treatment (12.36 ± 1.191 vs 4.88 ± 2.053, P < 0.01), intracellular glucose Glu levels were significantly reduced (0.268 ± 0.049 vs 1.841 ± 0.397, P < 0.01), reflecting enhanced uptake but rapid downstream shunting. Gluconeogenesis exhibited an “activated but ineffective” state, as evidenced by significantly increased G6PC levels (123.7 ± 8.553 vs 65.87 ± 6.804, P < 0.001) alongside a paradoxical decrease in fructose-1,6-diphosphate (FDP) (0.314 ± 0.088 vs 0.681 ± 0.134, P < 0.05). Pentose phosphate pathway (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.

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