Metabolomics reveals that tumor xenografts induce liver dysfunction

Abstract

Metabolomics, based on ultraperformance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry, was used to explore metabolic signatures of tumor growth in mice. Urine samples were collected from control mice and mice injected with squamous cell carcinoma (SCCVII) tumor cells. When tumors reached ∼2 cm, all mice were killed and blood and liver samples collected. The urine metabolites hexanoylglycine, nicotinamide 1-oxide, and 11β,20α-dihydroxy-3-oxopregn-4-en-21-oic acid were elevated in tumor-bearing mice, as was asymmetric dimethylarginine, a biomarker for oxidative stress. Interestingly, SCCVII tumor growth resulted in hepatomegaly, reduced albumin/globulin ratios, and elevated serum triglycerides, suggesting liver dysfunction. Alterations in liver metabolites between SCCVII-tumor-bearing and control mice confirmed the presence of liver injury. Hepatic mRNA analysis indicated that inflammatory cytokines, tumor necrosis factor α, and transforming growth factor β were enhanced in SCCVII-tumor-bearing mice, and the expression of cytochromes P450 was decreased in tumor-bearing mice. Further, genes involved in fatty acid oxidation were decreased, suggesting impaired fatty acid oxidation in SCCVII-tumor-bearing mice. Additionally, activated phospholipid metabolism and a disrupted tricarboxylic acid cycle were observed in SCCVII-tumor-bearing mice. These data suggest that tumor growth imposes a global inflammatory response that results in liver dysfunction and underscore the use of metabolomics to temporally examine these changes and potentially use metabolite changes to monitor tumor treatment response.

Fig. 1.

SCCVII tumor growth and metabolomics analysis of urinary metabolites. A, growth curve of SCCVII tumors from day 6 to day 13 (n = 10 mice/group). B, PLS-DA model of urinary metabolites between SCCVII (n = 10, solid squares) and control (n = 10, solid triangles). C, loadings scatter plot of PLS-DA.

Fig. 2.

Quantitation of urinary hexanoylglycine, nicotinamide 1-oxide, DHOPA, ADMA, and citrate. Urinary levels of hexanoylglycine (A), nicotinamide 1-oxide (B), DHOPA (C), ADMA (D), and citrate (E) in SCCVII-tumor-bearing and control mice at various times following tumor cell injection. The levels of metabolites were normalized according to the concentration of urinary creatinine. Statistical analysis was performed using two-tailed Student's t test (n = 10 in each group). *, p < 0.05; **, p < 0.01.

Fig. 3.

Body weights, liver weights, and serum parameters after SCCVII tumor xenograft. A, B, body weight and liver weight of control and SCCVII-tumor-bearing mice at day 13. C–E, serum triglyceride, C-reactive protein, and TGFβ levels in control and SCCVII-tumor-bearing mice at day 13. Statistical analysis was performed using two-tailed Student's t test (n = 10 in each group). *, p < 0.05; **, p < 0.01.

Fig. 4.

Hepatic levels of metabolites and gene expression in SCCVII mice. A, liver levels of nicotinamide, phosphocholine, and AMP. B, hepatic mRNA levels of genes associated with inflammation and P450s. The mRNA levels were normalized to those of β-actin mRNA and subsequently normalized to those of control mice. C, hepatic mRNA levels of genes associated with fatty acid oxidation. D, hepatic mRNAs from genes associated with the TCA cycle. The mRNA levels were normalized to those of β-actin mRNA and subsequently normalized to those of control mice. Statistical analysis was performed using two-tailed Student's t test (n = 10 in each group). *, p < 0.05; **, p < 0.01.

Fig. 5.

Activation of phospholipid metabolism during SCCVII tumor growth. A, PLS-DA model of serum metabolites in SCCVII (n = 10, solid squares) and control (n = 10, solid triangles). B, loadings scatter plot of PLS-DA. C, serum levels of LPC 18:1 in SCCVII and control mice. D, serum levels of LPC 16:1 in SCCVII-tumor-bearing and control mice. The levels of LPC were normalized to those of control mice and were expressed as the relative abundance. E, serum phospholipid in SCCVII-tumor-bearing and control mice. F, hepatic mRNA levels of genes associated with LPC metabolism and synthesis. The mRNA levels were normalized to those of β-actin mRNA and subsequently normalized to those of control mice. Statistical analysis was performed using two-tailed Student's t test (n = 10 in each group). *, p < 0.05; **, p < 0.01.

Fig. 6.

Proposed mechanism of liver dysfunction during SCCVII tumor growth. During SCCVII tumor growth, oxidative stress can be increased in the body, which can activate the inflammatory cytokines TNFα and TGFβ. The enhanced TNFα suppresses fatty acid oxidation and CYP450s. The suppression of fatty acid oxidation contributes to the disruption of the TCA cycle. The enhancement of TGFβ activates phospholipid metabolism and can also suppress P450s. The disruption of all these metabolic pathways contributes to liver dysfunction. ↑, up-regulation; ↓, down-regulation.