Metabolomics Reveals That Tumor Xenografts Induce Liver Dysfunction

Fei Li,Andrew D Patterson,Kristopher W Krausz,Changtao Jiang,Huichang Bi,Anastasia L Sowers,John A Cook,James B Mitchell,Frank J Gonzalez

doi:10.1074/mcp.m113.028324

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.

Highlights

From the ‡Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892; §Department of Veterinary and Biomedical Sciences and the Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania 16802; ¶Radiation Biology Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
Nicotinamide 1-oxide concentrations were increased from 27.5 ␮mol/mmol creatinine in control mice to 49.1 ␮mol/mmol creatinine in SCCVII-tumorbearing mice at day 13 (p Ͻ 0.05) (Fig. 2B), and dihydroxy-3-oxopregn-4-en-21-oic acid (DHOPA) was increased from 191 ␮mol/mmol creatinine in control mice to 405 ␮mol/mmol creatinine in SCCVII-tumor-bearing mice (p Ͻ 0.05) (Fig. 2C)
The results indicated that carnitine palmitoyltransferase II deficiency (Cpt2), acyl-CoA thioesterase 1 (Acot1), acyl-CoA thioesterase 2 (Acot2), medium-chain acyl-CoA dehydrogenase (Mcad), and cluster of differentiation 36 (Cd36) mRNAs were reduced in SCCVII-tumor-bearing mouse liver (Fig. 4C)

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—Nicotinamide, nicotinamide 1-oxide, citrate, asymmetric dimethylarginine (ADMA), hexanoylglycine, phosphocholine, and adenosine monophosphate (AMP) were purchased from Sigma Ultraperformance Liquid Chromatography Coupled with Electrospray Ionization Quadrupole Mass Spectrometry—Urine samples were prepared by adding 20 ␮l of urine to 180 ␮l 50% aqueous acetonitrile (50:50 water:acetonitrile). A 5-␮l aliquot of supernatant was chromatographed via ultra-performance liquid chromatography (Waters Corp., Milford, MA) using a 2.1 ϫ 50-mm Waters BEH C18 1.7-␮m column and introduced via electrospray into a quadrupole time-of-flight mass spectrometer (UPLC-ESIQTOFMS). Quantification of Metabolites in Urine, Serum, and Liver Homogenate—Quantitation of the endogenous metabolites was performed using an ACQUITY UPLC system coupled to a Xevo triple quadrupole tandem mass spectrometer (Waters Corp.). Gene Expression Analysis—Total RNA was prepared from frozen liver using TRIzol reagent (Invitrogen, Carlsbad, CA). Two-tailed Student’s t test was used to compare the levels of metabolites and changes in gene expression between wild-type mice and SCCVII-tumor-bearing mice. Experimental values are presented as the mean Ϯ S.E. p values of less than 0.05 are considered significant

RESULTS

Fold change

TABLE II Clinical serum chemistry of SCCVII

DISCUSSION