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. 2016 Jan 26:4:e1624.
doi: 10.7717/peerj.1624. eCollection 2016.

The metabolomic profile of gamma-irradiated human hepatoma and muscle cells reveals metabolic changes consistent with the Warburg effect

Min Wang  1 , Adrian Keogh  2 , Susan Treves  3 , Jeffrey R Idle  4 , Diren Beyoğlu  4
Affiliations

The metabolomic profile of gamma-irradiated human hepatoma and muscle cells reveals metabolic changes consistent with the Warburg effect

Min Wang et al. PeerJ. .

Abstract

The two human cell lines HepG2 from hepatoma and HMCL-7304 from striated muscle were γ-irradiated with doses between 0 and 4 Gy. Abundant γH2AX foci were observed at 4 Gy after 4 h of culture post-irradiation. Sham-irradiated cells showed no γH2AX foci and therefore no signs of radiation-induced double-strand DNA breaks. Flow cytometry indicated that 41.5% of HepG2 cells were in G2/M and this rose statistically significantly with increasing radiation dose reaching a plateau at ∼47%. Cell lysates from both cell lines were subjected to metabolomic analysis using Gas Chromatography-Mass Spectrometry (GCMS). A total of 46 metabolites could be identified by GCMS in HepG2 cell lysates and 29 in HMCL-7304 lysates, most of which occurred in HepG2 cells. Principal Components Analysis (PCA) showed a clear separation of sham, 1, 2 and 4 Gy doses. Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA) revealed elevations in intracellular lactate, alanine, glucose, glucose 6-phosphate, fructose and 5-oxoproline, which were found by univariate statistics to be highly statistically significantly elevated at both 2 and 4 Gy compared with sham irradiated cells. These findings suggested upregulation of cytosolic aerobic glycolysis (the Warburg effect), with potential shunting of glucose through aldose reductase in the polyol pathway, and consumption of reduced Glutathione (GSH) due to γ-irradiation. In HMCL-7304 myotubes, a putative Warburg effect was also observed only at 2 Gy, albeit a lesser magnitude than in HepG2 cells. It is anticipated that these novel metabolic perturbations following γ-irradiation of cultured cells will lead to a fuller understanding of the mechanisms of tissue damage following ionizing radiation exposure.

Keywords: GCMS; Gamma-irradiation; HMCL-7304 myotubes; HepG2 cells; Metabolomics; Warburg effect.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Fluorescence immunohistochemistry for γH2AX foci in γ-irradiated HepG2 cells.
(A) Sham irradiated HepG2 cells showing no γH2AX foci after 4 h culture and therefore no signs of DNA damage. (B) HepG2 cells γ-irradiated with 4 Gy showing γH2AX foci (yellow arrows) after 4 h culture and therefore DNA double-strand breaks.
Figure 2
Figure 2. Cell cycle analysis of γ-irradiated HepG2 cells.
Flow cytometry of HepG2 cells after different doses of γ-irradiation. (A) Sham irradiated. (B) 1 Gy irradiation. (C) 2 Gy irradiated. (D) 4 Gy irradiated. (E) Relationship between radiation dose (Gy) and proportion of cells in G2/M. There occurs a slight and saturable shift from G1 to G2/M with increasing dose of γ-irradiation.
Figure 3
Figure 3. GCMS chromatograms for irradiated HepG2 hepatocellular carcinoma cells and HMCL-7304 myotubes.
Cell lysates after γ-irradiation were derivatized with BSTFA/TMCS and MOX (see text). (A) HepG2 cell chromatogram. (B) HMCL-7304 myotube chromatogram. I.S. means internal standard (4-chlorophenylacetic acid). 1 = lactic acid; 2 = L-alanine; 3 = ethanolamine; 4 = phosphate; 5 = glyceric acid; 6 = L-threonine; 7 = L-5-oxoproline; 8 = glycerol 3-phosphate; 9 = citric acid; 10 = D-fructose; 11 = D-glucose; 12 = palmitic acid; 13 = myo-inositol; 14 = oleic acid; 15 = vaccenic acid; 16 = stearic acid; 17 = cholesterol; X = background contaminating peaks from reagents and laboratory plasticware.
Figure 4
Figure 4. Metabolomic analysis on lysates of γ-irradiated HepG2 cells.
(A) Unsupervised principal components analysis (PCA) scores plot showing separation of the sham irradiated (green symbols), 1 Gy γ-irradiated samples (blue symbols), the 2 Gy γ-irradiated (turquoise symbols) and 4 Gy γ-irradiated samples (red symbols). There was one outlier from the Hotelling’s T2 ellipse (95% confidence interval), belonging to the 1 Gy group, which has been removed. (B) Supervised projection to latent structures-discriminant analysis (PLS-DA) scores plot showing a clear separation of the sham, 1 Gy, 2 Gy and 4 Gy γ-irradiated samples. (C) Validation of the PLS-DA model using 100 iterations. The data are not over-modelled because the correlation coefficient (R2) fell below 0.3 and the predictability coefficient (Q2) fell below zero after data permutation. (D) Orthogonal PLS-DA (OPLS-DA) loadings S-plot for sham versus 2 Gy γ-irradiation of HepG2 cells. Each symbol represents a metabolite and its contribution to the separation between sham and 2 Gy γ-irradiated samples in panel B. Filled symbols are those metabolites selected for univariate statistical analysis. 1 = D-glucose; 2 = L-lactate; 3 = L-5-oxoproline; 4 = L-alanine; 5 = D-fructose; 6 = D-glucose 6-phosphate; 7 = succinate.
Figure 5
Figure 5. Univariate data analysis on metabolomic markers of γ-irradiated HepG2 cells.
Data are presented as means ± standard deviation (S.D.). Data were analyzed by one-way ANOVA for each metabolite with Bonferroni’s correction for multiple comparisons. ANOVA was significant at P < 0.0001. There were no statistically significant differences between the sham irradiated and 1 Gy γ-irradiated samples for any metabolite. * means P < 0.05; ** means P < 0.01; *** means P < 0.001; n.s. means not statistically significant. Relative concentrations were calculated as the peak area of each metabolite divided by the peak area of the internal standard and are based upon 4 × 106 cells.
Figure 6
Figure 6. Metabolomic analysis on lysates of γ-irradiated HMCL-7304 cells.
(A) Unsupervised PCA scores plot showing a clustering of the sham irradiated (green symbols), 1 Gy γ-irradiated samples (blue symbols) and 4 Gy γ-irradiated samples (red symbols) with the clear separation from the 2 Gy γ-irradiated (turquoise symbols). There were no outliers from the Hotelling’s T2 ellipse (95% confidence interval). (B) Supervised PLS-DA scores plot showing a clustering of the sham irradiated (green symbols), 1 Gy γ-irradiated samples (blue symbols) and 4 Gy γ-irradiated samples (red symbols) with the clear separation from the 2 Gy γ-irradiated (turquoise symbols). As for the PCA analysis, there were no outliers. (C) Validation of the PLS-DA model using 100 iterations. The data are not over-modelled because the correlation coefficient (R2) fell below 0.3 and the predictability coefficient (Q2) fell below zero after data permutation. (D) OPLS-DA loadings S-plot for sham versus 2 Gy γ-irradiation of HMCL-7304 cells. Each symbol represents a metabolite and its contribution to the separation between sham and 2 Gy γ-irradiated samples in panel (B). Filled symbols are those metabolites selected for univariate statistical analysis. 1 = phosphate; 2 = D-glucose; 3 = L-lactate. All metabolites measured in HMCL-7304 cells were correlated significantly to the OPLS-DA model, except ethanolamine and L-5-oxoproline.
Figure 7
Figure 7. Univariate data analysis on metabolomic markers of γ-irradiated HMCL-7304.
Data are presented as means ± S.D. Data were analyzed by one-way ANOVA for each metabolite with Bonferroni’s correction for multiple comparisons. ANOVA was significant at P < 0.0001. There were no statistically significant differences between the sham irradiated and either the 1 Gy γ-irradiated or 4 Gy γ-irradiated samples for any metabolite. *** means P < 0.001. Relative concentrations were calculated as the peak area of each metabolite divided by the peak area of the internal standard and are based upon 4 × 106 cells.
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