Dose-Dependent Metabolic Alterations in Human Cells Exposed to Gamma Irradiation

<div><p>Radiation exposure is a threat to public health because it causes many diseases, such as cancers and birth defects, due to genetic modification of cells. Compared with the past, a greater number of people are more frequently exposed to higher levels of radioactivity today, not least due to the increased use of diagnostic and therapeutic radiation-emitting devices. In this study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based metabolic profiling was used to investigate radiation- induced metabolic changes in human fibroblasts. After exposure to 1 and 5 Gy of γ-radiation, the irradiated fibroblasts were harvested at 24, 48, and 72 h and subjected to global metabolite profiling analysis. Mass spectral peaks of cell extracts were analyzed by pattern recognition using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The results showed that the cells irradiated with 1 Gy returned to control levels at 72 h post radiation, whereas cells irradiated with 5 Gy were quite unlike the controls; therefore, cells irradiated with 1 Gy had recovered, whereas those irradiated with 5 Gy had not. Lipid and amino acid levels increased after the higher-level radiation, indicating degradation of membranes and proteins. These results suggest that MS-based metabolite profiling of γ-radiation-exposed human cells provides insight into the global metabolic alterations in these cells.</p></div

PCA score plots were derived from UPLC-QTOF-MS.

<p>A and B were from positive mode, C and D were from negative mode, and E and F were from the integration of positive and negative modes. Blue arrows and red arrows indicate the flow of cells irradiated with 1 and 5 Gy, respectively. Triangles and squares represent samples irradiated with 1 and 5 Gy, respectively. Yellow diamond marks indicate the averages of each group. Cont, control; 24L, 48L, and 72 L samples irradiated with 1 Gy at 24, 48, and 72 h post radiation, respectively.; 24H, 48H, and 72H samples irradiated with 5 Gy at 24, 48, and 72 h post radiation, respectively.</p

PLS-DA score plots from UPLC-QTOF-MS.

<p>A is from positive mode, B is from negative mode, C is from the integration of positive and negative modes. (A. R²X: 0.417, R²Y: 0.182, Q²: 0.105; B. R²X: 0.429, R²Y: 0.203, Q²: 0.139; C. R²X: 0.554, R²Y: 0.276, Q²: 0.204) Blue and red arrows indicate the flow of cells irradiated with 1 and 5 Gy, respectively. Triangles and squares indicate samples irradiated with 1 and 5 Gy, respectively. Yellow diamond marks indicate the averages of each group. Other symbols follow those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113573#pone-0113573-g001" target="_blank">Figure 1</a>.</p

Relative quantification of metabolites identified in human fibroblasts.

<p>Units: peak area.</p><p>Data are given as means ± standard deviations.</p><p>Cont, control; 24L, samples irradiated with 1 Gy at 24 h post radiation; 48L, samples irradiated with 1 Gy at 48 h post radiation; 72L, samples irradiated with 1 Gy at 72 h post radiation; 24H, samples irradiated with 5 Gy at 24 h post radiation; 48H, samples irradiated with 5 Gy at 48 h post radiation; 72H, samples irradiated with 5 Gy at 72 h post radiation.</p>#, and *<p> indicate significant differences between groups irradiated with 1 Gy, and 5 Gy, respectively. Letters in the <i>post hoc</i> column indicate significant differences between each group (a, 48 h vs 24 h; b, 72 h vs 48 h; c, Cont vs 24 h; d, 72 h vs 48 h; e, Cont vs 48 h; f, Cont vs 72 h) (<i>P</i>>0.05). The <i>P</i>-values were calculated by using the Kruskal-Wallis test and Tukey test using rank as <i>post hoc</i> test.</p><p>Relative quantification of metabolites identified in human fibroblasts.</p

Heat map of interesting clusters from <i>k</i>-means clustering analysis.

<p>A: The clusters show the peaks that recovered from the decrease caused by radiation of 1 Gy. B: The clusters show peaks that increased 48 or 72 h after radiation with 5 Gy.</p

Major metabolic pathways for the metabolites that are significantly altered in urine and serum from <i>ob</i>/<i>ob</i> mice compared to lean controls, observed by ¹H NMR spectroscopy.

<p>The pathways were analyzed on the basis of the KEGG pathway database (<a href="http://www.genome.jp/kegg/pathway.html" target="_blank">http://www.genome.jp/kegg/pathway.html</a>). The levels of the identified metabolites that were elevated and depleted in obese mice, compared to lean mice, are color coded as red and blue, respectively, and the metabolites in gray were not identified in our study. Direction of change is shown for all obese mice including males and females. For the direction of change based on gender, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075998#pone-0075998-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075998#pone-0075998-t003" target="_blank">3</a>. The metabolites with asterisk are shown several times in this diagram to avoid the complexity of the pathways. The metabolites with sharp indicate the significant ones in male or female group only. Solid and dashed arrows indicate the single and multiple steps involved, respectively, between two metabolites.</p

Gender-Specific Metabolomic Profiling of Obesity in Leptin-Deficient <i>ob</i>/<i>ob</i> Mice by ¹H NMR Spectroscopy

<div><p>Despite the numerous metabolic studies on obesity, gender bias in obesity has rarely been investigated. Here, we report the metabolomic analysis of obesity by using leptin-deficient <i>ob</i>/<i>ob</i> mice based on the gender. Metabolomic analyses of urine and serum from <i>ob</i>/<i>ob</i> mice compared with those from C57BL/6J lean mice, based on the ¹H NMR spectroscopy in combination with multivariate statistical analysis, revealed clear metabolic differences between obese and lean mice. We also identified 48 urine and 22 serum metabolites that were statistically significantly altered in obese mice compared to lean controls. These metabolites are involved in amino acid metabolism (leucine, alanine, ariginine, lysine, and methionine), tricarbocylic acid cycle and glucose metabolism (pyruvate, citrate, glycolate, acetoacetate, and acetone), lipid metabolism (cholesterol and carnitine), creatine metabolism (creatine and creatinine), and gut-microbiome-derived metabolism (choline, TMAO, hippurate, <i>p</i>-cresol, isobutyrate, 2-hydroxyisobutyrate, methylamine, and trigonelline). Notably, our metabolomic studies showed distinct gender variations. The obese male mice metabolism was specifically associated with insulin signaling, whereas the obese female mice metabolism was associated with lipid metabolism. Taken together, our study identifies the biomarker signature for obesity in <i>ob</i>/<i>ob</i> mice and provides biochemical insights into the metabolic alteration in obesity based on gender.</p></div

Summary of significant serum metabolites differentiating between ob/ob and B6 mice.

<p>All the metabolites that have p<0.05 in <i>t</i>-test and VIP score >1 from OPLS-DA are shown for all, male, and/or female. The p values of the individual metabolic concentration in <i>t</i>-test are symbolized as *, **, and ***, indicating p<0.05, p<0.01, and p<0.005, respectively and the metabolites with no asterisk are less significant, having p>0.05.</p>a<p>Direction of change is shown for obese mice compared to lean controls.</p>b<p>Other metabolites that do not belong to any of the metabolic categories listed above.</p>c<p>Total number of significant metabolites with VIP score >1 and p<0.05 for all, male, and female.</p

Summary of pathway enrichment analysis of urine (A) and serum (B) samples.

<p>Each pathway is ranked from low (red) to high (white) Holm p-value, corresponding to the most and the least significant values, respectively.</p

OPLS-DA score plots derived from the ¹H NMR spectra of urine (A) and serum (B) samples from lean and obese mice based on gender.

<p>Data for obese (left panels) and lean (right panels) are demonstrated. Closed and open circles represent male and female mice, respectively.</p