Sedoheptulose-1,7-bisphospate Accumulation and Metabolic Anomalies in Hepatoma Cells Exposed to Oxidative Stress

Abstract

We have previously shown that GSH depletion alters global metabolism of cells. In the present study, we applied a metabolomic approach for studying the early changes in metabolism in hydrogen peroxide- (H₂O₂-) treated hepatoma cells which were destined to die. Levels of fructose 1,6-bisphosphate and an unusual metabolite, sedoheptulose 1,7-bisphosphate (S-1,7-BP), were elevated in hepatoma Hep G2 cells. Deficiency in G6PD activity significantly reduced S-1,7-BP formation, suggesting that S-1,7-BP is formed in the pentose phosphate pathway as a response to oxidative stress. Additionally, H₂O₂ treatment significantly increased the level of nicotinamide adenine dinucleotide phosphate (NADP⁺) and reduced the levels of ATP and NAD⁺. Severe depletion of ATP and NAD⁺ in H₂O₂-treated Hep G2 cells was associated with cell death. Inhibition of PARP-mediated NAD⁺ depletion partially protected cells from death. Comparison of metabolite profiles of G6PD-deficient cells and their normal counterparts revealed that changes in GSH and GSSG per se do not cause cell death. These findings suggest that the failure of hepatoma cells to maintain energy metabolism in the midst of oxidative stress may cause cell death.

Figure 1

Early changes in metabolism of H₂O₂-treated hepatoma cells. Cells were treated with 5 mM H₂O₂ for 0, 15, 30, 60, 90, and 120 min and harvested for metabolomic analysis. Molecular features were identified by Progenesis QI, and the data were processed and analyzed using SIMCA-P. (a) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot for various treatment groups are shown. (b) OPLS-DA score plot of the 0 min- and 15 min-treated groups. The ellipse shown in the model (a, b) represents the Hotelling's T ² with 95% confidence. (c) Variable importance in projection (VIP) plot of the OPLS-DA model for the 0 min- and 15 min-treated groups. Selected metabolites with a VIP value > 1.0 are presented. (d) The data were subjected to metabolite pathway analysis. A summary plot for metabolite set enrichment analysis (MSEA), in which metabolite sets are ranked according to the p value, is shown. The bar plot is color coded according to the calculated p values.

Figure 2

Oxidative stress-induced accumulation of Fru-1,6-BP and S-1,7-BP. The extracted ion chromatograms of Fru-1,6-BP (a) and S-1,7-BP (b) in Hep G2 cell treated with 5 mM H₂O₂ for 0, 15, 30, 60, 90, and 120 min are shown. Metabolite abundance is indicated by the peak intensity. A representative experiment is shown. The identities of Fru-1,6-BP (c, d) and S-1,7-BP (e) were validated by MS/MS analysis. The MS/MS spectrum of Fru-1,6-BP in cell specimen (d) was matched against that of standard (c). The spectrum of S-1,7-BP reveals a parent cation (369.0001 m/z) and a fragment ion (271.0227 m/z). The latter differs from the fragment ion of Fru-1,6-BP (241.0125 m/z) by a CHOH unit (30.01 m/z). For (c–e), the chemical structures of the fragment ions and their experimentally obtained m/z values are shown alongside the corresponding peaks.

Figure 3

Temporal changes in metabolism in G6PD-deficient hepatoma cells. (a) G6PD activities in Sc and Gi cells were measured and are expressed in U/mg of cell lysate. Data are means ± SD, n = 6. (b) Sc and Gi cells were untreated or treated with 0.5 and 5 mM H₂O₂ for 4 and 24 hr, and their viabilities were determined. Data are means ± SD, n = 6. (c) Sc and Gi cells were treated with 5 mM H₂O₂ for 0, 15, 30, 60, 90, and 120 min and collected for metabolomic analysis. Data were analyzed as described in the legend of Figure 1. The OPLS-DA score plot of Sc and Gi cells treated for various times is shown.

Figure 4

Changes in intermediates in PPP and glutathione metabolism. Analysis of metabolomic data that were obtained as described in the legend of Figure 3(c) reveals that a number of metabolites in PPP and glutathione metabolism change with time after H₂O₂ treatment. The levels of metabolites are expressed relative to that of untreated Sc cells. Data are means ± SD, n = 6. ^$ p < 0.05, Sc cells vs Gi cells; ^∗ p < 0.05, treated vs untreated Gi cells; and ^# p < 0.05, treated vs untreated Sc cells.

Figure 5

Oxidative stress-induced changes in energy and redox metabolism. Sc and Gi cells were treated with 5 mM H₂O₂ for 0, 15, 30, 60, 90, and 120 min. The cells were harvested, and cellular levels of ATP (a), ADP (b), AMP (c), NAD⁺ (d), and NADP⁺ (e) were determined by LC-MS. The levels of metabolites are expressed relative to those of untreated Sc cells. Data are means ± SD, n = 6. (f) Sc and Gi cells were untreated or treated with 5 mM H₂O₂ for 0, 15, and 30 min and harvested for immunoblotting with antibodies to p-AMPK, AMPK, NADK, and actin. A representative experiment out of three is shown. ^$ p < 0.05, Sc cells vs Gi cells; ^∗ p < 0.05, treated vs untreated Gi cells; and ^# p < 0.05, treated vs untreated Sc cells.

Figure 6

ATP and NAD exhaustion accounts for death of hepatoma cells subject to oxidative stress. Sc and Gi cells were treated with 0.5 or 5 mM H₂O₂ for 0, 15, 30, 60, 90, and 120 min and were extracted for LC-MS analysis of ATP (a) and NAD⁺ (b). The levels of metabolites are expressed relative to that of untreated Sc cells. Data are means ± SD, n = 6. ^$1 p < 0.05 or ^$2 p < 0.05, Sc cells vs Gi cells treated with 0.5 mM or 5 mM H₂O₂; ^∗1 p < 0.05 or ^∗2 p < 0.05, treated vs untreated Gi cells upon treatment with 0.5 mM or 5 mM H₂O₂; and ^#1 p < 0.05 or ^#2 p < 0.05, treated vs untreated Sc cells upon treatment with 0.5 mM or 5 mM H₂O₂.

Figure 7

Inhibition of PARP partially rescues hepatoma cells from death. Hep G2 cells were pretreated with 30 μM PJ34 for 2 hr prior to treatment with 5 mM H₂O₂. Cell viability was determined and is expressed relative to control. Data are means ± SD, n = 6. ^∗∗∗ p < 0.005 and ^∗∗∗∗ p < 0.001.