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. 2023 Jun 9;51(10):5056-5072.
doi: 10.1093/nar/gkad305.

Changes in metabolic landscapes shape divergent but distinct mutational signatures and cytotoxic consequences of redox stress

Natalya P Degtyareva  1 , Victoria C Placentra  1 , Scott A Gabel  2 , Leszek J Klimczak  3 , Dmitry A Gordenin  4 , Brett A Wagner  5 , Garry R Buettner  5 , Geoffrey A Mueller  2 , Tatyana I Smirnova  6 , Paul W Doetsch  1
Affiliations

Changes in metabolic landscapes shape divergent but distinct mutational signatures and cytotoxic consequences of redox stress

Natalya P Degtyareva et al. Nucleic Acids Res. .

Abstract

Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress.

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Figures

Figure 1.
Figure 1.
Potassium bromate induces mutations in ssDNA and has a distinct mutational signature/motif. (A) Exposure to potassium bromate leads to an increase in frequencies of CanR and CanR Red mutations. All experiments were carried out as described in Materials and Methods. Mutation frequencies for 8–12 independent cultures were measured in each experiment. ‘ns’ stands for ‘non-significant’, P > 0.05 (Mann–Whitney test). Whiskers depict 95% confidence intervals. The number above the bar is the median frequency. (B) Spectra of mutations identified by sequencing of the entire triple subtelomeric reporter of CanR Red mutants in wt (n = 226) and rev3 (n = 102) strains. (C) Overrepresented motifs flanking C to A substitutions induced by potassium bromate identified by pLOGO analysis. The height of each letter corresponds to the magnitude of the overrepresentation. The enrichment for the nucleotides above the red arrow is statistically significant, P< 0.05. The sequence is shown in the conventional format of presenting the mutational target as a pyrimidine at a fixed position. The sequence of the mutational target is complimentary to the sequence shown in the figure. Fold enrichment for specific motifs is calculated as in (59). The capital C and A indicate the site of a C to A mutation. The sequences are shown in the conventional format of presenting pyrimidines at a fixed position of the mutational target and are complimentary to the mutated target sequence. The sequence of the mutational target is complimentary to the sequence shown in the figure. (D) Evidence for potassium bromate-related mutational motifs in human cancer genomes. Tumor types with higher than 10% of samples with non-zero mutational loads of mutations in tgC to tgA motifs from the Pan-Cancer Analysis of Whole Genomes (PCAWG) catalogs of mutations are shown. The numbers above the bars indicate the total number of analyzed samples of a specific cancer type. The summaries for all the 39 tumor types are presented in Supplementary Figure S1C and S1 Data.
Figure 2.
Figure 2.
The chemical reaction of potassium bromate with glutathione in vitro generates a reactive species, different from superoxide and hydroxyl radical, as detected by EPR spin trapping. (A) Background trace of a reaction of 100 mM KBrO3 in phosphate buffer mixed with 50mM DMPO (spin trap), and 0.1 mM EDDA (a chelating agent). (B and C) EPR spectra of reactive species generated in a reaction of 100 mM KBrO3 with 1 mM GSH and 50 mM DMPO in presence (B) or absence (C) of 0.1 mM EDDA. (D) EPR spectrum of reactive species generated in a reaction of 100 mM KBrO3 with 1 mM GSH and 50mM DMPO in presence of 500 u/ml superoxide dismutase (SOD). The analysis of SOD activity is described in Supplemental Materials and Supplemental Figure 2D. (E) EPR spectrum reactive species generated in a reaction of 100 mM KBrO3 with 1 mM GSH and 50mM DMPO in presence 10% DMSO. Positive control for DMSO activity is presented in Supplementary Figure S2E. Reaction mixtures were prepared at room temperature, EPR spectra were collected at 3 min after mixing. Empty circles indicate background peaks and are typical for degradation product of DMPO. Red arrows are above peaks that represent the reactive species adduct with DMPO.
Figure 3.
Figure 3.
Principal component analysis (PCA) of the metabolites of yeast cells exposed to different oxidative agents reveals distinct metabolic landscapes. (A) PCA of NMR- derived metabolomes of yeast cells exposed to potassium bromate and hydrogen peroxide. (B) PCA of NMR- derived metabolomes of wild type yeast cells, sod1Δ, and cta1Δ mutant strains. Yeast cells were exposed to 100 mM potassium bromate, 5mM hydrogen peroxide or mock exposed as described in Materials and Methods. Metabolite identification and quantification were performed by 1H NMR spectroscopy and analyzed using Mataboanalyst 4.0 software. Each dot represents one of the biological replicates for each experiment. 95% confidence intervals are depicted as ovals. PC, principal component. (C) Concentrations of intracellular levels of GSH under conditions of exogenous and endogenous redox stress. Each dot represents a single measurement, whiskers depict standard deviation, short horizontal lines indicate mean of the measurements. GSH concentration was measured by NMR as described in Materials and Methods. Levels of GHS in gsh2Δ strains, which lack glutathione synthetase, served as a positive control. N of total experiments = 16. The ordinate is the normalized concentration of GSH, auto-scaled (mean-centered and divided by the standard deviation, s). Mann-Whitney tests were used to analyze the data. Asterisk (*) signifies difference (P< 0.05) in GSH levels compared to wild type, non-exposed cells, pound sign (#) signify difference between GSH levels in cells exposed to potassium bromate and hydrogen peroxide.
Figure 4.
Figure 4.
Exposure of yeast cells to potassium bromate leads to the highest increase in intracellular fluorescence from oxidized DHEt, as compared to the increases following exposure to equitoxic doses of PQ or hydrogen peroxide. (A) Flow cytometry measurements of fluorescence signals generated by the interaction of DHEt and endogenous superoxide radical following exposure of wild type and sod1Δ cells to 100 mM potassium bromate. (B) Flow cytometry measurements of fluorescence signals generated by the interaction of DHEt and endogenous superoxide and oxidants derived via superoxide following exposure of wild type and sod1Δ cells to 150 uM paraquat. (C) Flow cytometry measurements of fluorescent signal generated by interaction of DHEt and endogenous superoxide radical following exposure of wild type and sod1Δ cells to 5 mM hydrogen peroxide. Yeast cells were harvested and incubated with DHEt as described in Materials and Methods. Each contour plot represents the relative fluorescence intensity of 10 000 cells plotted with FlowJo v10.6.1 software. One representative sample is shown for each condition. The fluorescence of mock-exposed cells of each genotype was gated as background fluorescence (indicated by black line across the plot). Fluorescence intensity of any cell above the line is higher than background and considered to be contributed by increased levels of superoxide. Relevant data for independent experiments can be found in the source file for Figure 4. Quantification of data is presented in Supplementary Figure S4.
Figure 5.
Figure 5.
Sod1 and NAC potentiate mutagenicity of potassium bromate. (A) In strains lacking Sod1, frequencies of potassium bromate–induced CanR and CanR Red mutations are lower, than in wild type strains. (B) Exposure of yeast cells to NAC leads to increased frequencies of potassium bromate–induced CanR and CanR Red mutations. All experiments were carried out as described in Materials and Methods. Mutation frequencies for 6–12 independent cultures were measured in each experiment. Whiskers represent 95% confidence limits for the median frequency of mutations. KBrO3 stands for potassium bromate; NAC stands for N-acetylcysteine.
Figure 6.
Figure 6.
NAC enhances cytotxicity of potassium bromate. Yeast cells were exposed to solutions of 100 mM KBrO3 with increased concentrations of NAC as decribed in Materials and Methods. Survival was calculated as a number of cells that grew on complete media following 2 h of exposure divided by number of mock-exposed cells grown on the same type of media.
Figure 7.
Figure 7.
Unique metabolic and mutational consequences of exposure to potassium bromate in yeast. Interaction of KBrO3 with GSH leads to generation of unique, uncommon reactive species (X.) and depletion of intracellular GSH. Exposure of the cells to KBrO3 leads to G to T mutations and increases endogenous levels of superoxide (O2.−). Mutagenicity and cytotoxicity of KBrO3 is augmented (black horizontal arrows) by antioxidants, superoxide dismutase (Sod1) and N-acetylcysteine (NAC). Blue arrows signify direct consequences of KBrO3 and GSH interactions. Dashed blue lines indicate potentially indirect or independent outcomes of such interaction. In black font are proven consequences of exposure to potassium bromate, in blue font – hypothetical consequences of exposure to potassium bromate.
Figure 8.
Figure 8.
Integral scheme of the processes defining biological consequences of redox stress in vivo. (A) Mutagenesis by potassium bromate, hydrogen peroxide and paraquat are differentially modulated by exogenous and endogenous antioxidants. Left panel. Major type of substitutions induced by specific redox stress agent is shown in white font for each of the oxidizing agents. Vertical black arrows signify an increase, blocked horizontal lines signify a decrease in mutation frequencies. Sod1 stands for superoxide dismutase; Cta1 stands for catalase; NAC stands for N-acetylcysteine. Asterisk indicates that the increase in mutational frequencies caused by paraquat and its mutational signature was detected in sensitized, sod1Δ strains. Right panel. Mutational motifs detected for each redox stress agent are shown with the target nucleotide in upper case. (B) Dashboard of the impact of different types of redox stress on intracellular processes. Biological readouts of the consequences of exposure of yeast cells to potassium bromate, hydrogen peroxide and paraquat are presented. Increases or decreases in intracellular levels of superoxide radical or GSH, or toxicity in the presence of antioxidants are shown as arrow inclinations toward + or – segment of a dashboard respectively. Mutational targets impacted by these endogenous changes shown next to DNA cartoon. ND stand for not determined. X. stands for reactive species generated by interaction of potassium bromate with thiol-containing antioxidants. O2.− stands for superoxide radical.
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