CO2 protects cells from iron-Fenton oxidative DNA damage in E. coli and humans

Abstract

Whereas hydroxyl radical is commonly named as the Fenton product responsible for DNA and RNA damage in cells, here we demonstrate that the cellular reaction generates carbonate radical anion due to physiological levels of bicarbonate. Analysis of the metabolome, transcriptome and, in human cells, the nuclear genome shows a consistent buffering of H₂O₂-induced oxidative stress leading to one common pathway, namely guanine oxidation. Particularly revealing are nanopore-based studies of direct RNA sequencing of cytosolic and mitochondrial ribosomal RNA along with glycosylase-dependent qPCR studies of oxidative DNA damage in telomeres. The focusing of oxidative modification on one pathway is consistent with the highly evolved base excision repair suite of enzymes and their involvement in gene regulation in response to oxidative stress.

Fig. 1.. Changes in redox-active metabolite concentrations after adding H₂O₂ to the medium with dependency on the bicarbonate concentration.

HPLC-MS/MS provided quantification of metabolite concentrations in HEK293T cells pre-incubated in PBS medium with 0, 5, or 20 mM HCO₃⁻ for 1 h at 37 °C under atmospheric CO₂ before adding 100 μM H₂O₂. The reactions were allowed to progress for 15 min before quenching and analysis. Ratios for reduced vs. oxidized states of (A) GSH:GSSG and (B) NADH:NAD⁺ are reported, and the mass spectrometry (MS) intensities are provided for the nucleotide-monophosphates (C) AMP, (D) CMP, (E) GMP, and (F) UMP. The analyses were conducted with 3–6 replicates and the levels of statistical significance are represented by * P < 0.05, ** P < 0.01, and *** P < 0.001 calculated by a student’s T-test.

Fig. 2.. Profile of RNA oxidation products or sites upon adding H₂O₂ to E. coli or human cells showing dependency on the bicarbonate concentration.

The redox-active products from RNA base oxidation ho⁵C, OA, ho⁵U, and OG were profiled in (A) E. coli or (B) HEK293T total RNA via nuclease/phosphatase digestion of the polymers to nucleosides and HPLC-UV-ECD quantification. RNA direct nanopore sequencing of the LSU and SSU rRNA from (C) E. coli, (D) HEK293T cytosolic rRNA, and (E) HEK293T mitochondrial rRNA were profiled from the oxidized cells to measure changes in base miscalls with ELIGOS2 that report on modification sites in the strands (20). The base miscall analysis for the E. coli and mitochondrial rRNAs was conducted by comparison of the cellular RNAs against a synthetic RNA of the same sequence without modification made by in vitro transcription (IVT). This approach permitted a profile of those RNAs in cells not exposed to oxidant (i.e., background (bkgd)). In contrast, the human cytosolic rRNAs are too G/C rich to allow synthesis of the RNAs without modifications via IVT; therefore, the comparison for the oxidized samples was against the rRNA from the non-oxidized cells resulting in no background being reported. Prior work and controls reported herein identified oxidation sites in RNA yield base call errors. The background for the E. coli was obtained from cells grown for 24 h at 37 °C in LB Miller medium under atmospheric CO₂ levels, and the HEK295T cells were grown to ~80% confluency in DMEM medium in a humidified incubator with 5% CO₂ at 37 °C. The cells were placed in PBS with 0, 5, or 20 mM bicarbonate for 1 h (HEK293T) or 2 h (E. coli) before adding 100 μM H₂O₂. The oxidations proceeded for 15 min before quenching and harvesting the total RNA for analysis. The analyses were conducted in triplicate for HPLC-UV-ECD and duplicate trial for direct RNA nanopore sequencing with levels of statistical significance represented by * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 calculated by a student’s T-test. *The OA values from the HPLC-UV-ECD analysis are an overestimation resulting from A and OA coeluting in the HPLC.

Fig. 3.. Bicarbonate dependency in telomere oxidation sites upon addition of H₂O₂ assayed by qPCR telomere length measurements.

(A) Scheme to illustrate telomere DNA oxidation types analyzed via glycosylase removal of the damaged site. Direct measurement of the telomere length before and after oxidation reports on frank strand breaks that occur upon oxidation. Pyrimidine oxidation sites are revealed by EndoIII, an enzyme for which the preferred substrate is Tg, while the glycosylase can also remove 5hoC and 5hoU from DNA. Sites of sugar oxidation and abasic sites are substrates for EndoIV to yield strand breaks that are quantified by telomere-specific qPCR. Purine oxidation sites are found by Fpg, a glycosylase that favorably removes OG and Fapy-G from DNA, while the enzyme can also remove OA and Fapy-A. The bicarbonate dependency in H₂O₂-mediated oxidation of the telomeres was followed by qPCR to quantify (B) strand breaks, (C) EndoIII-sensitive sites, (D) EndoIV-sensitive sites, and (E) Fpg-sensitive sites. The oxidations were conducted by adding 100 μM H₂O₂ to HEK293T cells pre-equilibrated for 1 h in PBS medium with 0, 5, or 20 mM bicarbonate at 37 °C under atmospheric CO₂ levels, followed by reaction quenching and harvesting of the gDNA. The background (bkgd) measurements were obtained from cells not exposed to oxidant. The analyses were conducted in triplicate with levels of statistical significance represented by ** P < 0.01 and *** P < 0.001 calculated by a student’s T-test.

Fig. 4.. Iron dependency in oxidatively derived damage to HEK293T nucleic acids upon H₂O₂ addition.

(A) Colorimetric assay determination of labile iron relative concentration before and after a 16 h treatment with the ferroptosis-inducing compound erastin. (B) Change in OG nucleoside levels in total RNA measured by HPLC-UV-ECD before and after erastin treatment, as a function of added H₂O₂ and bicarbonate to the culture medium. (C) Telomere lengths were measured by qPCR before and after erastin treatment and as a function of bicarbonate concentration during H₂O₂-mediated oxidation. qPCR quantification in telomeres of (D) EndoIII-, (E) EndoIV-, or (F) Fpg-sensitive sites in the high vs. basal iron level cells before and bicarbonate-dependent H₂O₂ oxidation. In all cases, background (bkgd) refers to cells grown under a 5% CO₂ atmosphere in DMEM medium without exposure to H₂O₂. In the oxidation studies, the cells were equilibrated with 0, 5, or 20 mM bicarbonate in PBS for 1 h before adding 100 μM H₂O₂ for 15 min at 37 °C under atmospheric CO₂ followed by quenching the reaction and harvesting the nucleic acids to be analyzed. The analyses were conducted in triplicate.

Scheme 1.

Products of HO^• or CO₃^•− (dashed box) oxidative modification of nucleic acids.