Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA

Abstract

Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50-100-fold higher in mtDNA than in nuclear DNA in several different human cell lines. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/-) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.

Figure 1.

Formation of M₁dG from MDA and base propenals and the oxidation of M₁dG to 6-oxo-M₁dG.

Figure 2.

M₁dG levels in mtDNA in RKO cells after treatment with adenine propenal (400 μM) for 1 h. The electrophile was removed, and cells were lysed at the indicated times for mtDNA isolation and analysis. Data represent the mean ± S.D. of triplicate determinations.

Figure 3.

M₁dG levels in the mtDNA of RKO cells (panel A), HEK293 cells (panel B), HepG2 cells (panel C) and RAW264.7 cells (panel D) after treatment with rotenone (100 nM), and/or mitoTEMPO (10 μM), and/or TEMPOL (10 μM) for 24 h. Basal levels of M₁dG in mtDNA were determined to be two adducts per 10⁶ dG, 2.3 adducts per 10⁶ dG, two adducts per 10⁶ dG and one adduct per 10⁵ dG in RKO cells, HEK293 cells, HepG2 cells and RAW 264.7 macrophages respectively. Data represent the mean ± S.D. of triplicate determinations.

Figure 4.

M₁dG levels in nuclear DNA in RKO cells after a 24-h treatment with rotenone (100 nM), mitoTEMPO (10 μM), or TEMPOL (10 μM) alone or with rotenone (100 nM) and either mitoTEMPO (10 μM) or TEMPOL (10 μM). Data represent the mean ± S.D. of triplicate determinations. In RKO cells, basal levels of M₁dG in genomic DNA was determined to be 1.5 adducts per 10⁸ dG.

Figure 5.

(A) Mitochondrial M₁dG levels in mtDNA in PMVECs isolated from transgenic BMPR2 (bone morphogenetic protein receptor 2) mutant mice (BMPR2 ^R899X) and wild type mice. Data represent the mean ± S.D. of quadruplicate determinations. (B) Mitochondrial M₁dG levels in mtDNA in PMVECs isolated from BMPR2 heterozygous null (BMPR2^+/−) cells compared to wild-type. Data represent the mean ± S.D. of quadruplicate determinations.

Figure 6.

Workflow for the preparation of mtDNA from RKO cells for sequencing analysis following treatment with adenine propenal (400 μM) for 1 h.

Figure 7.

Representative coverage of a 2 kb region of the mitochondrial genome. The signal corresponding to mtDNA from cells treated with adenine propenal and enriched with the M₁dG antibody (blue) shows increased sequence reads but does not show enrichment of distinct regions when compared to the signal corresponding to mtDNA from cells not treated with adenine propenal and enriched (red).