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. 2023 Apr 12;24(8):7148.
doi: 10.3390/ijms24087148.

Chronological Age and DNA Damage Accumulation in Blood Mononuclear Cells: A Linear Association in Healthy Humans after 50 Years of Age

Nikolaos I Vlachogiannis  1 , Panagiotis A Ntouros  1 , Maria Pappa  1 , Evrydiki Kravvariti  1   2 , Evangelia Georgia Kostaki  3 , Georgios E Fragoulis  1 , Christina Papanikolaou  4 , Dimitra Mavroeidi  4 , Vasiliki-Kalliopi Bournia  1 , Stylianos Panopoulos  1 , Katerina Laskari  1 , Aikaterini Arida  1 , Vassilis G Gorgoulis  5 , Maria G Tektonidou  1 , Dimitrios Paraskevis  3 , Petros P Sfikakis  1   2 , Vassilis L Souliotis  1   4
Affiliations

Chronological Age and DNA Damage Accumulation in Blood Mononuclear Cells: A Linear Association in Healthy Humans after 50 Years of Age

Nikolaos I Vlachogiannis et al. Int J Mol Sci. .

Abstract

Aging is characterized by the progressive deregulation of homeostatic mechanisms causing the accumulation of macromolecular damage, including DNA damage, progressive decline in organ function and chronic diseases. Since several features of the aging phenotype are closely related to defects in the DNA damage response (DDR) network, we have herein investigated the relationship between chronological age and DDR signals in peripheral blood mononuclear cells (PBMCs) from healthy individuals. DDR-associated parameters, including endogenous DNA damage (single-strand breaks and double-strand breaks (DSBs) measured by the alkaline comet assay (Olive Tail Moment (OTM); DSBs-only by γH2AX immunofluorescence staining), DSBs repair capacity, oxidative stress, and apurinic/apyrimidinic sites were evaluated in PBMCs of 243 individuals aged 18-75 years, free of any major comorbidity. While OTM values showed marginal correlation with age until 50 years (rs = 0.41, p = 0.11), a linear relationship was observed after 50 years (r = 0.95, p < 0.001). Moreover, individuals older than 50 years showed increased endogenous DSBs levels (γH2Ax), higher oxidative stress, augmented apurinic/apyrimidinic sites and decreased DSBs repair capacity than those with age lower than 50 years (all p < 0.001). Results were reproduced when we examined men and women separately. Prospective studies confirming the value of DNA damage accumulation as a biomarker of aging, as well as the presence of a relevant agethreshold, are warranted.

Keywords: Comet assay; DNA damage response; apurinic/apyrimidinic sites; chronological age; double-strand breaks repair capacity; endogenous DNA damage; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Endogenous SSBs ± DSBs in PBMCs. (A) Representative alkaline comet assay images of untreated PBMCs from a healthy individual under the age of 50 years and an individual over the age of 50 years (scale bar: 20 μm). (B) Correlation of DNA damage measured by alkaline comet assay with age in 243 apparently healthy humans (men and women). To reduce variance, we calculated the median OTM value per each age including only those ages with at least 4 observations. (C,D) Correlation of DNA damage measured by alkaline comet assay with age in 34 men older than 50 years (C), and in 53 women older than 50 years (D). Correlation coefficients were calculated using Pearson’s test for individuals aged ≥50 years and Spearman’s rank correlation coefficient (rs) for individuals younger than 50 years due to non-normal data distribution.
Figure 2
Figure 2
Endogenous DNA double-strand break levels in PBMCs. (A) Representative confocal microscopy images showing γH2AX staining from an individual under the age of 50 years (37 years old) and an individual over the age of 50 years (61 years old). Upper images, immunofluorescence γH2AX staining; middle, cell nuclei labeled with DAPI; bottom, merged (scale bar: 25 μm). (B–D) Endogenous DNA double-strand breaks assessed by γH2AX immunofluorescence staining in PBMCs of individuals younger or older than 50 years in the whole study population (mean ± SEM: <50 = 8.9 ± 0.3; ≥50 = 15.6 ± 0.4) (B), only in men (mean ± SEM: <50 = 8.8 ± 0.5; ≥50 = 15.4 ± 0.6) (C), and only in women (mean ± SEM: <50 = 8.9 ± 0.4; ≥50 = 15.8 ± 0.6) (D). The p-values are derived from Mann–Whitney U test. Bars and error bars represent mean and standard error of the mean (SEM).
Figure 3
Figure 3
Oxidative stress, abasic sites and DSBs repair capacity in PBMCs. Bar graphs showing (A) the oxidative stress levels expressed as the ratio of reduced-to-oxidized glutathione (lower ratio is an indication of higher oxidative stress levels) (GSH/GSSG mean ± SEM: <50 = 71.6 ± 1.1; ≥50 = 60.2 ± 1.7), (B) the apurinic/apyrimidinic (AP) sites (AP sites/105 nucleotides mean ± SEM: <50 = 5.7 ± 0.2; ≥50 = 8.3 ± 0.3) and (C) the melphalan-induced accumulation of γH2AX foci (expressed as AUC over 24 h incubation) in healthy individuals younger and older than 50 years (γH2AX AUC over 24 h mean ± SEM: <50 = 105.4 ± 3.1; ≥50 = 157.1 ± 5.6). The experiments shown were based on a minimum of 3 independent repeats. The p-values are derived from Mann–Whitney U test. Bars and error bars represent mean and standard error of the mean (SEM).
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