Decreased integrity of exercise-induced plasma cell free nuclear DNA - negative association with the increased oxidants production by circulating phagocytes.
Author(s): Stawski R, Walczak K, Perdas E, Wlodarczyk A, Sarniak A, Kosielski P, Meissner P, Budlewski T, Padula G, Nowak D
Publication: Sci Rep, 2019, Vol. 9, Page 15970
PubMed ID: 31685910 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of exercise on the levels and integrity of cell-free nuclear DNA (cfn-DNA) and cell-free mitochondrial DNA (cfmt-DNA), and the production of reactive oxygen species (ROS).
Conclusion of Paper
Exercise resulted in significant increases in cfn-DNA levels and declines in cfn-DNA integrity, as determined by the ratio of a 229 bp GAPDH amplicon to a 97 bp amplicon (I229.97), when compared to respective specimens collected immediately before exercise. While exercise also resulted in increased levels of cfmt-DNA, the magnitude of increase was more modest than observed for cfn-DNA, and cfmt-DNA integrity (the ratio of a 218 bp to a 97 bp product) was not significantly affected. Reactive oxygen species generation, as measured by resting luminol enhanced whole blood chemiluminescence (rLBBCL) and n-formyl-methionyl-leucyl-phenylalanine stimulated LBCL (fMLP-LBCL), increased significantly after each exercise session compared to pre-exercise levels in each respective patient. Although no conclusive correlations between variables were found when data was analyzed independently, pooling data identified negative correlations between I229/97 and fMLP-LBCL and rLBCL. Even after data was pooled cf mt-DNA integrity (I218/78) was not significantly correlated with either rLBCL or fMLP-LBCL. The authors hypothesize the increase in ROS from exercise may be responsible for increasing neutrophil extracellular traps (NETS) formation and the resultant increases in cfn-DNA and decreases in cfnDNA integrity.
Studies
-
Study Purpose
This paper investigated the effects of exercise on the concentration and integrity of cfn-DNA and cfmt-DNA and ROS production. Blood was collected from eleven healthy average-fitness trained men at each timepoint. On day 1 of the study each participant completed a treadmill test to determine their maximal oxygen uptake (V02 max). Blood was collected into EDTA vacutainer tubes ≤ 5 min before and after exercise to exhaustion at the predetermined 70% VO2 max on day 7, 10 and 13. Aliquots of blood were immediately removed for rLBCL and fMLP-LBCL analysis. The remainder of the blood was then immediately centrifuged at 1600 × g for 10 min at 4°C, and the resultant plasma was recentrifuged at 1600 × g for 5 min at 4°C. Plasma was then stored at -80°C for <4 weeks before cfDNA extraction using the QIAamp DNA Blood Mini Kit. cfn-DNA and cfmt-DNA were quantified by real-time PCR amplification. Integrity of cfn-DNA was determined by the ratio of a 229 bp GAPDH amplicon to a 97 bp amplicon (I229/97). Similarly, the integrity of cfmt-DNA was assessed by the ratio of a 218 bp amplicon of mitochondrial ATPase 6 gene to a 78 amplicon of the ATPase 8 gene (I218/78). To measure rLBCL and fMLP-LBCL, blood was mixed with a solution containing luminol, placed in a luminometer and incubated for 15 min in the dark before addition of fMLP solvent (rLCBL) or fMLP (fMLP-LBCL) and quantification of light emission for 2 min.
Summary of Findings:
Exercise resulted in significant increases in cfn-DNA levels (11.3-, 11.8- and 17.3 -fold after the first second and third exercise session, respectively), and decreases in cfn-DNA integrity as determined by the ratio of a 229 bp GAPDH amplicon to a 97 bp amplicon (I229.97): 0.59 pre-exercise, 0.48 after the first session (P<0.05), 0.53 before the second session, and 0.44 after the second session (P<0.05). In contrast, cfmt-DNA concentration did not increase after the first session and only increased 2- and 2.3-fold after the second and third exercise session. Further, cfmt-DNA integrity (ratio of a 218 bp to a 97 bp product) was stable over the study period and was not affected by exercise. Reactive oxygen species generation, as measured by resting luminol enhanced whole blood chemiluminescence (rLBBCL), increased 2.1-, 2.6- and 2.4-fold (P<0.05, all) after the first, second and third exercise session, respectively, compared to each individual's pre-exercise level. Similarly, n-formyl-methionyl-leucyl-phenylalanine stimulated LBCL (fMLP-LBCL) increased 1.7-, 1.8-, and 1.5-fold (P<0.05, all) after the first, second, and third exercise session, respectively, compared to each individual's pre-exercise level. Importantly, rLBCL and fMLP-LBCL baseline levels were stable throughout the study. The authors report no conclusive correlations were observed between variables due to low sample size (11 men); but after pooling, weak negative correlations were observed between pre-exercise I229/97 and pre-exercise fMLP-LBCL (ρ= −0.36, p < 0.05). When pre- and post-exercise data were combined weak negative correlations were also identified between I229/97 and both measures of whole blood chemoluminescence (ρ = −0.37, p < 0.05 for rLBCL and ρ = −0.40, p < 0.05 for fMLP-LBCL). The integrity of cf mt-DNA (I218/78) was not significantly correlated with either rLBCL or fMLP-LBCL. The authors hypothesize the increased ROS from exercise is responsible for increasing neutrophil extracellular traps (NETS) formation and the resultant increases in cfn-DNA and decreases in cfn-DNA integrity.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Small molecule Chemiluminescence Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qPCR Specific Targeted nucleic acid cfn-DNA (GAPDH)
cfmt-DNA (ATPase 6 and ATPase 8)
Biospecimen Acquisition Time of biospecimen collection Pre-exercise day 7
Post-exercise day 7
Pre-exercise day 10
Post-exercise day 10
Pre-exercise day 14
Post-exercise day 14