Protective role of extracellular vesicles against oxidative DNA damage

Abstract

Background: Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators of cellular processes and may be involved in maintaining cellular redox balance. Herein, we aimed to develop a method to assess the effects of EVs on DNA oxidation using porcine seminal plasma extracellular vesicles (sEVs).

Results: The technique was set using a cell-free plasmid DNA to avoid the bias generated by the uptake of sEVs by sperm cells, employing increasing concentrations of hydrogen peroxide (H₂O₂) that generate DNA single-strand breaks (SSBs). Because SSBs contain a free 3'-OH end that allow the extension through quantitative PCR, such extension -and therefore the SYBR intensity- showed to be proportional to the amount of SSB. In the next stage, H₂O₂ was co-incubated with two size-differentiated subpopulations (small and large) of permeabilized and non-permeabilized sEVs to assess whether the intravesicular content (IC) or the surface of sEVs protects the DNA from oxidative damage. Results obtained showed that the surface of small sEVs reduced the incidence of DNA SSBs (P < 0.05), whereas that of large sEVs had no impact on the generation of SSBs (P > 0.05). The IC showed no protective effect against DNA oxidation (P > 0.05).

Conclusions: Our results suggest that the surface of small sEVs, including the peripheral corona layer, may exert a protective function against alterations that are originated by oxidative mechanisms. In addition, our in vitro study opens path to detect, localize and quantify the protective effects against oxidation of extracellular vesicles derived from different fluids, elucidating their function in physiopathological states.

Keywords: DNA fragmentation; DNA oxidation; Extracellular vesicles; Peripheral corona layer; Seminal plasma; Single-strand breaks.

Fig. 1

Schematic overview of the procedure conducted to generate oxidative DNA single-strand breaks, and their detection through a quantitative PCR-based method and a TUNEL assay. First, a treatment with hydrogen peroxide (H₂O₂) causes oxidation of nitrogenous bases, which are excised by the formamidopyrimidine DNA glycosylase (FPG) enzyme. The generated nicks can serve as priming sites for the quantitative PCR-based method and can be detected with the TUNEL assay

Fig. 2

Set up and validation of the method. A Dose-dependent generation of DNA single-strand breaks (SSBs) by different concentrations of hydrogen peroxide (H₂O₂) detected by assessing separate amounts of DNA in the quantitative PCR-based approach. B Validation of the dose-dependent generation of DNA SSBs through the TUNEL assay. The straight line in each figure represents the linear regression equation (Simple linear regression analysis, least squares method). Dotted lines represent the 95% of the confidence interval

Fig. 3

Characterization of small and large extracellular vesicle (EV) samples isolated from porcine seminal plasma. A Total protein concentration measured by Nanodrop. B Particle size distribution as measured by dynamic light scattering. C Representative cryo-electron micrographs showing the morphology of seminal EVs. D Representative flow cytometry plots showing the gating of EV and the identification of seminal EVs by labeling with carboxyfluorescein succinimidyl ester (CFSE), EV protein markers (tetraspanin CD81 and cytosolic protein HSP70/HSC70), and a non-vesicular extracellular particle marker (albumin; BSA). (****) show statistically significant differences (Mann–Whitney U test, P < 0.0001)

Fig. 4

Evaluation of the protective effect of the surface of porcine seminal extracellular vesicles (sEVs) upon oxidative DNA single-stranded breaks generated by different concentrations of hydrogen peroxide (H₂O₂). In this experiment, the sEVs were not permeabilized. Green indicates controls without sEVs (control), red indicates co-incubations with large sEV samples, and blue indicates co-incubations with small sEV samples. (**) show statistically significant differences (P < 0.01) (Two-way repeated measures ANOVA including sEV subtype and H₂O₂ concentration as factors; Tukey’s post-hoc test)

Fig. 5

Evaluation of the protective effect of the intravesicular content of porcine seminal extracellular vesicles (sEVs) upon oxidative DNA single-stranded breaks (SSBs) generated by different concentrations of hydrogen peroxide (H₂O₂). In this experiment, the sEVs were permeabilized using 0.1% Triton X-100. Green indicates controls without sEVs (control), red indicates co-incubations with large sEV samples, and blue indicates co-incubations with small sEV samples. (n.s.) indicate no significant differences (P > 0.05) (Two-way repeated measures ANOVA including sEV subtype and H₂O₂ concentration as factors; Tukey’s post-hoc test)

Fig. 6

A Enzymatic (thiol-reactive antioxidant molecules, THIOLs) and B non-enzymatic (cupric ion reducing antioxidant capacity, CUPRAC) antioxidant capacity of small and large porcine seminal extracellular vesicles (sEV). Results of 6 samples of small sEVs and 6 samples of large sEVs, with three technical replicates per each sEV sample. Results are expressed as nmol per mg of total protein. (*) show statistically significant differences (Unpaired T-tests, P < 0.05)

Fig. 7

Model showing that molecules located at the surface of small porcine seminal extracellular vesicles are able to counteract oxidative compounds, providing greater protection against oxidation