Application of the CometChip platform to assess DNA damage in field-collected blood samples from turtles

Abstract

DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc.

Keywords: CometChip; DNA damage; comet assay; erythrocytes; vertebrates.

Figure 1. Assessment of nucleated erythrocytes using the CometChip Platform

Whole blood from a T. scripta was exposed to (A) H₂O₂, (B) Etoposide or (C) MMS for 30 minutes and measured using the CometChip assay. The CometChip was able to measure DNA damage accumulation, which increased with the concentration of DNA damaging agent. (D) Representative images of the cells after loading into the CometChip and the extent of DNA damage after exposure. (n=3964–4277 from duplicate experiments). Error bars represent mean ± 95% confidence interval.

Figure 2. Determination of CometChip suitability for field-based research

(A) Plan for the collection and utilization of blood samples collected in the field. Steps in green are conducted in the field, the steps in blue are conducted in the laboratory. (B) Cell counts were taken from each of the four turtles: T. scripta (TS), T. carolina (TC) and C. serpentina (CS). All turtle species had a high cell count. Each count was measured in triplicate. (C) Morphology of erythrocytes from the different turtle species. Magnified image of the cells after 4, 10 and 24 hours on ice. Storage on ice did not visibly effect cellular morphology. Erythrocytes from the CS animal had a distinct morphology compared to TS and TC cells. Scale bar represents 200 μm. Inserted box is 20 μm.

Figure 3. Effect of various storage conditions on levels of endogenous DNA damage

Adaptability of erythrocytes to freezing and thawing. Cells were frozen under multiple treatments and the effect on the levels of endogenous DNA damage was evaluated. As expected, PBS alone and no treatment resulted in high levels of DNA damage. The results suggest that any of the three interventions adequately protect the cell. The commercial freezing medium had the greatest protective effect closely followed by samples frozen in 10% DMSO (n= 350–1642). Error bars represent mean ± 95% confidence interval.

Figure 4. Measurement of DNA damage levels after exposure to DNA damaging agents when cells are stored on ice

Levels of DNA damage measured after 4 and 10 hours on ice. The capacity to repair induced DNA damage (exposure period of 30 minutes) was measured following incubation for 30 minutes at RT. (A) Levels of DNA damage in cells kept on ice for 4 hours (from each turtle type) were evaluated by the CometChip Platform. The levels of endogenous DNA damage, DNA damage following a 30-minute exposure to H₂O₂ (100 μM) and the levels of DNA damage following an additonal 30-minute repair period are shown. (B) Levels of DNA damage in cells kept on ice for 10 hours, as in panel A. (C) Levels of DNA damage in cells kept on ice for 4 hours (from each turtle type) were evaluated by the CometChip Platform. The levels of endogenous DNA damage, DNA damage following a 30-minute exposure to MMS (100 μM) and the levels of DNA damage following an additional 30-minute repair period are shown. (D) Levels of DNA damage in cells kept on ice for 10 hours, as in panel C. (****= p<0.001, n= 488–1272). Error bars represent mean ± SEM. All experiments carried out in unfrozen samples.

Figure 5. DNA damage analysis in cells from T. scripta

(A) Spread of endogenous DNA damage in the cohort. All data points are shown. Error bars are mean ± 95% confidence interval. Female animals are represented in grey, males represented in black. (B) Female animals trended towards having a higher endogenous level of DNA damage, n= 9859–12852 comets analyzed. (C) Samples were exposed to H₂O₂ and DNA damage measured immediately and after 30 minutes of repair. (D) The amount of DNA damage accumulated was normalized by subtracting the level of endogenous damage. There were no sex-linked differences in damage accumulation. (E) The % of DNA damage that was repaired was also compared. The amount of repaired substrate was also normalized by subtracting the level of endogenous damage. Both sexes repaired the induced DNA damage poorly. (Male n=9, Female n=9). All experiments were technical duplicates using un-frozen samples. Error bars represent mean ± SD.

Figure 6. DNA damage analysis in cells from T. carolina

(A) Spread of endogenous DNA damage in the cohort, data shows all points. Females are in grey, males in black. Error bars are mean ± 95% confidence interval. (B) Female animals have a statistically higher endogenous level of DNA damage, n=13457–11412 comets analyzed. (C) Samples were exposed to H₂O₂ and DNA damage measured immediately and after 30 minutes of repair. (D) The amount of DNA damage accumulated was normalized by subtracting the level of endogenous DNA damage. There were no sex-linked differences in damage accumulation but (E) the % of DNA damage that was repaired was statistically lower in the female group. Normalization: the % repair data has had the endogenous DNA damage levels subtracted from the values. (male n=9, Female n=8), ** = p< 0.01. All experiments carried out in unfrozen samples in technical duplicate. Error bars represent mean ± SD.

Figure 7. Comparative analysis of T. carolina and T. scripta

(A) The endogenous DNA damage levels of the two species T. carolina (TC) and T. scripta (TS) were compared. Data were compared at the single comet level. Individual comets from duplicate assays were evaluated (TC, n= 24869; TS, n=22711). The comparison of the level of accumulated DNA damage shows that (B) the cells from T. scripta animals accumulated less DNA damage than cells from T. carolina animals after H₂O₂ exposure and (C) the cells from T. scripta animals repaired a greater percentage of the H₂O₂ damaged DNA than cells from T. carolina animals. (TS n=19, TC n= 17), **** p< 0.001, Error bars represent mean ± SD. (D) The cells from the T. scripta had a correlation (r= 0.419) between the length of the animal and the capacity to repair DNA damage. (E) This correlation was less evident in the cells from the T. carolina cohort (r= 0.078).