Comet assay 51

The alkaline version of the comet assay [110]was performed to assess repair capacity of SSB, alkaline labile sites (ALS), and DSB in LEC and LE cells. At alkaline pH (pH > 13) increased DNA migration results from conversion of ALS into SSB. The method was performed at the facilities of BFS (Institute for Radiation Hygiene, Neuherberg), following the protocol developed by Singh, 1995 [111] with modifications introduced by Maria Gomolka [112].

Preliminary experiments, which were conducted using fibroblasts, showed large heterogeneity in measured values and it was decided to measure repair in lymphocytes of whole blood. Investigations of Chuang et al., 2004 [113] show that the use of whole blood creates comet images, which where not different from those obtained from isolated lymphocytes. Three animals were bleeded and repair capacity was evaluated at 0, 15, 30 and 60 min after 4 Gy of IR.

Collection of blood and irradiation of samples

The blood samples were taken from the sublingual vein of rats following the protocol developed by Zeller et al., 1998 [114]. Unfasted rats were anaesthetized with isofluorane in an

inhalation chamber. One person held the unconscious animal and the second person pulled forward the tongue and punctured one of the sublingual veins with a gauge. Blood samples of volume about 0.5 ml were collected into tube containing anticoagulant (heparin).

20 µl of the whole blood was aliquoted in 1.5 ml Eppendorf tubes and irradiated on ice with 4 Gy of γ-irradiation. Irradiation was performed with HWM D2000 (Cs-137) at dose rate of

1.7 Gy/min).

Repair incubation, cell lysis and slides preparation

Immediately after irradiation the samples were placed in an incubator at 370C. At 0, 15, 30 and 60 min after irradiation 10 µl of an irradiated and for chosen time points of a control sample was removed and embedded in a second layer of agarose, prepared as the first layer (described later) and stored at 500C until use. The irradiated samples (10 µl) were mixed gently with 100 µl of the liquid agarose, placed on the slide and covered with a cover slide (dipped

before in 0.1% Triton-X/H2O and dried) to create a uniform surface without included air-bubbles.

The slides were placed for 5 min on a cold plate (40C), before removing the cover slip.

After removing the cover glass, the slides were placed in 50 ml of freshly prepared Lysis Buffer I and were incubated overnight at 40C. To remove proteins slides were placed in 50 ml of Lysis Buffer II and incubated at 370C for 1 hour.

Glass slides were prepared in advance. Slides were covered with a first layer of 0.1% low- melting agarose (Serva). 0.1% low-melting agarose was suspended in 0.9% NaCl, vortexed and permitted to rest for 10 min at 500C. Afterwards, the suspension was vortexed and microwaved briefly until boiling, checked for clumps. The process of vortexing and microwaving was repeated three times and agarose was aliquoted into Eppendorf tubes, which were agitated at 500C for 1 hour. Slides were put onto a warming plate at 450C and 200 µl aliquots of liquid agarose were pipetted onto the slides and evenly distributed with a spatula. The slides were dried completely at least for 1 hour.

Electrophoresis. Neutralization, dehydration and precipitation

1 litre of Electrophoresis Buffer was set up prior to electrophoresis and was precooled at 40C. Before electrophoresis slides were placed and fixed in a special modified electrophoresis chamber to allow DNA unwinding for 20 min at 40C. Slides were exposed to electrophoresis at 40C, for 30 min, at 24 V with stirring of buffer in the electrophoresis chamber.

For neutralization, dehydration and precipitation slides were placed in 50 ml of the neutralization buffer in Coplin Staining Jar and incubated for 30 min at RT in the dark. For further dehydration slides were placed in absolute EtOH overnight at RT.

Before drying slides covered with agarose were rehydrated in 70% EtOH for 5 min at RT preferably in the darkness. Dried slides were stored in a closed box until staining.

Staining of slides

For staining slides were placed twice into H2Odist, and 50 µl of the staining solution were

applied. The staining steps were performed in the dark as SYBRGreen is sensitive to light. Evaluation of results was done immediately following the staining.

Image acquisition

60-120 SYBR Green stained electrophoregrams (comets) in the central part of each slide were examined under epifluorescence microscope (Axiovert 135, Zeiss, Germany; 40 x air objective) equipped with filters for SYBRGreen and a monochromator (T.I.L.L. Photonics, Munich, Germany) as a light source for the image analysis with excitation at 461 nm and emission at 510 nm. Images were acquired with a Sony Video Camera (XC-7500) and evaluated by VisCOMET Software. Acquisition was done with preliminary adjustment of image saturation.

Figure 2.8. VisCOMET program interface with pictured example of acquired comet with defined regions of interest - tail, head, background

The image pictured by VisCOMET represents an electrophoregramm of the single cell DNA with extended ‘tail’ containing DNA fragments of different size, stained with SYBR Green. The program features for comet analysis include automatical and manual settings of head and tail regions, adjusting exposure and image parameters. The measured values include 24 characteristics of the acquired comet with statistical evaluation within the sample (mean, maximum, minimum, standard deviation).

Comet analysis

The regions of interests (head and tail) were determined automatically by VisCOMET from comet images (see Figure 2.8.). The list of evaluated parameters included eleven parameters

characterizing comet in total, eight parameters – head of comet, and six - % of tail DNA. From all comet characteristics measured the Tail (Olive) moment as commonly measured representative parameter was selected for further analysis.

Tail (Olive Moment) is defined as the product of the tail length and the fraction of total DNA in the tail. Tail moment incorporates a measure of both the smallest detectable size of migrating DNA (reflected in the comet tail length) and the number of relaxed / broken pieces (represented by the intensity of DNA in the tail):

Olive Tail Moment = (|CG – CG H|) x DNA/100

where:

CG = center of gravity of the tail or body weighted by gray values CGH = center of gravity of the head weighted by gray values DNA = tail or body DNA

Statistical analysis

Tail Olive Moment values were estimated in 3 animals in 60 – 120 cells each at each time point and data were presented in box and whiskers plots. A box and whiskers plot shows quartiles: One quarter of the values lie between the top whisker and the top of the box; one quarter (25th percentile) in above the median line (the 50th percentile) within the box; one quarter below the median line within the box (75th percentile); whiskers show the +/- 1.5 x IQR (interquartile range). The outliers are pictured too.

The values of Tail (Olive) moment for repair were compared at different times. Tail moment is known to have a non-Gaussian distribution [115]. In this case comparison between groups was done with Mann-Whitney U-test and nonparametric Kruskal-Wallis ANOVA rank model, using the Prizm 3.0 Software [116]. The work of Duez et al., 2003 [115] on statistical evaluation of comet data show that commonly used Mann-Witney test and Kruskal-Wallis

statistics are oversensitive and show the statistical significance even when the difference is not relevant biologically because of the large number of measurements in one sample (100). They recommend reduction of data to representative non-parametric statistics (medians, 75th percentiles) for further statistical analysis. Considering these recommendations, the statistical analysis of produced data included firstly testing for a difference between samples taken from different animals of one rat strain and measured at one time point applying Mann-Whitney U-test. Further statistical analysis included comparison of the means, medians and 75th percentiles, estimated for each measured animal, applying ANalysis Of VAriance (ANOVA).