Abstract

During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many ‘real-life’ studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate ‘tissues’ such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 μM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.

Introduction

The efficiency of DNA repair processes is suggested to be an important determinant in the ageing process and development of human degenerative diseases such as cancer. There is evidence that genetic and dietary factors may influence DNA repair (1,2), but in order to elucidate these effects, simple, fast and reliable assays to measure DNA repair are required. In the past decade, three types of in vitro repair assays have been developed and used widely to measure DNA repair and/or incision activity. The assays depend on the capacity of a cell extract to recognise and incise substrate DNA-containing specific lesions. One of these approaches uses a closed circular plasmid that contains a specific lesion as substrate. When incubated with the whole cell extract, repair can be estimated either as nicking of the plasmid followed by separation on gel (3) or by the incorporation of radioactive precursors into a repair patch (4). In this way, the plasmid assay measures the overall repair starting from incision through repair synthesis. In a second approach, the cell extract is incubated with an oligonucleotide that is constructed with a specific DNA lesion and a terminal radioactive or fluorescent tag (5,6). The repair enzymes in the extract will incise the oligonucleotide at the damaged site, causing the release of the label, which can be measured as an indicator for DNA repair using conventional gel electrophoresis.

The third type of in vitro repair assay is a modified version of the alkaline comet assay (single-cell gel electrophoresis) that was first developed by Collins et al. (7,8) to measure base excision repair (BER). This alternative approach involves measurement of the capacity of cell extracts to perform the initial steps of BER, i.e. damage recognition and incision, on DNA substrates carrying 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesions. Moreover, this modified comet-based in vitro repair assay was modified for the measurement of nucleotide excision repair (NER)-related DNA incision activity, using substrates containing UV- (8,9) or benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced (10) DNA lesions. These comet assay-based methods are versatile and can be readily adapted to measure either DNA incision activity or DNA repair capacity as a whole via the addition of dNTPs to the cell extract (8).

In most studies, these 3 types of in vitro repair assays have been used to study DNA repair in cell extracts from cultured cells or surrogate tissues (e.g. lymphocytes or semen). However, to further substantiate the role of DNA repair in the development of degenerative diseases and to investigate the influence of gene–environment interactions on DNA repair, it would be a considerable advantage to be able to measure DNA repair in the tissues of interest. For several years, the oligonucleotide and plasmid-based assays have been used extensively and with success to measure DNA repair/incision activity in tissue extracts (11–15). Although comet-based assays are easy to use, sensitive, versatile and relatively inexpensive, to the best of our knowledge, there are only a few reports that describe the use of animal tissue extracts in the comet-based assay to measure NER (16)- or BER (17)-related DNA incision activity in vitro. Rodents, in particular mice, are the preferred animal model for studies of longevity and of age-related diseases as well as of the effects of interventions to delay or prevent the development of such diseases (18,19). Therefore, various attempts have been made by different laboratories to use the comet-based in vitro assays with extracts from rodent tissues [discussed at the International Comet Assay Workshop (ICAW) and other meetings]. However, similar to reports on the other two in vitro repair assays (20–23), attempts with the comet-based in vitro assays (for assessing BER or NER activity) have been frustrated by low repair activity and/or low detection sensitivity due to the presence of non-specific nuclease activity (unpublished results and from discussions at the ICAW and other meetings). Although, increasing the salt concentrations of the buffers (>100 mM KCl and addition of 1 mM EDTA) has previously been shown to reduce the interference of non-specific nucleases substantially (22,23), still some tissue extracts showed some non-specific nuclease background activity. Therefore, our aim was to further optimise the comet-based in vitro repair assay for use with rodent tissues extracts. In this study, we focussed on the measurement of DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage, which can be used as an indicator for BER.

To overcome the problem of interference by non-specific nuclease/cleavage activity, various optimisation steps were undertaken, including optimisation of the protein concentration in the tissue extract. Furthermore, the use of aphidicolin (APC) has been reported to increase the sensitivity of other comet-based assays (24–26) and to improve determination of DNA repair capacities (27,28). Although, APC is generally used as a DNA polymerase inhibitor, it was also shown to inhibit certain nuclease activities (29–31). Therefore, we used APC in an attempt to increase the specificity of the repair assay. The improved assay was validated by using tissues from BER-deficient Ogg1 knockout mice. This newly developed assay has been proven to be sensitive and specific and was shown to be useful in determining the effect of ageing and of dietary factors on DNA repair in various mouse tissues.

Materials and methods

All chemicals and reagents were purchased from Sigma (Dorset, UK) unless specified.

Animal husbandry and tissue processing

Mice were obtained from a long-established colony of the C57/BL(ICRFa) strain that had been selected for use in studies of intrinsic ageing because it is free from specific age-associated pathologies and thus provides a good general model of ageing (32). Mice were housed in standard cages of different sizes depending on housing density (between one and six mice per cage). Mice were housed at 20 ± 2°C under a 12-h light/12-h dark photoperiod with lights on at 7 am. Mice were provided with sawdust and paper bedding and had ad libitum access to water and food [CRM (P) Special Diet Services; BP Nutrition Ltd, Essex, UK]. All work complied with the UK Home Office Animals (Scientific procedures) Act of 1986.

To study the effects of ageing, brains were collected from ad libitum fed male mice at ages 3 and 30 months (four per age group). A subgroup of animals from the ageing colony was included in a dietary restriction (DR) study, where the food intake of DR mice was restricted by 26% compared with ad libitum fed male mice (33). DR was initiated at 14 months of age, and after 3 months, both DR (n = 7) and ad libitum mice (n = 6) were killed and livers collected.

For validation purposes, we obtained liver and brain tissues from two wild-type (C57BL/6) and two Ogg1−/− (also with C57BL/6 genetic background) (34) 16-month-old female mice from Dr Bernd Epe and Dr Markus Fußer (Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University, Mainz, Germany). The Ogg1−/− genotype was confirmed using polymerase chain reaction (PCR) amplification specific to the wild-type and targeted Ogg1 allele. The mice were housed in standard cages under a 12-h light/12-h dark cycle according to the German animal welfare act and had ad libitum access to water and a standard diet (Altromin in Lage, Germany).

In all experiments, tissues were immediately snap frozen in liquid nitrogen and stored at −80°C. Before analyses, tissues were ground under liquid nitrogen, weighed, aliquoted, and stored at −80°C.

Measuring DNA repair incision activity

To get a measure of the BER activity in mouse tissues, we modified a comet-based in vitro repair assay originally developed by Collins et al. (7) and used it to quantify the DNA repair incision activity. For clarity of the paper and to assist new users of the in vitro repair assay, each step of the assay is described below.

Principle of the assay.

The DNA substrate consists of gel-embedded nucleoids (i.e. protein-depleted nuclei of supercoiled DNA that remain after cell lysis) from cells that were pre-treated with the photosensitiser Ro 19-8022 plus light. The predominant form of DNA damage induced by this treatment is 8-oxodG [∼75% of the formamidopyrimidine DNA glycosylase (FPG)-sensitive base modifications] (35), which are to a large extent generated via singlet oxygen. Incubation of these substrate nucleoids with tissue extracts allows the initial steps of BER to occur; (i) repair is initiated by a DNA glycosylase that recognises specifically the 8-oxodG lesions and removes it from the sugar-phosphate-backbone, generating an AP (apurinic/apyrimidinic) site; (ii) followed by incision of this phosphodiester backbone by an AP endonuclease enzyme or by the glycosylase itself. This will result in single-strand breaks that can be determined by subsequent single-cell alkaline gel electrophoresis. Therefore, increased %DNA in the tail (TI) and tail moments (TMs) are proportional to the BER-related DNA repair incision activity of the tissue extracts. A schematic overview of the assay is shown in Figure 1.

Fig. 1

Overview of comet-based in vitro repair assay to measure BER-related incision activity. Substrate cells are exposed to the photosensitiser Ro 19-8022 to induce 8-oxodG lesions (filled cross). After lysis, gel-embedded nucleoids are incubated with tissue extracts. Subsequent standard single-cell gel electrophoresis reveals the single-strand breaks introduced by the BER enzymes.

Fig. 1

Overview of comet-based in vitro repair assay to measure BER-related incision activity. Substrate cells are exposed to the photosensitiser Ro 19-8022 to induce 8-oxodG lesions (filled cross). After lysis, gel-embedded nucleoids are incubated with tissue extracts. Subsequent standard single-cell gel electrophoresis reveals the single-strand breaks introduced by the BER enzymes.

Preparation of substrate cells.

HeLa cells were grown in T175 plates in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin until ∼80% confluency. Cells were washed in ice-cold phosphate-buffered saline (PBS) and subsequently exposed on ice to 1 μM photosensitiser Ro 19-8022 (Hoffmann-La Roche, Basel) in PBS (hereafter called Ro cells) or PBS alone (here after called noRo cells) for 5 min while being irradiated by a 500 W halogen lamp at a distance of 33 cm. After washing with PBS, cells were harvested, gently re-suspended and diluted to 3 × 106 cells/ml in freezing medium (i.e. DMEM containing 10% dimethyl sulfoxide (DMSO)). The cell suspensions were divided in aliquots of 200 μl and frozen slowly overnight in a thick polystyrene box at −80°C. Frozen aliquots were then stored in −80°C until use in the repair assay.

To check the total level of substrate in the Ro cells, nucleoids were incubated with FPG (New England Biolabs) 1/3000 in reaction buffer F (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8). FPG is a bacterial DNA glycosylase that recognises/repairs oxidised purines, including 8-oxodG, resulting in DNA strand breaks when incubated with oxidised DNA substrates (7,36). In our assay, the level of substrate in the Ro cells assessed as FPG-sensitive sites corresponded to ∼60% DNA in the tail after 25 min of incubation at 37°C.

To study the effect of low-melting point (LMP) agarose concentration on DNA migration and the tissue extract/enzyme incubation, a subset of HeLa cells was exposed to 50 μM of H2O2 in DMEM or DMEM only for 15 min on ice. After exposure, cells were washed with PBS, harvested and diluted to 3 × 106/ml in PBS for immediate use.

Preparation of tissue extracts.

Prior to an assay, ∼50 mg aliquots of ground tissue were thawed. Ground liver was washed briefly with ice-cold PBS and spun down at 700 g for 5 min to remove non-liver cell material such as haemoglobin and bilirubin, which may lead to overestimation of the cellular protein concentration (37,38). Subsequently, 200 μl extraction buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, pH 7.8) were added to the washed liver and 100 μl to the unwashed brain tissue aliquots. Resulting suspensions were vortexed vigorously, snap frozen in liquid nitrogen and immediately thawed. To each sample, 30 μl of 1% Triton X-100 in buffer A were added per 100 μl of aliquot, vortexed vigorously and incubated for 5 min on ice. If necessary, larger particles of tissue were homogenised with a microtube pestle. After centrifugation at 14 000 g for 5 min at 4°C to remove cell debris, the supernatant was collected and protein concentrations were determined by the BIO-RAD DC Protein Assay Kit using bovine serum albumin as a standard and controlling for the presence of Triton X-100. This Lowry-based protein assay measures protein concentrations at 650–750 nm. At these wavelengths, absorbance of haemoglobin (high absorption at ∼250–600 nm) and bilirubin (high absorption at ∼400–500 nm) is negligible, especially when samples are well diluted (39–41), which suggests that it is unlikely that contamination by these substances caused overestimation of liver protein in our studies. Protein extracts from liver tissues were diluted with 0.23% Triton X-100 in buffer A to a concentration of 5 mg/ml, whereas protein extracts from brain tissues were diluted to 1 mg/ml. For validation purposes, we also used the same protocol to prepare extracts from liver and brain tissues from wild-type and Ogg1−/− mice. Diluted protein extracts were stored at −80°C until use in the repair assay. Tissues extracts with a protein concentration of ∼20–30 mg/ml can be obtained from ∼50 mg of tissue, which is sufficient material for several assays (∼20 assays when running samples in duplicate in a 2 gel/slide format).

Incubation reaction and quantification of DNA incision activity.

One the day of the repair assay, 200 μl aliquots of noRo and Ro cells (3 × 106 cells/ml) were thawed and washed with 1 ml of ice-cold PBS spinning at 700 g for 5 min at 4°C. The supernatant was removed and 3 ml of 0.65% LMP agarose (dissolved in PBS) were added to each cell pellet. A 2 gels/slide format was used. Thus, from each suspension (containing noRo or Ro cells), 75 μl were transferred to each microscope slide, which were pre-coated with 1% agarose. Gels were covered with 22 × 22 mm coverslips and kept at 4°ºC for 10 min to solidify. Subsequently, cover slips were removed and gel-embedded cells were lysed for 1 h at 4°ºC in lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 0.25 M NaOH, pH 10, and 10% dimethyl sulphoxide and 1% Triton X-100 were added just before use). After lysis, slides were washed 2× for 15 min with reaction buffer F.

Prior to the repair incubation, diluted tissue extracts were thawed and four volumes of reaction buffer F were added. Similarly, a control solution was prepared, i.e. buffer A containing 0.23% of Triton X-100 (used to dilute the extracts) to which buffer F was added (1:4). Extracts and control solution were kept on ice until use. To assess DNA repair incision activity, 50 μl of tissue extract or control solution were added to each gel (containing nucleoids of either Ro or noRo cells), which were covered with 24 × 50 mm coverslips and incubated in a moist slide moat (VWR, Boekel Scientific) for 25 min at 37°ºC. Samples were tested in two independent incubations within each single experiment. After the incubation, slides were immediately put on ice to stop the enzyme reactions and kept on ice during transportation to the cold room, where denaturation of the DNA was performed by immersion of the slides in cold electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, ca. pH 13) for 40 min, followed by 30 min of electrophoresis at ∼1.14 V/cm. Next, slides were neutralised by washing for 10 min in ice-cold PBS and 10 min in ice-cold milipure water. Slides were either dried overnight and stained the day before analysis or immediately stained with SYBR Gold (10 000× stock in DMSO from Invitrogen, Molecular Probes, UK) diluted to 1× in DMSO and analysed the day after. Comets were visualised using an Olympus BX51 fluorescence microscope and 50 comets/slide selected at random were analysed using the Comet assay IV software program (Perceptive Instruments, Haverhill, UK). Resulting data were presented as TM and %DNA in the tail [also known as tail intensity (TI)] ± standard error. After subtracting background levels (TInoRo/Buffer) from all data, the final DNA repair incision activity was calculated using a formula similar to that published previously (10): 

graphic
in which Ro/Extract refers to nucleoids containing 8-oxodG lesions that were incubated with the tissue extract, noRo/Extract refers to nucleoids containing no lesions that were incubated with tissue extract, and Ro/Buffer represents the nucleoids containing 8-oxodG lesions that were incubated with the buffer control solution only.

Metal determination by inductively coupled plasma mass spectrometry

Protein extracts, diluted to 10 mg/ml total protein in extraction buffer A, were diluted to final protein concentration of 2 mg/ml (Table I) or 1 mg/ml (Table I) in 65% HNO3 (Merck) and incubated at room temperature for 48 h to decompose. These samples were diluted 1:10 into 2.5% HNO3 (w/v) containing 20 μg/l cobalt and 20 μg/l silver as internal standards (BDH) and analysed by inductively coupled plasma mass spectrometry (Thermo X-series). Each mass ion (24Mg, 59Co, 66Zn and 107Ag) was measured 100 times (20 ms integration time, 5 channels, 0.02 AMU separation) using the peak-jump method, in triplicate for each sample, and detector pulse counts were converted to concentration by comparison with matrix-matched metal standards (BDH). Resulting data were presented as μg/mg protein in the tissue extract.

Table I

Magnesium (Mg) and Zinc (Zn) content in tissue extracts after standard protein extraction procedurea and for comparison of washing effectb.

Metal concentration (μg/mg protein) Braina (unwashed) Livera (washed) Brainb
 
Liverb
 
Washed Unwashed Washed Unwashed 
Mg 6.95 ± 0.549* 4.51 ± 0.557* 10.40 ± 0.068 10.34 ± 0.204 7.35 ± 0.044 10.13 ± 0.113 
Zn 0.45 ± 0.030 0.47 ± 0.056 1.06 ± 0.001 0.89 ± 0.002 1.05 ± 0.001 1.25 ± 0.001 
Metal concentration (μg/mg protein) Braina (unwashed) Livera (washed) Brainb
 
Liverb
 
Washed Unwashed Washed Unwashed 
Mg 6.95 ± 0.549* 4.51 ± 0.557* 10.40 ± 0.068 10.34 ± 0.204 7.35 ± 0.044 10.13 ± 0.113 
Zn 0.45 ± 0.030 0.47 ± 0.056 1.06 ± 0.001 0.89 ± 0.002 1.05 ± 0.001 1.25 ± 0.001 

Data are presented as microgram of metal per milligram of protein ± standard error.

a

Analysis performed on protein extracts (n = 3) prepared from tissues samples using the standard protocol, including a washing step in cold 1× PBS during preparation of liver extracts but no washing step for brain extracts. SEMs of three independent biological replicates are shown.

b

Analysis performed on separate protein extracts after tissue samples were either washed or not washed in cold 1× PBS before protein extraction (n = 1 for each condition, standard errors of three experimental replicates are shown).

*P = 0.035 for Mg concentration in brain versus liver extracts.

Statistical analysis

The effect of LMP agarose concentration and heat inactivation on the detected DNA repair incision activity was examined by analysis of variance (ANOVA) test using post hoc Dunnett's t-test or Tukey's honest significance test (HSD) test, respectively. General linear model univariate analysis of variance (GLMUA) using Dunnett's t-test for between-treatment comparisons was used to study the influence of APC and/or DMSO as well as the effect of Ogg1−/−. Differences in DNA repair incision activity due to ageing and nutrition as well as differences in Mg concentrations were analysed by Student's t-tests. Homogeneity of variance was assessed by Levene's test. Statistical analysis was performed using SPSS v.17.0. and P < 0.05 was considered statistically significant. Data presented on the effect of ageing, nutrition and Mg concentrations are shown as mean values of biological replicates (n ≥ 3) repeated in two independent experiments with standard error of the mean (SEM). All other results are reported as mean values of biological replicates (n = 2) repeated in two independent experiments with the pooled SEM.

Results and discussion

Although Collins et al. (7) did not observe any non-specific endonuclease activity in cell extracts, the use of tissue extracts in various in vitro repair assays has often been reported to be associated with high levels of non-specific nuclease activity (20–23), which makes it difficult to obtain a robust estimate of BER-related DNA incision activity. In this study, we performed various optimisation tests to minimise any non-specific signals and thereby maximise the specificity of the assay. Tissue extracts from both male and female mice were prepared and used at random during the optimisation steps.

Importance of incubation time and protein concentration of tissue extracts

Based on previously reported time curves of the incision activity (7) and our own results (data not shown), 25 min was chosen as the preferred incubation time for the extracts since this allows us to detect the DNA repair incision activity in the linear part of the activity-with-time curve. A typical curve shows an initial linear increase in DNA incision activity after which it reaches a plateau. Ideally an incubation time would be selected that is still on the linear part of the time-incisions curve, detecting a high enough number of incisions before reaching the plateau.

In addition to the incubation temperature, the incision activity of an extract is also dependent on its protein concentration. When the protein concentration of an extract is too low, it is difficult to select a reliable incubation time (data not shown). On the other hand, a high protein concentration can result in higher non-specific damage recognition and decreased specificity of the assay (10). To identify the protein concentration at which there is a maximal difference between the total and non-specific damage recognition and incision by a given tissue extract, we recommend running a dilution curve for the tissue of interest before starting the main experiments. In this study, protein extracts from liver and brain tissues from two mice were diluted in buffer A with 0.23% Triton X-100 to various concentrations ranging from 2.5 to 7.5 mg/ml and 0.25 to 2 mg/ml, respectively. No dilutions of protein extracts lower than 2.5 mg/ml (for liver) or 0.25 mg/ml (for brain) were tested to prevent the TIRo/Extract values from becoming too close to the background levels. For protein extracts prepared from mouse brain, a protein concentration of 1 mg/ml was selected. At this protein concentration, the non-specific damage recognition was relatively low (TInoRo/Extract = 3.38 ± 1.03%) and specific damage recognition was high (TIRo/Extract = 10.05 ± 1.07%), with a difference of 6.66 (Figure 2A). The preferred protein concentration for tissue extracts from mouse liver was 5 mg/ml, showing a non-specific signal of 1.53 ± 0.41% and a difference of 9.48% DNA in the tail (Figure 2B). Although protein extracts with a concentration of 7.5 mg/ml showed a higher difference (14.97%) with a similar low non-specific signal (TInoRo/Extract = 1.32 ± 0.41%), we chose a concentration of 5 mg/ml to minimise the required amount of protein extract and thus reduce the amount of tissue needed. Moreover, these selected protein concentrations are able to incise ∼10–15% of the substrate DNA during 25 min of incubation at 37°C, which falls on the linear part of the time curve. In conclusion, it is advisable to run dilution curves for each tissue and different mouse strain in addition to time curves.

Fig. 2

Protein dilution curves of brain (A) and liver (B) extracts. The incision activity is related to the extract concentration and different for each tissue. Data from two biological replicates are presented as the mean of two independent experiments, calculated based on TI values. Bars indicate the pooled SEMs.

Fig. 2

Protein dilution curves of brain (A) and liver (B) extracts. The incision activity is related to the extract concentration and different for each tissue. Data from two biological replicates are presented as the mean of two independent experiments, calculated based on TI values. Bars indicate the pooled SEMs.

Effect of LMP agarose

Previous studies reported the use of various concentrations of LMP agarose in the comet assay (42,43). It is uncertain whether the different gel concentrations only affect the migration of the DNA or if they could affect penetration of enzymes from the tissue protein extract into the gel as well. Therefore, we tested the effect of a range of concentrations (0.65–1%) of LMP agarose on the sensitivity of the assay (Figure 3). Substrate cells exposed to H2O2 or unexposed controls cells were either used in a standard comet assay to detect H2O2-induced DNA damage or incubated with a liver extract, FPG enzyme or buffer control solution to detect the DNA repair incision activity. The results are presented as the difference in DNA incisions between the unexposed (average background level = 1.734 ± 0.44) and H2O2-esposed substrates. Significantly different (Pt-test = 0.009) slopes of the relationships between the % of LMP agarose and the DNA incisions were detected for both the liver extract (slope = 130.70) and the FPG enzyme (slope = 122.83), compared with incubation with buffer (slope = 73.42) and the standard comet assay (slope = 79.18) that both represent the H2O2-induced (background) damage. Moreover, ANOVA analysis showed that the LMP agarose concentration affected significantly the number of DNA incisions detected after incubation with liver extract (PANOVA = 0.020; PDunnett = 0.014 and PDunnett = 0.026 for 0.9 and 1% versus 0.65%, respectively) or FPG enzyme (PANOVA = 0.032; PDunnett = 0.044 for 1% versus 0.65%), while the migration of the DNA, as detected via the standard comet assay (PANOVA = 0.131) and buffer incubation (PANOVA = 0.171), are not affected significantly by the % of LMP agarose (Figure 3). To summarise, the concentration of the LMP agarose gel seemed to influence the penetration of DNA repair proteins from the tissue extracts through the gel to reach the nucleoids and the optimum concentration appeared to be ≤0.8%.

Fig. 3

The effect of the LMP agarose concentration on the DNA migration (PANOVA ≥ 1.131) and detection of DNA repair incision activity (PANOVA ≤ 0.032). To study DNA migration, H2O2-induced DNA damage was detected via standard comet assay (filled diamonds, slope = 79.18) or after buffer incubation (filled triangles, slope = 73.42). DNA incisions were detected after incubation with liver extract (filled squares, slope = 130.7; ***PDunnett = 0.014 and **PDunnett = 0.026 versus 0.65%) or FPG enzyme (filled circles, slope = 122.83; *PDunnett = 0.044 versus 0.65%). The results are presented as the mean difference (n = 2) in DNA incisions between the unexposed and H2O2-exposed substrates. Bars indicate the pooled SEMs.

Fig. 3

The effect of the LMP agarose concentration on the DNA migration (PANOVA ≥ 1.131) and detection of DNA repair incision activity (PANOVA ≤ 0.032). To study DNA migration, H2O2-induced DNA damage was detected via standard comet assay (filled diamonds, slope = 79.18) or after buffer incubation (filled triangles, slope = 73.42). DNA incisions were detected after incubation with liver extract (filled squares, slope = 130.7; ***PDunnett = 0.014 and **PDunnett = 0.026 versus 0.65%) or FPG enzyme (filled circles, slope = 122.83; *PDunnett = 0.044 versus 0.65%). The results are presented as the mean difference (n = 2) in DNA incisions between the unexposed and H2O2-exposed substrates. Bars indicate the pooled SEMs.

Effect of protease inhibitors

To prevent degradation of the proteins in the tissue extract and thereby to maximise the detection of DNA repair incision activity, we tested the effect of addition of a cocktail of protease inhibitors (Complete, Roche, Germany) to different aliquots of the same ground liver tissue during the preparation of the tissue extracts as follows; either extraction buffer A or 1× protease inhibitors in buffer A was added. The rest of the extraction procedure was the same as described above. No significant difference in DNA repair incision activity was observed when protease inhibitors were added; DNA repair incision activity was 9.45 ± 3.45 versus 10.36 ± 0.72 with and without protease inhibitors, respectively. This lack of effect of addition of protease inhibitors may be due to our maintenance of samples on ice during the tissue extract preparation, which will minimise the activity of endogenous proteases.

The presence of non-specific nuclease activity

To test the tissue extracts for the presence of non-specific nuclease activity, non-exposed substrate cells in parallel to cells exposed to light (noRo) and light plus Ro 19-8022 were incubated with buffer control solutions, liver extracts, brain extracts or FPG enzyme solutions that were either heat inactivated (HI) for 10 min at 100°C or not (Figure 4). Focussing first on the Ro data, it is clear that the heat inactivation had worked because enzyme activity in the liver (PTukey HSD = 0.016) and brain extracts (PTukey HSD = 0.002) as well as the FPG enzyme solutions (PTukey HSD < 0.001) was significantly reduced to background levels. Similar observations were reported by Mikkelsen et al. (17) who detected no incision activity in liver tissues or FPG enzyme activity after the samples had been HI. Non-specific nuclease activity was only detected in the brain extracts after incubation with both non-exposed and noRo substrates (PTukey HSD = 0.047 and PTukey HSD = 0.001 non-HI versus HI, respectively). Ro-exposed substrates incubated with HI brain extracts seemed to show still some residual non-specific, non-enzymatic cleavage activity. However, the levels of %DNA in the tail was not significantly higher when compared with the HI buffer control (PTukey HSD = 0.998), indicating that it might represent background damage (e.g. due to handling of the cells) rather than non-specific cleavage activity.

Fig. 4

DNA repair incision activity of HI and non-HI samples and controls. Data are presented as the mean (n = 2) of two independent experiments, calculated based on TI values. Bars indicate the pooled SEMs (aP = 0.047, bP = 0.001, cP = 0.016, dP = 0.002, eP < 0.001 non-HI versus HI, post hoc Tukey HSD).

Fig. 4

DNA repair incision activity of HI and non-HI samples and controls. Data are presented as the mean (n = 2) of two independent experiments, calculated based on TI values. Bars indicate the pooled SEMs (aP = 0.047, bP = 0.001, cP = 0.016, dP = 0.002, eP < 0.001 non-HI versus HI, post hoc Tukey HSD).

Effect of APC

Previously, the DNA polymerase inhibitor APC has been shown to increase the sensitivity of comet-based assays in detecting DNA damage responses and DNA repair capacities (24–28). Moreover, APC may have an inhibitory effect on various nucleases; it had been demonstrated to inhibit Herpes Simplex virus DNA polymerase-associated nuclease activity (29), as well as the 3′ → 5′ exonuclease activity of eukaryotic polymerases δ and ϵ (30,31), although it had no detectable effect on the exonuclease activity of Escherichia coli DNA polymerase II (44). In our study, we used APC mainly to inhibit non-specific nucleases as well as any remaining DNA polymerase activity present in the tissue extract (see supplementary data, available at Mutagenesis Online), thereby increasing the detectable DNA repair incision activity. Prior to the incubation step, 0 (vehicle control DMSO), 0.6, or 1.5 μM of APC (Merck) was added to brain and liver extracts. These particular APC concentrations were chosen because they were shown previously (24,27) not to be genotoxic when assessed by the standard comet assay, which was confirmed by our own results (Figure 5A). Importantly, we found that APC addition (1.5 μM) to the tissue extracts increased significantly (PDunnett = 0.018) the detection of specific DNA damage recognition/incision (Figure 5B and C). This APC concentration was selected as the optimal concentration for further experiments.

Fig. 5

The effect of APC on (A) the background DNA damage and on the BER-related incision activity detected in (B) liver and (C) brain extracts. Data are shown as mean values (n = 2) of two independent experiments, and bars indicate the pooled SEMs. Addition of 1.5 μM APC significantly affected the sensitivity of the assay (*P = 0.018, brain and liver data combined, GLMUA post hoc Dunnett t-test).

Fig. 5

The effect of APC on (A) the background DNA damage and on the BER-related incision activity detected in (B) liver and (C) brain extracts. Data are shown as mean values (n = 2) of two independent experiments, and bars indicate the pooled SEMs. Addition of 1.5 μM APC significantly affected the sensitivity of the assay (*P = 0.018, brain and liver data combined, GLMUA post hoc Dunnett t-test).

We then repeated the dilution curves for the extracts with and without the addition of APC. [Note: no vehicle control DMSO was added to the extracts without APC as an attempt to compare the standard BER assay protocol with our modified assay (discussed below)]. Although, APC is mainly known as a DNA polymerase inhibitor, in these studies, it also significantly reduced the non-specific nuclease activity in brain extracts (Figure 6A, white bars; PGLMUA = 0.002), which was most pronounced in the undiluted (∼20 mg/ml) and 2× diluted extracts. There was no similar effect when using liver extracts (Figure 6B). Indeed, undiluted liver extracts (∼50 mg/ml) did not show significantly higher non-specific nuclease activity than the diluted extracts. Although the same extraction buffer, with the same concentration of EDTA (Mg chelator), was used to prepare the various brain and liver extracts, the differences in detected nuclease activity could be related to differences in Mg content (Table I); Mg concentration was significantly higher in the brain extracts than in the liver extracts (PANOVA = 0.035).

Fig. 6

Protein dilution curves showing the effect of 1.5 μM APC on the non-specific nuclease activity in (A) brain (PGLMUA = 0.002, white bars with versus without APC) and (B) liver extracts. Mean values (n = 2) of two independent experiments are shown. Bars indicate the pooled SEMs.

Fig. 6

Protein dilution curves showing the effect of 1.5 μM APC on the non-specific nuclease activity in (A) brain (PGLMUA = 0.002, white bars with versus without APC) and (B) liver extracts. Mean values (n = 2) of two independent experiments are shown. Bars indicate the pooled SEMs.

We further investigated whether this effect could be due to the additional washing step used during the preparation of liver extracts (not used in the preparation of brain extracts) because this was the only difference between sample preparation. Although this additional washing step seemed to be responsible for the lower Mg concentrations in the preparation of liver extracts (Table I), similar non-specific damage recognition was detected in undiluted liver extracts (TInoRo/Extract = 14.91 ± 2.13% without versus 15.50 ± 0.19% with washing step). However, adding a washing step during the preparation of brain extracts did not alter the Mg concentration in the extracts (Table I), nor reduce the non-specific nuclease activity in undiluted extracts (TInoRo/Extract = 33.01 ± 1.61% without versus 36.26 ± 0.94% with washing step). This suggests that the washing step mainly removes Mg originating from residual blood, present on frozen liver tissues but not on brain tissues, rather than Mg derived from the tissue itself.

Alternatively, it was possible that the solvent DMSO had an effect on the non-specific nuclease activity since we had compared extracts with the addition of APC dissolved in DMSO (Figure 6) without including a DMSO control. Furthermore, studies have shown that the solvent DMSO can inhibit the oxidative DNA cleavage activity of redox active transition metal complexes (45,46) and food compounds such as anthocyanins and their aglycone anthocyanidins (47). Therefore, we repeated part of these experiments analysing undiluted tissue extracts with and without the addition of DMSO in parallel to extracts supplemented with 1.5 μM APC in DMSO (Figure 7). Although, not statically significant (PDunnett = 0.112), the addition of DMSO indeed appeared to be partially responsible for the increase in detectable BER activity. Importantly, the addition of 1.5 μM APC in DMSO produced a further increase of the detectable BER activity that enhanced significantly (PDunnett = 0.017) the sensitivity of the assay compared with buffer incubation only (Figure 7).

Fig. 7

The effect of DMSO on the non-specific cleavage activity, without or in combination with 1.5 μM APC (*P = 0.002 versus buffer control, brain and liver data combined, GLMUA post hoc Dunnett t-test). Data are presented as the mean (n = 2) of two independent experiments, calculated based on TI values. Bars indicate pooled SEMs.

Fig. 7

The effect of DMSO on the non-specific cleavage activity, without or in combination with 1.5 μM APC (*P = 0.002 versus buffer control, brain and liver data combined, GLMUA post hoc Dunnett t-test). Data are presented as the mean (n = 2) of two independent experiments, calculated based on TI values. Bars indicate pooled SEMs.

In summary, addition of APC in DMSO increased the specificity of the assay, by inhibiting non-specific nuclease activity and any residual DNA polymerase activity. Although the addition of APC in DMSO makes it possible to use undiluted tissue extracts, running dilution curves and selecting a reliable protein concentration is still advisable because (i) the assay reaches saturation when using undiluted extracts (∼80–90% of DNA in tail) and (ii) smaller amounts of tissue are required if diluted tissue extracts are used. Nonetheless, the addition of APC in DMSO makes it possible to use higher protein concentrations than those selected initially. For instance, a protein concentration of 5 mg/ml could be used for brain extracts as well as for liver extracts, to allow comparison of DNA repair incision activity between tissues.

Effect of long-term cryopreservation

To evaluate the effect of long-term storage of tissues, we compared the DNA repair incision activity of tissue extracts freshly prepared from different aliquots of the same ground tissue at distinct time points after the initial storage of the tissue in −80°C (Table II, columns 2 and 3). Even after long-term storage of the tissues (up to ∼1.5 year), no significant difference in DNA repair incision activity could be detected. This indicates that the assay is applicable in long-term studies, making it possible to analyse frozen samples at a later time point.

Table II

The effect of long-term cryopreservation on the DNA repair incision activity.

Tissue Storage tissue in −80°C (weeks) Incision activity of fresh extracta (±SEM) Storage extract in −80°C (weeks) Incision activity after storageb (±SEM) 
Liver 16.40 ± 4.52 16.40 ± 4.52 
 21.27 ± 0.60 5.5 21.91 ± 4.52 
 18.73 ± 0.26 18.55 ± 4.52 
Brain 80 7.49 ± 0.41 9.34 ± 3.27 
 45 NA 43 10.88 ± 3.27 
Tissue Storage tissue in −80°C (weeks) Incision activity of fresh extracta (±SEM) Storage extract in −80°C (weeks) Incision activity after storageb (±SEM) 
Liver 16.40 ± 4.52 16.40 ± 4.52 
 21.27 ± 0.60 5.5 21.91 ± 4.52 
 18.73 ± 0.26 18.55 ± 4.52 
Brain 80 7.49 ± 0.41 9.34 ± 3.27 
 45 NA 43 10.88 ± 3.27 

NA, not analysed. Freshly prepared tissue extracts were compared with frozen extracts and extracts form different aliquots of the same ground tissue. Data are shown as mean values of two independent incubations within one experiment.

a

Extracts were freshly prepared from different aliquots of the same ground tissue at distinct time points after the initial storage of the tissue in −80°C.

b

Tissue extracts stored from 3 up to 43 weeks at −80°C were compared with freshly prepared extracts.

Next, the stability of the extract during storage was tested. Tissue extracts were analysed before and after long-term (from 3 up to 43 weeks) storage at −80°C (Table II, column 5 versus 3). The effect of long-term freezing of the extracts was also studied between extracts prepared from different aliquots of the same ground tissue (Table II, column 5). We observed that the tissue extracts did not lose their capacity to recognise and incise oxidative DNA lesions after long-term storage at −80°C, which is consistent with the report by Collins et al. (7) showing that BER-related incision activity in lymphocyte extracts was stable at −80°C.

DNA repair incision activity of tissues from Ogg1-/- mice

To validate the assay, we measured the DNA repair incision activity in liver and brain tissues from Ogg1-/- and corresponding wild-type control mice. Results are presented as percentages of the DNA repair incision activity in tissues from wild-type animals (Figure 8). The capacity of Ogg1-/- mice to repair 8-oxodG lesions was significantly lower (PGLMUA = 0.001), i.e. only about one-third of that observed in wild-type animals for both brain and liver. These data are similar to the ∼3-fold lower DNA repair incision activity detected by Guarnieri et al. (48) in livers of Ogg1−/− mice compared with their wild-type controls. Our data are supported further by the lower incision activity seen in Ogg1−/− mouse fibroblasts by Collins et al. (7) using the comet-based in vitro repair assay and in Ogg1−/− mouse testes and brain tissue using an oligonucleotide based in vitro repair assay (34,49), confirming the specificity of our assay.

Fig. 8

DNA repair incision activity in Ogg1−/− and corresponding wild-type mice liver and brain extracts. Mean values (n = 2) of two independent experiments are presented as the percentage of the incision activity in wild-type tissues and were significantly lower in Ogg1−/− mouse tissues (*PGLMUA = 0.001). Bars indicate pooled SEMs.

Fig. 8

DNA repair incision activity in Ogg1−/− and corresponding wild-type mice liver and brain extracts. Mean values (n = 2) of two independent experiments are presented as the percentage of the incision activity in wild-type tissues and were significantly lower in Ogg1−/− mouse tissues (*PGLMUA = 0.001). Bars indicate pooled SEMs.

DNA repair incision activity in ageing and dietary restricted mice

Finally, the optimised assay was tested on tissue samples from an ageing mouse colony and in DR mice. DNA repair incision activity was 60% lower in brains from old (30 months) compared with young adult (3 months) ad libitum fed mice (Figure 9A; Pt-test = 0.018). In contrast, DNA repair incision activity was ∼50% higher in liver from DR compared with ad libitum fed controls (Figure 9B; Pt-test = 0.008). Although the outcomes of studies on the effect of ageing and of DR on DNA repair have been inconsistent (11,50,51), as in the present study, several have reported that DNA repair is plastic with lower activities in older animals (13,15) and increased activity in response to DR (11,13).

Fig. 9

The effect of age (A) and 26% DR (B) on the DNA repair incision activity in mice brain and liver tissue, respectively. Mean repair activities of two independent experiments (n = 4/age group and n = 6–7/diet group) are presented. Bars represent SEMs (*P = 0.018 and **P = 0.008, t-test). Note: Incision activity of the different tissues should not be compared since tissues originated from different mice and different protein concentrations were used (i.e. 1 mg/ml for brain and 5 mg/ml for liver).

Fig. 9

The effect of age (A) and 26% DR (B) on the DNA repair incision activity in mice brain and liver tissue, respectively. Mean repair activities of two independent experiments (n = 4/age group and n = 6–7/diet group) are presented. Bars represent SEMs (*P = 0.018 and **P = 0.008, t-test). Note: Incision activity of the different tissues should not be compared since tissues originated from different mice and different protein concentrations were used (i.e. 1 mg/ml for brain and 5 mg/ml for liver).

Differences in study outcomes might be a result of different in vitro repair assays used. Although other assays have their strengths, the main advantage of our comet-based assay is that we study DNA repair incision activity on nucleoids rather than oligonucleotides or plasmids that may emulate more closely the in vivo situation.

Our adapted and optimised assay for quantification of BER-associated incision activity in rodent tissues opens opportunities for further studies of the effects of ageing on BER activity and of the effects of dietary and other interventions aimed at enhancing cell function during ageing.

Conclusions

We have adapted the comet-based in vitro repair assay for quantification of BER-related DNA incision activity in mouse tissues. We have shown that the assay is sensitive, specific for BER and is able to detect differences in DNA repair incision activity as a result of physiological changes (ageing) and dietary manipulation. The problem of non-specific nuclease/cleavage activity was overcome by a combination of the addition of APC in DMSO and selection of a reliable protein concentration, allowing specific detection of DNA repair incision activity. Although we have not fully validated the assay by multiple-laboratory testing of the same samples and controls, this assay will be useful for a wide range of in vivo studies on BER capacity including effects of environmental exposures (such as toxins, dietary factors and pharmaceutical agents) and of physiological processes including growth, development, degenerative diseases and ageing.

Supplementary data

Supplementary data are available at Mutagenesis Online.

Funding

Biotechnology and Biological Sciences Research Council (BBSRC) and Engineering and Physical Sciences Research Council (EPSRC) (to Centre for Integrated Systems Biology of Ageing and Nutrition); Lifelong Health and Wellbeing cross council initiative by the Medical Research Council, BBSRC, EPSRC and Economic and Social Research Council (to Centre for Brain Ageing and Vitality).

We thank Hoffmann-La Roche (Basel) for supplying Ro 19-8022 and the laboratory of Dr Bernd Epe (Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University, Mainz, Germany) for providing tissues from Ogg1−/− and corresponding wild-type (C57BL/6) mice. Further thanks go to Mr Tom E. L. Bawin and colleagues for providing us with custom made equipment. Finally, we also like to thank Dr Markus Fußer for performing dissections of the Ogg1−/− mice and Adele Kitching, Satomi Miwa, Liz Nicolson and Julie Wallace for assistance with all other mouse dissections.

Conflict of interest statement: None declared.

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Supplementary data