Mutagenesis Advance Access originally published online on April 5, 2006
Mutagenesis 2006 21(3):185-190; doi:10.1093/mutage/gel019
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hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII
Genetic Toxicology, Safety Assessment AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK
The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to detect oxidative DNA damage. In the present study, FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for the ability to modify the sensitivity of the comet assay. Mouse lymphoma L5178Y cells were treated with dimethyl sulphoxide (DMSO) as a standard solvent or reference agents known to induce oxidative damage (gamma irradiation and potassium bromate) or alkylation (methyl methanesulfonate, MMS; ethylnitrosurea, ENU). Using DMSO even up to toxic concentrations, no increase in breaks was seen with FPG, ENDOIII or hOGG1. With gamma irradiation (1–10 Gy), dose-related increases in breaks were seen with all three enzymes. FPG and hOGG1 gave similar increases in breaks after potassium bromate treatment between 0.25 and 2.5 mmol/l, but ENDOIII showed an increase only at the highest concentration, 2.5 mmol/l. Following MMS treatment (5–23 µmol/l), FPG induced a dramatic increase in breaks compared with control levels and ENDOIII also showed a significant but smaller increase; in marked contrast, hOGG1 gave no increase. With ENU (0.5–2.0 mmol/l), increases in breaks were seen with FPG and ENDOIII at 1 and 2 mmol/l but, again, no increase was observed with hOGG1. These data indicate that all three endonucleases recognize oxidative DNA damage and, in addition, FPG and ENDOIII also recognize alkylation damage. Therefore, caution should be taken when using FPG and ENDOIII in the comet assay with an agent that has an unknown mode of action since any additional strand breaks induced by either enzyme cannot necessarily be ascribed to oxidative damage. The use of hOGG1 in the modified comet assay offers a useful alternative to FPG and is apparently more specific for 8-oxoguanine and methyl-fapy-guanine.
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Introduction |
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The comet assay is a rapid method used in genotoxicity testing in vitro and in vivo, and in biomonitoring studies for detecting low levels of DNA damage (1
). However, the standard alkaline method gives limited information on the type of DNA damage being measured as it is not possible to determine whether these are a consequence of direct effects of the damaging agent, or of indirect effects, such as oxidative damage, apurinic/pyrimidinic (AP) sites or DNA repair. The sensitivity and specificity of the assay can be improved by incubating the lysed cells (nucleoids) with lesion-specific endonucleases, which recognize particular damaged bases and create additional breaks. In theory, any lesion for which a specific repair endonuclease exists can be detected this way.
To date, the endonucleases most commonly used in the modified comet assay are the bacterial enzymes, formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), which recognize different types of oxidative damage. FPG is specific for oxidized purines, including 8-oxo-7,8-dihydroguanine (8-oxoGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FaPyGua) and 4,6-diamino-5-formamidopyrimidine (FaPyAde) and other ring-opened purines (2,3). ENDOIII recognizes oxidized pyrimidines, including thymine glycol and uracil glycol (4,5).
The comet assay using FPG was included in the European Standards Committee on Oxidative DNA Damage (ESCODD) trial, set up to optimize methods for measuring background levels of oxidative damage, particularly 8-oxoGua, in man (6). The trial concluded that the FPG-based methods (of which the comet assay was one) seemed less prone to spurious oxidation than other methods including high-performance liquid chromatography with electrochemical detection (HPLC–ECD), gas chromatography with mass spectrometry (GC–MS) or HPLC-tandem mass spectrometry (HPLC-MS/MS). However, previous studies have proven that when FPG is used in the comet assay, in addition to oxidized purines, AP sites and various ring-opened purine adducts are also detected (7). Therefore caution should be taken when using this enzyme to assess compounds with an unknown mode of action. Consequently, due to the apparent lack of specificity of FPG, it was decided to investigate the use of an alternative endonuclease to detect oxidized purines, specifically 8-oxoGua. The nuclease selected was human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1), the primary enzyme for the repair of 8-oxoGua (8,9).
In the present study FPG, ENDOIII and hOGG1 were compared for their ability to detect DNA damage induced in mouse lymphoma cells by agents with different modes of action. The agents were: dimethyl sulphoxide (DMSO), a routinely used solvent; gamma irradiation and potassium bromate (KBrO3), inducers of oxidative DNA damage; methyl methanesulfonate (MMS) and ethylnitrosourea (ENU), reference alkylating agents.
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Materials and methods |
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FPG and ENDOIII were kindly donated by Prof. Andrew Collins (Department of Nutrition, University of Oslo, Norway). hOGG1 was purchased from New England Biolabs (Herts, UK). All other reagents, except where specified, were purchased from Sigma–Aldrich (Dorset, UK).
Cell line and culture conditions
Mouse lymphoma L5178Y cells, clone 3.7.2c, were originally obtained from Dr J. Cole, MRC Cell Mutation Unit, University of Sussex, Brighton, UK. Stock cell cultures were screened to confirm the absence of mycoplasma and karyotyped. Cells were cultured in RPMI 1640 medium (Invitrogen, Paisley, UK) 2 mmol/l L-glutamine, 2 mmol/l sodium pyruvate, 200 IU/ml penicillin, 200µg/ml streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air (complete RPMI) supplemented with 10% heat-inactivated donor horse serum (DHS).
Cell treatment
L5178Y cells (107 in 20 ml) were treated with test agents in solution in DMSO or water in complete RPMI with 5% DHS for 3 h at 37°C (except gamma irradiation in PBS, 4.64 Gy per min). Treatments were: DMSO (1–6% v/v), KBrO3 (0.3–2.5 mmol/l, in water), MMS (5.4–22.7 µmol/l, in DMSO), ENU (0.5–2.0 mmol/l, in water) and gamma irradiation (1–10 Gy). Following treatment, a 500 µl cell sample was taken from each treatment flask and prepared for the comet assay. The remaining cells were used to calculate relative suspension growth (RSG).
Modified comet assay
The cell samples (500 µl) were centrifuged (700 g, 3 min) and the supernatant was discarded. The cell pellets were washed in PBS at 4°C and centrifuged again. The supernatant was removed leaving a residual amount of PBS and 300 µl 0.5% low melting-point agarose (LMA) was added. The cells were gently resuspended and 40 µl was added directly to a slide that had been pre-coated with 0.5% agarose (two gels per slide). The gels were covered with a cover slip and allowed to set on a cold plate. The cover slips were then removed and the slides immersed in lysis solution [2.5 mol/l NaCl, 100 mmol/l Na2EDTA, 10 mmol/l Tris buffer (pH 10), 10% DMSO, 1% Triton X-100] and stored at 4°C overnight. Following lysis, the slides were immersed in two changes of buffer F [40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA and 0.2 mg/ml (pH 8.0)] for 5 min, each time at room temperature. FPG, ENDOIII or hOGG1, was added to the gel in 50 µl of buffer F at dilutions previously shown by titration to give the highest percentage of breaks following gamma irradiation (1:3000, 1:300, 1:1000, respectively). For hOOG1 this was equivalent to 0.08 U per gel, but for FPG and ENDOIII this data was not known. Gels were covered with a cover slip and incubated in a humidified chamber for 10 min (hOGG1) or 45 min (FPG and ENDOIII) at 37°C. The cover slips were removed and the slides were placed on an electrophoresis platform, covered with electrophoresis buffer [1 mmol/l Na2EDTA, 0.3 mol/l NaOH (pH 13)] and DNA was allowed to unwind for 20 min before electrophoresis at 0.7 V/cm, 300 mA for a further 20 min. DNA unwinding and electrophoresis was performed in a cold unit at 4–8°C. The slides were removed and immersed in three changes of neutralizing buffer [0.4 mol/l Tris–HCl (pH 7.5)] for 5 min at room temperature, then stained with 50 µl propidium iodide (10 mg/ml) and scored 20 min later. Slides were scored using a Comet IV capture system (Perceptive Instruments) and 50 nuclei were scored per gel. The tail intensity (TI), defined as the percentage of DNA migrated from the head of the comet into the tail, was measured for each nucleus scored. Tail moment (TM) was not used for data analysis, as it has no recognized units. In addition, the equation used to calculate TM uses tail length, which tends to increase rapidly with concentration at low levels of damage (10).
RSG
Following treatment, the remaining cells were centrifuged and resuspended in 50 ml complete RPMI with 10% DHS and incubated for 1 day. They were then counted and adjusted to 1.5 x 105 cells/ml in 30 ml complete RPMI with 10% DHS and incubated for a further day. RSG was calculated as the increase in cell number expressed as a percentage of the concurrent solvent or water control.
Data analysis and statistics
All experiments were independently performed at least three times. The background TI values in the control groups were different for each enzyme. In order to determine specific changes with each of the enzymes following compound treatment, the mean background TI values for each enzyme were subtracted from the treatment TI values. The adjusted mean TI values were then used for statistical analysis to confirm a significant increase in FPG, ENDOIII or hOGG1 recognized break sites following compound treatment. Each treatment was analysed separately. For each treatment a two-way ANOVA was performed, with enzyme and treatment concentrations fitted as categorical variables. Since increases in DNA strand breaks were of interest a one-sided test was applied with P-values of less than 5% considered significant. No adjustment for multiple testing was performed. The overall TI means and standard deviations using non-adjusted values from multiple testing are presented graphically and in tables. For gamma irradiation both the means and adjusted means are presented.
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Results |
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Background control TI values
The background TI levels in the water controls varied with each endonuclease. There was no significant difference in TI in the presence or absence of hOGG1. However, mean increases of 3.1% and 7.1% in TI were seen after treatment with FPG and ENDOIII, respectively (Table I). In addition, collation of the background levels of ENDOIII for all experiments shows a higher level of variability in TI (SD = 4.01) compared to FPG and hOGG1.
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DMSO
No increase in TI was seen in the absence or presence of any of the endonucleases after treatment of L5178Y cells with DMSO (Table II and Figure 1a). Furthermore, since treatment with 6% DMSO reduced survival to 55%, none of the enzymes appeared to detect non-specific DNA damage that might have been associated with cytotoxicity (Table II).
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Gamma irradiation
Treatment with gamma irradiation alone gave a dose-dependent increase in comet TI from 3% (1 Gy) to 15% (10 Gy). In addition there was evidence of cytotoxicity at 5 and 10 Gy with RSG giving 22 and 16% survival, respectively (Table II). There were significant increases in strand breaks following treatment with each of the endonucleases at 5 and 10 Gy (P
0.05) compared with background levels (Table II and Figure 1e and f). Specifically, TI increased from 4 to 28% with FPG, from 11 to 24% with ENDOIII and from 1 to 20% for hOGG1 at 0 Gy and 10 Gy, respectively.
KBrO3
KBrO3 treatment alone gave no increase in comet TI, although there was evidence of cytotoxicity at the highest concentration (2.5 mmol/l) giving 39% RSG as with DMSO (Table II). FPG and hOGG1 induced significant increases (P < 0.001) in strand breaks at all concentrations tested, with 64 and 38% TI at 0.3 mmol/l and 91 and 90% TI at 2.5 mmol/l, respectively (Table II and Figure 1b). There was also a smaller, significant increase in strand breaks following incubation with ENDOIII at the highest concentration (2.5 mmol/l) with a TI of 42% (P < 0.001) (Table II and Figure 1b).
MMS
MMS treatment alone induced no cytotoxicity and no increase in comet TI at the concentrations tested (5.4–22.7 µmol/l) (Table II and Figure 1c); it should be noted that these concentrations are also significantly lower than those known to induce mutation at the tk locus in these cells in this laboratory (M. Fellows, personal communication). There was a dramatic increase in strand breaks compared with background levels following incubation with FPG at all concentrations tested, with 5.4 µmol/l MMS giving 63% TI and 22.7 µmol/l giving 94% TI (P < 0.001) (Table II and Figure 1c). Incubation with ENDOIII also induced a significant increase in strand breaks, but to a lesser extent with 11.8 µmol/l and 22.7 µmol/l MMS giving 29 and 48% TI, respectively (P < 0.001) (Table II and Figure 1c). In marked contrast, hOGG1 resulted in no significant increase in strand breaks at any concentration tested (Table II and Figure 1c).
ENU
Following treatment with ENU in the absence of any of the endonucleases there were increases in TI of 7 and 15% at 1 and 2 mmol/l, respectively (Table II and Figure 1d). In addition there was evidence of cytotoxicity at these concentrations giving RSG of 46 and 26%, respectively (Table II). Following incubation with FPG and ENDOIII there was a similar, significant increase in strand breaks at 1 mmol/l (37 and 40% TI, respectively) and 2 mmol/l (63 and 62% TI, respectively) (P < 0.001) (Table II and Figure 1d). FPG also gave a significant increase in strand breaks at the lowest concentration, 0.5 mmol/l giving 14% TI (P < 0.05). Again in marked contrast, incubation with hOGG1 gave no significant increase in strand breaks following treatment with ENU at any concentration tested (Table II and Figure 1d).
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Discussion |
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Modification of the alkaline comet assay to incorporate lesion-specific endonucleases increases its sensitivity and specificity through the recognition of damaged bases and introduction of additional breaks. However, the bacterial FPG most commonly used for the detection of oxidized purines is not very specific and also detects AP sites and ring-opened N7 guanine adducts (7
). In the present study, two other endonucleases ENDOIII and hOGG1, were compared with FPG for their ability to increase DNA strand breaks in the comet assay after treatment of mouse lymphoma cells with compounds that cause oxidative or alkylation damage.
Gamma irradiation and KBrO3 were chosen as specific inducers of oxidative DNA damage. Following gamma irradiation, there was a concentration dependent increase in comet TI and a significant increase in break sites with all three enzymes. It is known to damage DNA through (i) radiolysis of water leading to the formation of reactive oxygen species (ROS), such as the hydroxyl radical (•OH) and (ii) direct interaction with DNA giving double strand breaks (11). The •OH radical causes base modifications by attack at sites of high electron density. Interactions with guanine preferentially occur at the C8 and C4 positions, producing 8-oxoGua or FaPyGua, respectively. With pyrimidines, the major product is thymine glycol (11). David and Williams (12) have reviewed the differences in enzyme substrate specificity. FPG recognizes a number of oxidized purine bases and ENDOIII is able to recognize several oxidized pyrimidines, whereas hOGG1 is specific for 8-oxoGua and methyl FapyGua (MeFapyGua). hOGG1 can repair these oxidized bases only when paired with cytosine and, therefore, is more specific than FPG, which recognizes 8-oxoGua paired with cytosine, guanine or thymidine. OGG1 is structurally related to ENDOIII but functionally related to FPG; thus it also has AP-lyase activity, albeit weaker compared to FPG, in addition to its glycosylase activity (12). The data show different magnitudes of response to gamma irradiation with FPG > ENDOIII > hOGG1. This is consistent with the affinity of adduct recognition by the three endonucleases.
The mechanism of DNA oxidation by KBrO3 is not clear although it is reported to cause an increase in 8-oxoGua adducts (13,14). Intracellular reduced glutathione (GSH) is thought to be involved in the production of the reactive species (possibly the hydroxyl radical) because, after treatment of L1210 mouse leukaemia cells and LLC-PK1 porcine kidney cells with KBrO3, an increase in strand breaks with FPG was seen in the presence of GSH (15). In the present study, similar, dramatic increases in both FPG and hOGG1 recognized break sites were induced in cells following treatment with KBrO3, with almost the entire DNA in the tail at the highest concentration, 2.5 mmol/l. These data are consistent with KBrO3 forming 8-oxoGua, recognized by both FPG and hOGG1. The response with ENDOIII was smaller, and significant strand breaks were formed only at the highest concentration. It is possible that increasing concentrations produce more general damage, perhaps including thymine glycol, which is recognized by ENDOIII. Thus, it appears from the data that KBrO3 induces damage predominantly to purines at non-cytotoxic concentrations and would be a useful positive control for the induction of hOGG1-recognizible damage.
MMS and ENU were chosen as well established compounds known to induce alkylation damage in DNA. It has been shown by Speit and co-workers (7) that FPG is not specific for oxidative DNA lesions in the comet assay, but also recognizes alkylation damage, notably alkylation of N7 guanine. In human whole blood, isolated lymphocytes and V79 cells treated with alkylating agents, FPG strongly enhanced MMS- and ethyl methanesulfonate (EMS)-induced DNA damage but had no significant effect on ENU-induced damage.
The results presented here confirm that, after treatment of L5178Y cells with MMS, large increases in strand breaks are seen with FPG and, to a lesser extent, with ENDOIII. MMS is known to methylate predominantly nitrogens in purine rings, which can lead to the formation of AP sites (16). Methylation of N7 guanine can also destabilize the imidazole ring of the purine making it prone to alkali catalysed opening and generation of FaPyGua, recognized by FPG (http://www/ich.ucl.ac.uk/cmgs/mechmut.htm). In the present study, concentrations were chosen not to induce an increase in TI following treatment of MMS in the absence of repair endonucleases. The results shown in this study are very similar to the results observed in lymphocytes treated with MMS and processed for the comet assay using AlkA endonuclease for the detection of alkylation damage (17). A concentration related increase in AlkA induced breaks was observed following MMS treatment with maximal DNA breaks at 0.1 mmol/l and saturation at 0.2 mmol/l. It has also shown that, following treatment of MMS in the presence of antioxidants, there was no suppression of damage measured by the comet assay (18), indicating the damage is not due to ROS. The results in the present study show that there is no increase in strand breaks with hOGG1 following treatment with MMS, indicating that hOGG1 does not recognize alkylation damage and therefore, is more specific than FPG and ENDOIII. Furthermore, hOGG1 does not recognize AP sites produced from spontaneous depurination of methylated guanines.
ENU is an alkylating agent that predominately causes the formation of phosphotriesters (50–60%) and alkylation of N7- (13–20%) and O6- (9–17%) guanine in mammalian cultured cells (19). ENU is the only alkylating agent to significantly bind to O-alkyl pyrimidines (19). It was shown by Speit and co-workers (7) that FPG does not recognize ENU-induced DNA damage in the comet assay using peripheral blood lymphocytes treated for 2 h at 37°C. In contrast, the data in the present study clearly show that there is a similar increase in strand breaks with both FPG and ENDOIII following ENU treatment at similar concentrations in L5178Y cells treated for 3 h at 37°C. This difference in response could be due to differences in cell cycle kinetics or repair capacities of the two cell types. As with MMS, there was no increase in hOGG1 recognized break sites following ENU treatment, further supporting the suggestion that hOGG1 does not recognize alkylation damage in these conditions.
In conclusion, the addition of lesion-specific endonucleases increases the sensitivity of the comet assay as expected. The overall comparison shows that both FPG and ENDOIII are capable of recognizing and cleaving alkylation damage in addition to oxidative DNA damage. Therefore, additional strand breaks induced by either FPG or ENDOIII after treatment with an agent that has an unknown mode of action, cannot necessarily be ascribed to oxidative damage. In contrast, hOGG1 is more specific for oxidative DNA damage and may be a more useful alternative for elucidating the mode of action of a novel agent in the comet assay and other techniques, such as alkaline elution.
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Acknowledgments |
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The authors would like to thank Andrew Collins for his kind donation of FPG and ENDOIII, and Karen Oldman and Debbie Godwin for the statistical analyses.
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Notes |
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*To whom correspondence should be addressed. Tel: +44 1625 231279; Fax: +44 1625 231281; E-mail: elizabeth.martin{at}astrazeneca.com
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Received on January 26, 2006; revised on March 14, 2006; accepted on March 14, 2006.
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