Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide metabolite glycidamide

Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide... Abstract The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult. Introduction The traditional comet assay gives information on strand breaks arising in cellular DNA after a genotoxic treatment. Higher assay sensitivity and some information on the nature of the DNA lesions are achieved by the recent inclusion of DNA repair enzymes to treat nucleoids after cell lysis. We here report that varying the lysis conditions (duration and pH) gives additional information on DNA adducts induced by certain mutagens. Acrylamide (AA) is a genotoxic industrial compound, but it is also formed in starch-rich foods upon heating hence representing a genotoxic load to the general population (1). The estimated dietary daily intake of AA is 0.4–1 µg/kg bodyweight (2–4). In humans, ~12% of the urinary metabolites of AA arise via the formation of glycidamide (GA) (5) which is the ultimate DNA-reactive metabolite. Using chromatographic analysis, GA has been shown to form DNA adducts, i.e. N7-(2-carbamoyl-2-hydroxyethyl)-deoxyguanosine (N7-GA-dG), and to a lesser extent N3-(2-carbamoyl-2-hydroxyethyl)-deoxyadenosine (N3-GA-dA), both in vivo and in vitro (6–8). In addition, the N1-adenine adducts of GA (analysed after conversion to N⁶-GA-deoxyadenosine-5′-monophosphate) were detected in DNA reacted with GA and in DNA from cells exposed to GA but not in DNA from mice treated with AA (9). In reactions with pure nucleosides, GA forms adducts not only with the purines but also with cytidine and thymidine (10,11). GA induces DNA damage as single-strand breaks (SSB) and alkali-labile sites (ALS), detected in the comet assay in various human and rodent cell types after in vitro exposure (12–15), and in vivo (16,17). The GA-induced SSB and ALS were detected at moderate to high doses and concentrations (in the millimolar range). Recently, chronic exposure of mice to very low AA concentrations (1 µg/ml in drinking water) gave increased levels of DNA damage in spermatozoa (18). In addition to the GA-modified bases which are likely to lead to base loss and subsequent formation of apurinic/apyrimidinic sites (AP sites) (6), modification of DNA could arise via alkylation of the DNA phosphate backbone (19). High sensitivity of detection of AA-related genotoxicity in cells and tissues is essential for evaluation of a potential health risk. In general, the comet assay allows measurement of DNA damage in single cells at biologically relevant levels of damage. The sensitivity of this assay depends on whether the initial DNA modification leads to the formation of a strand break. Single-strand DNA breaks may occur either directly or during endogenous DNA repair, or they may represent chemically unstable lesions such as AP sites leading to DNA strand breakage during the alkaline treatment conditions (pH 13) normally used in the assay. A major advance in the comet assay was the introduction of lesion-specific enzymes with the ability to cleave agarose-embedded lysed and histone-depleted DNA, resulting in a substantially increased sensitivity and specificity (20,21). DNA glycosylases in base excision repair (BER) pathways thus recognise various oxidised or alkylated base modifications (20,22–24). In particular, formamidopyrimidine-DNA glycosylase/AP-lyase (Fpg) was found to detect oxidised guanines (8-oxo-7,8-dihydroguanine, 8-oxoG) more reproducibly than various analytical methods (25). For a variety of other DNA adducts, methods based on liquid chromatographic (LC) separation and mass spectrometric (MS) detection are considered more sensitive and specific for the detection of modified DNA bases [reviewed in (26)]. This has in the past also been the case for GA; in the in vivo study by Maniere et al. (17), significant formation of DNA adducts was detected by LC-MS analysis for the lowest GA dose used, in contrast to insignificant DNA damage detected by the traditional comet assay at the same dose. Thielen and co-workers and also our group, using the comet assay, reported that Fpg markedly increases the detection of GA-induced DNA damage (13,27). In general, this bifunctional enzyme is known to recognise and cleave not only oxidised purines such as 8-oxoG, but also substituted and unsubstituted imidazole ring-opened guanines (Fapy) (28–32). We previously reported that the human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is more specific towards oxidised DNA lesions than Fpg in the comet assay (33), does not detect GA-induced DNA lesions (13). We therefore suggested that the Fpg-sensitive GA-induced lesions result from N7-GA-dG adducts being ring-opened during the lysis step (pH 10), ultimately resembling 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-FapyG). Such adducts and oxidised purines may have different genotoxic outcomes, and knowledge of the exact nature of the DNA lesions induced by GA—and their repair—is highly important for risk assessment of GA. In this paper, we have investigated the ability of the comet assay to detect these adducts. We explored various parameters of the lysis step in the comet assay (incubation time, pH and temperature) and their importance for Fpg-sensitive lesion detection. Parallel experiments were carried out with GA and also methyl methane sulfonate (MMS; giving mainly methylation of N-purines), X-rays (inducing SSB, ALS, oxidised pyrimidines and purines and double strand breaks) and a phototoxic compound primarily inducing 8-oxoG. Furthermore, we studied the nature of the GA-induced lesions by subjecting GA-treated naked DNA to Fpg in a cell-free system and analysing the ‘fall-off’ products by means of LC-MS. Overall, the results strongly suggest that the high efficiency of detection of GA-induced lesions with Fpg is due to N7-GA-dG adducts which are ring-opened during the alkaline lysis step (pH 10). The data demonstrate a novel use of the comet assay not only to detect DNA damage but also to partially analyse their identity. Material and methods Induction of GA adducts in calf thymus DNA treated with Fpg Five milligrams of double-stranded calf thymus DNA (Sigma, St. Louis, MO) were dissolved in 0.1 M phosphate buffer, pH 7.6 overnight (ON, 16–18 h) at 4°C under circulation and light protection as described in the manufacturer’s protocol. The final concentration was determined spectrophotometrically (NanoDrop® ND-1000 spectrophotometer, Saveen & Werner A/S) and adjusted to 2 mg/ml. GA dissolved in sterile H2O at 0.5 M was added to 1 ml DNA solutions to give a final GA concentration of 21 mM, followed by incubation of samples in the dark at 37°C for 18 h (pH 7.6). Subsequently, enzyme buffer [1 mM EDTA, 0.1 M KCl, 20 mM Tris–HCl, pH 7.6 and 2 mg/ml BSA (final concentrations)] with or without 10 or 100 µg/ml Fpg was added to DNA aliquots with or without GA (stoichiometric ratio for GA versus Fpg approximately the same as in the comet assay). All samples were further incubated at 37°C for 1 h. NaCl was then added to a final concentration of 1 M, and DNA was precipitated with ethanol at −20°C for 3 days. Supernatants were collected after two centrifugations at 1150×g for 10 min; samples were stored at −20°C until chromatographic analysis. DNA samples incubated without GA and Fpg were included in the analysis for comparison. Chromatographic methods The LC separations were performed on Agilent 1100 and 1200 systems, both consisting of a binary pump, a vacuum degasser, an autosampler and a thermostated column oven. The 1100 system was connected to a triple quadrupole mass spectrometer (MS) and the 1200 system was hyphenated with the Q-ToF MS. The compounds in the DNA supernatants were separated on a reversed-phase C18 analytical column (5 µm, 4 × 125 mm, Hypersil BDS-C18). The column was eluted with a gradient consisting of 0.01 M aqueous ammonium acetate and acetonitrile starting from 1% acetonitrile and ending after 20 min at 30% acetonitrile at the flow rate 0.5 ml/min and with the injection volume 20 µl. A Micro Triple-Quadrupole MS (Micromass, Manchester, UK) equipped with an electrospray interface was used in the multiple reaction monitoring (MRM) mode. The source block temperature was 120°C and the desolvation temperature was 325°C. Nitrogen was used as the desolvation gas (616 l/h) and the cone gas (33 l/h), while argon was applied as the collision gas, at a collision cell pressure of 5.8 × 10−3 mbar. Positive ions were acquired with a dwell time of 0.25 s and inter-channel delay of 0.05 s. A microTOF-Q (Bruker Daltonics, Bremen, Germany) was used for screening of adducts in the DNA supernatants and for recording the exact mass of adducts. Ionisation was performed with electrospray ionisation in the positive mode and at a capillary voltage of 4500 V. Nitrogen was used as the nebulizer gas (pressure 1.4 bar) and drying gas (8.0 l/min, temperature 200°C). Masses of m/z 50–600 were recorded. The spectrometer was calibrated daily prior to the analyses using a sodium formate solution. A post-run internal mass scale calibration was performed at the end of each chromatographic run calibration. The instrument was operated at a resolution of about 10 000 (FWHM). Chemical treatment of human lymphocytes Human peripheral blood lymphocytes (hPBL) were isolated from one (the same in all experiments) healthy male non-smoking volunteer (in compliance with Norwegian legislation and with ethical permission) using LymphoprepTM tubes as described in the manufacturer’s protocol (Axis Shield, Oslo, Norway). Cells were finally suspended in ice-cold cell medium (BioWhittaker®RPMI 1640 medium with 25 mM HEPES and L-glutamine, Lonza Verviers, Belgium). Cell membrane integrity was measured with trypan blue exclusion (>95%) indicating high cell viability. Stock solutions of GA (Toronto Research Chemicals Inc., Canada, CAS no. 5694-00-8; the dry compound was stored under argon at −20°C) were made fresh for each experiment; GA was dissolved in phosphate-buffered saline (PBS). MMS (Sigma-Aldrich, Oslo, Norway) was diluted in dimethyl sulfoxide (DMSO). GA and MMS dilutions or their solvents were added to hPBLs (1–2 × 106 cells/ml). These cell samples were then incubated in Eppendorf tubes for 2 h at 37°C in the dark. The phototoxic compound Ro 12-9786 (a gift from Dr Elmar Gocke, Roche Ltd, Basel, Switzerland) was dissolved in DMSO and stored at −20°C; this stock solution was added at a final concentration of Ro 12-9786 of 3 µM (0.5% DMSO final) to cell suspensions on ice. After 1 min, the latter samples were exposed to visible light (18 000 lux) at 10 cm distance from a fibre-optic light source, for different time periods. The cell treatments were terminated by adding ice-cold cell medium plus 10% foetal bovine serum (PAA laboratories GMbH, Austria) followed by centrifugation (200×g, 4°C, 5 min). Cell pellets were gently resuspended in cell medium for comet analysis. X-ray exposure (260 kV, filtered through 0.5 mm Cu, dose rate 10 Gy/min (13) took place while cell samples were on ice, at doses 0, 6 and 10 Gy. Comet analysis with Fpg and various lysis conditions DNA SSB plus ALS and Fpg-sensitive sites were measured with a modified alkaline comet assay, as previously described (13). In brief, cells were mixed 1:10 with 0.75% low melting point agarose and added as 12–48 gels per GelBond film (Cambrex, Rockland, ME). Samples were placed for specified time periods in lysis solution at 4°C [2.5 M sodium chloride, 0.1 M Na2EDTA, 10 mM Trizma base, 1% Lauroylsarcosine sodium salt (≥94%; Sigma-Aldrich, Oslo, Norway), with 1% Triton X-100 (Sigma-Aldrich, Oslo, Norway) and 10% DMSO freshly added]. The normal pH of this standard lysing solution is pH 10; in some experiments it was adjusted to pH 7.5. After lysis films were transferred to an enzyme buffer [40 mM HEPES, 0.5 mM Na2EDTA, 0.1 M KCl and 0.2 mg/ml BSA, pH 7.6] for 1 h at 4°C, followed by incubation at 37°C for 1 h in fresh enzyme buffer with or without 1 µg/ml Fpg (prepared from an E. coli extract (13,24,34). After unwinding (40 min, 4°C) in electrophoresis solution (pH ≥13.2), electrophoresis was performed at 0.8 V/cm, 8–10°C for 20 min as described (35). Films were further neutralised and fixed in ethanol. SybrGold (Invitrogen, Oslo, Norway) stained gels were microscopically examined and comet tails were scored using the Comet assay IV software (Perceptive Instruments Ltd., Suffolk, UK). DNA damage was defined as %Tail DNA, i.e. the fluorescence intensity in the tail relative to the total intensity of the comet, as described (35). Samples were scored in triplicate each of 30 cells, and medians were calculated. Each experiment was repeated up to three times. The net increase in DNA damage measured with Fpg is denoted as Fpg-sensitive lesions. The activity of the Fpg prep used in this study has been tested using an Oligo Repair Chip (LXRepair SAS, Grenoble) showing high 8-oxoG and AP-site activity in vitro (data not shown, personal communication from Dr Sylvie Sauvaigo). Statistics Concentration–effect relationships of GA and each lysis condition were explored using linear regression analyses with goodness to fit; the %Tail DNA measurements were square root transformed to obtain normal distribution of the dependent variables and residuals. The slopes for the regression lines for GA were compared using the one hour lysis at pH 10 (1 h pH10) group as the reference value. Statistically significant differences between slopes are indicated in the result section and in figures and their legends. JMP Pro (JMPPRO WINDOWS 13, SAS) was used for the statistical analyses. Results Reaction of naked DNA with GA Chromatographic analyses with LC-MS were carried out to confirm the formation of GA-purine adducts, and to investigate whether Fpg treatment has any effect on the pattern of bases released from GA-treated DNA at neutral pH. The Fpg treatment step (pH 7.6) in the comet assay is in general carried out after an alkaline lysis step (pH 10) included to remove cellular proteins and histones from DNA. Assuming that the Fpg enzyme should be similarly active on naked double-stranded DNA in solution, reactions were set up in which similar relative amounts of GA, Fpg enzyme and calf thymus DNA were used. GA (21 mM) was mixed with calf thymus DNA and the reaction mixtures were thereafter incubated at 37°C with Fpg (pH 7.6) to allow enzymatic release of DNA adducts. Possible reaction products (DNA bases) released from ethanol precipitated DNA were subjected to LC-MS chromatographic analysis. The identification of the GA adducts was based on exact mass and the isotopic pattern determinations provided by the Q-ToF mass analyser and on mass spectra generated by MRM transitions in the triple quadrupole MS. The N7-GA-Gua and N3-GA-Ade nucleobase derivatives were detected in the supernatants from samples in which DNA was treated with GA. In the case of N7-GA-Gua, the chromatographic retention matched the retention time of pure N7-GA-Gua aglycon (prepared by reacting GA with dG and hydrolysis of the sugar unit). The MRM transition 239–152 and 223–136 for N7-GA-Gua and N3-GA-Ade, respectively (Figure 1), corresponded to the loss of the GA unit from the nucleobase (i.e. the aglycon). The masses of both adducts measured by the Q-ToF analyser differed from the calculated masses by less than 5 ppm corresponding to a mass error of only 0.7 mDa. LC-Q-ToF MS was applied for screening for the occurrence of additional adduct-identities, but no other adducts than N7-GA-Gua and N3-GA-Ade were observed. The MRM transitions generated a peak area three times higher for N7-GA-Gua than for N3-GA-Ade, in all samples analysed. Due to the lack of N3-GA-Ade standard, it was not possible to conclude from these experiments that N7-GA-Gua was the dominating adduct. However, the ratio of formed adducts was the same in all samples. Furthermore, the peak intensities were approximately the same in all samples. In conclusion, the data suggest that the addition of the Fpg enzyme did not affect the total adduct yield in the supernatant, and new products were not identified. No adducts could be detected in the supernatant from untreated DNA (data not shown). Fig. 1. View largeDownload slide Multiple reaction monitoring (MRM) chromatograms with retention times (minutes) for N7-GA-Gua and N3-GA-Ade from the analysis of the supernatant obtained by precipitation of calf thymus DNA incubated with (A) GA (21 mM) and the Fpg enzyme (10 µg/ml); and (B) GA in the absence of Fpg. Fig. 1. View largeDownload slide Multiple reaction monitoring (MRM) chromatograms with retention times (minutes) for N7-GA-Gua and N3-GA-Ade from the analysis of the supernatant obtained by precipitation of calf thymus DNA incubated with (A) GA (21 mM) and the Fpg enzyme (10 µg/ml); and (B) GA in the absence of Fpg. DNA damage detection in the comet assay and the effect of lysis time, temperature and pH A higher sensitivity for detection of DNA lesions induced by GA in cells is obtained when the comet assay includes Fpg treatment of nucleoids after cell lysis (often 1 h at pH 10). However, this increase was markedly higher after over-night lysis at pH 10 prior to the Fpg treatment (Figures 2 and 3A). Using this protocol, effects of approximately 20–50 times lower concentrations of GA can be measured, in whole blood or primary cell cultures from mice and human lymphocytes (13,27). In the present study, the level of GA-induced DNA lesions in human lymphocytes detected without Fpg was low (0–7% Tail DNA) and is subtracted so that Figures 2 and 3A show net levels of Fpg-sensitive lesions. The standard lysis condition used in our comet assay protocol is over-night (16–18 h) at 4°C in lysis buffer (pH 10). Figures 2 and 3A show that the level of GA-induced Fpg-sensitive lesions was significantly different (Figure 3A), approximately twice as high, when lysis lasted over-night rather than for 1 h. One hour lysis time at pH 10 is in accordance with the traditional protocol by Tice et al. (36). When we increased the lysis treatment to 40 h or 6 days, no further increase in the level of GA-induced DNA-damage was observed beyond that obtained with over-night lysis (Figure 2). The temperature did not seem to be important since lysis at room temperature for 6 days (144 h) gave similar levels of Fpg-sensitive sites as lysis at 4°C (Figure 2). The data in Figure 3A show that, in addition to the duration of the lysis step, the pH of the lysis solution was highly important for the detection of the GA-induced Fpg-sensitive sites. At pH 7.5, no Fpg-sensitive GA-induced lesions were detected. Fig. 2. View largeDownload slide Induction of Fpg-sensitive sites in human peripheral blood lymphocytes by glycidamide (GA), as a function of duration of alkaline lysis (hours, pH 10) in the comet assay. The shortest lysis time is 1 h. Open symbols: lysis performed at 4°C; median %Tail DNA values of all comets from three technical replicates for each experiment (n = 1 or 2). Closed symbols: Lysis at room temperature for 6 days (144 h) (n = 1). GA concentrations during incubation of lymphocytes are indicated in µM. Fig. 2. View largeDownload slide Induction of Fpg-sensitive sites in human peripheral blood lymphocytes by glycidamide (GA), as a function of duration of alkaline lysis (hours, pH 10) in the comet assay. The shortest lysis time is 1 h. Open symbols: lysis performed at 4°C; median %Tail DNA values of all comets from three technical replicates for each experiment (n = 1 or 2). Closed symbols: Lysis at room temperature for 6 days (144 h) (n = 1). GA concentrations during incubation of lymphocytes are indicated in µM. Fig. 3. View largeDownload slide Influence of lysis time (1 h or overnight (ON)) and pH in the comet assay on the detection of Fpg-sensitive sites and SSB/ALS, in human peripheral blood lymphocytes, exposed to either (A) glycidamide (GA, µM); (B) methyl methane sulfonate (MMS, µM); (C) 3 µM Ro 12–9786 + light exposure (1.5 or 3 min) or (D) X-rays (Gy). Data points represent various lysis conditions (pH and duration) as indicated in the figures. Net Fpg-sensitive sites are shown in (A–C), whereas in (D) the X-ray-induced lesions (SSB/ALS) were assayed without Fpg. (A) shows dose-response curves (versus GA concentration; ±SEM n = 3); stars denote lysis dependent regression lines significantly different from lysis at 1 h pH 10 (P < 0.001). (B–D) show mean %Tail DNA, calculated from medians of technical replicates, from at least two independent experiments. The concentration-response relationship in (B) (for MMS) was not tested by statistical methods, since n = 2. For (C) and (D), there were no significant effects of lysis conditions. Fig. 3. View largeDownload slide Influence of lysis time (1 h or overnight (ON)) and pH in the comet assay on the detection of Fpg-sensitive sites and SSB/ALS, in human peripheral blood lymphocytes, exposed to either (A) glycidamide (GA, µM); (B) methyl methane sulfonate (MMS, µM); (C) 3 µM Ro 12–9786 + light exposure (1.5 or 3 min) or (D) X-rays (Gy). Data points represent various lysis conditions (pH and duration) as indicated in the figures. Net Fpg-sensitive sites are shown in (A–C), whereas in (D) the X-ray-induced lesions (SSB/ALS) were assayed without Fpg. (A) shows dose-response curves (versus GA concentration; ±SEM n = 3); stars denote lysis dependent regression lines significantly different from lysis at 1 h pH 10 (P < 0.001). (B–D) show mean %Tail DNA, calculated from medians of technical replicates, from at least two independent experiments. The concentration-response relationship in (B) (for MMS) was not tested by statistical methods, since n = 2. For (C) and (D), there were no significant effects of lysis conditions. In order to examine how different types of lesions respond to the lysis conditions (37), cells were exposed to other genotoxic treatments. The detection of Fpg-sensitive sites induced by the methylating agent MMS was influenced by duration of lysis and pH in a similar way as for GA (Figure 3). In the case of treatment with Ro 12-9786 plus light which in analogy with similar compounds leads to the formation of 8-oxoG (25,38,39), alkaline lysis conditions had no significant effect on the total yield of Fpg-sensitive sites (Figure 3C). For X-rays, inducing SSB and ALS, the lesions were as expected detectable in the comet assay without Fpg; the different lysis conditions gave similar levels of damage (Figure 3D). X-rays are known to also induce frequent oxidative lesions at the doses applied (6 and 10 Gy), but sequestering of Fpg due to its high affinity to SSBs, and damage clustering of such lesions with DNA strand breaks (40), prevent their efficient cleavage by Fpg until the strand breaks have been largely repaired, as we have discussed previously (41). Accordingly, we observed very low levels of Fpg-sensitive lesions in the comet assay after X-rays (data not shown). In some of the experiments illustrated in Figure 3, the over-night lysis was carried out not at pH 10 but at pH 7.5, except for 1 h initial treatment at pH 10. Figure 3A–D show that no major increase in lesion detection was associated with extending the lysis time beyond 1 h at pH 7.5. Discussion When calf thymus DNA was incubated with GA at neutral conditions, N7-GA-Gua and N3-GA-Ade adducts were detected in the supernatant by LC-MS analysis implying that the modified bases had been released from DNA. These adducts are the same major GA-induced DNA-adducts as those detected in previous in vivo rodent studies (6–8,17,42). Incubation with Fpg prior to DNA precipitation did not change the occurrence of the adducts, nor were new types of adducts/modified bases detected in the supernatant. This strongly suggests that the N7-GA-dG and N3-GA-dA adducts per se were not major substrates for Fpg. The N7-GA-Gua and N3-GA-Ade adducts detected are likely to have been released from the sugar phosphates via spontaneous depurination. N3-alkylated adenosines are not known to be ring-opened or recognised by Fpg; the predominant reaction for labile N7-alkylated guanosines at physiological pH is depurination and not imidazole ring opening (reviewed in (43)). Ring opening of N7-alkylated guanosines is facilitated by strongly electron-withdrawing substituents (44); in some cases ring-opening rates may be comparable to depurination rates under physiologically relevant conditions (43). GA does possess electron-withdrawing groups; however, the N7-GA-adducts formed in the non-cellular reaction (Figure 1) seemed not to be ring-opened for recognition and cleavage by Fpg to detectable levels at these (neutral pH) conditions. Purified or crude extracts of the bacterial DNA glycosylase Fpg have in recent years been much used for detection of oxidised purines, i.e. 8-oxoG and unsubstituted FapyG (29,31), in the comet assay (23–25,45) and also with alkaline elution (34,46). This bifunctional enzyme has strong substrate affinity also to me-FapyG (28,30,47), and was previously shown to detect DNA damage induced by alkylating agents in the comet assay (33,48,49). Fpg is known to have high substrate affinity also to imidazole ring-opened guanosines containing larger substituents than methyl at the N7-position of the guanine, such as ethyl (50), aminofluorene (51), hydroxyethyl-thioethyl (52) and aminoethyl (53). The substituent at the N7-position in the imidazole ring-opened guanosine seems to be a determinant for the enzyme activity, with me-FapyG being more efficiently excised than ethyl-FapyG (50) but at a similar rate as the larger substituent hydroxyethyl-thioethyl (52). This implies that Fpg has broad substrate specificity towards various substituted N7-guanosines if they become imidazole ring-opened. It is hence likely that the enzyme would be able to recognise and cleave imidazole ring-opened N7-GA-dG. We hypothesise that this mechanism explains the Fpg sensitivity of DNA from GA-treated cells and also the characteristic dependence of the comet lysis conditions (Figures 2 and 3A). Fpg has previously been shown also to be able to cleave substrates from naked DNA such as oligonucleotides and double-stranded DNA (47,54). Fpg did not change the pattern of GA-induced DNA lesions in the chromatographic analysis of the supernatant from the non-cellular reaction of GA and naked DNA (Figure 1). Initially, we found this to be in contrast to the apparent high efficiency of Fpg in transforming GA-induced DNA lesions into SSB/ALS detectable in the comet assay (13,27,55). However, this is now explained by the pH dependence of the comet lysis step, during which the N7-GA-dG adduct is imidazole ring-opened producing a structure which can be recognised and cleaved by Fpg. This ring-opening is likely to be pH- and time-dependent, in accordance with the results presented in Figures 2 and 3A. Alkaline lysis (pH 10) for 1 h is apparently only partially sufficient for complete imidazole ring-opening of the N7-GA-dG adduct. Similar patterns were seen for MMS-induced Fpg-sensitive sites (Figure 3B). The t1/2 for imidazole ring-opening of N7-methylated deoxyguanosine (N7-me-dG) in double-stranded DNA is reported to be more than 54 h at pH 8.9 and 37°C (reviewed in (43); for the free methylated nucleoside, t1/2 = 4 h at pH 10 and 24°C (56). N7-me-dG and N3-me-dA constitute 84 and 8%, respectively, of the methylated adducts induced by in vitro exposure of mammalian cells to MMS (reviewed in (57)). The reaction scheme which we propose for the conversion of GA-induced DNA lesions into the ring-opened structure is illustrated in Figure 4. From the comet analysis it seemed that 1 h, pH 10 and 4°C gave only partial ring-opening (Figure 2), whereas the reaction seemed complete after over-night lysis at pH 10 at 4°C since no more lesions were detected by Fpg by extending the alkaline lysis period to a maximum of 6 days. Without Fpg, very little increase in GA-induced lesions were detected even after 6 days of alkaline lysis (data not shown), suggesting that spontaneous N3-GA-dA and N7-GA-dG depurination must be very slow even at pH 10; such depurination would have produced ALS detectable in the comet assay without Fpg treatment. Some spontaneous depurination does apparently occur also at neutral pH, not seen in the comet assay but reflected by adducts more sensitively detected with LC-MS in the supernatant from GA-treated naked DNA (Figure 1). Fig. 4. View largeDownload slide A possible mechanism for ring-opening of the N7-GA-dG adduct under alkaline conditions (pH 10), yielding a formamidopyrimidine dG adduct. Fig. 4. View largeDownload slide A possible mechanism for ring-opening of the N7-GA-dG adduct under alkaline conditions (pH 10), yielding a formamidopyrimidine dG adduct. Using the comet assay to study specific adducts such as N7-GA-dG and N3-GA-dA formed in vivo may be particularly relevant for evaluation of the human health risk of AA. The N7-position of guanine is not involved in the hydrogen bonds for base pairing; similar to the N7-me-dG (58), the N7-GA-dG adduct is not expected to block DNA replication or lead to miscoding during replication. However, slow depurination of N7-alkylated guanosines under physiological conditions results in apurinic sites which may block DNA replication if not repaired (58,59). The t1/2 of the N7-GA-dG adduct is 40–80 h in rodents (6,17). If N7-GA-dG is ring-opened over time in vivo, a process which has been observed for N7-me-dG (60,61), this secondary form of the adduct may also cause DNA replication block (31,62). As discussed above, ring-opening of N7-GA-dG would be expected to be slow at physiological pH. The N3-GA-dA is formed at 50–100 times lower rates than N7-GA-dG but the former adduct is more labile and depurinates at a higher rate (6,17). The N3-GA-dA adduct probably blocks DNA replication similar to N3-me-dA (58) since the N3-purine position is important for DNA polymerase function (reviewed in (63)). In fact, we have observed a clear accumulation in S-phase of lymphoblastoid cells following GA-exposure (E. Ansok and K.B. Gutzkow, personal communication). N-methylpurines, if not successfully repaired by a methylpurine DNA glycosylase, may cause strand breaks and chromosomal aberrations as a consequence of DNA replication blockage and potentially damaging BER intermediates (reviewed in (63,64)). Indeed, AA exposure of rodents through drinking water for 30 days resulted in GA-induced structural DNA damage (in mice, not in rats) including micronuclei, but no somatic mutations as measured using the Pig-A mutation assay (65). This all adds to the conclusion that AA (and GA) is clastogenic and carcinogenic (66–68), with dominant lethality and reproductive toxicity as well as carcinogenicity as likely outcomes (69–71). In this study it was apparent that the alkaline lysis conditions did not influence the detection of lesions induced by the phototoxic compound Ro 12–9786 inducing mostly 8-oxoG lesions (25,38,39) which are excellent substrates for Fpg (31) (Figure 3C). Lesions induced by X-rays, i.e. SSB and ALS, and detected without Fpg, were as expected not affected by the lysis conditions (Figure 3D). Taken together, the different types of DNA lesions (GA-induced and MMS-induced (alkylations), vs 8-oxoG and SSB/ALS), show strikingly different patterns of comet alkaline lysis dependence (Figure 3A–D). In general, this can give information on the nature of the DNA damage, thus extending the applicability of the comet assay. Most importantly, it highlights the importance of using relevant comet assay protocols when testing new chemicals for genotoxicity. Conclusions This study shows that the comet assay may give specific information on genotoxic DNA adducts using DNA repair enzymes and specific assay protocols. The chemical modification of adducts during the alkaline lysis step represents a novel application of the assay. These results also emphasise the need for strict control of steps in the comet assay protocol, which may affect the stability of DNA lesions and lead to erroneous conclusions on genotoxicity. Funding This work was financed by the Research Council of Norway in project 159572/I20, and through its Centres of Excellence funding scheme, project number 223268/F50 CERAD. Further financial support was from the European Commission, Priority 5 on Food Quality and Safety, ‘Heat-generated food toxicants—identification, characterisation, and risk minimisation’, [FOOD-CT-2003–506820 Specific Targeted Project]. This publication reflects the authors’ views and not necessarily those of the EC. The information in this document is provided as is with no guarantee or warranty that the information is fit for any particular purpose. The user, therefore, uses the information at their sole risk and liability. Acknowledgements We thank Fredrik N. Fritzøe, Minh Hoang, Solveig M. B. Lakså and Anne Graupner for valuable technical assistance, and Dr Dag Markus Eide for assistance with statistical tests. The contribution of Dr Nur Duale in preparing the Fpg enzyme is gratefully acknowledged. 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Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide metabolite glycidamide

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Abstract

Abstract The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult. Introduction The traditional comet assay gives information on strand breaks arising in cellular DNA after a genotoxic treatment. Higher assay sensitivity and some information on the nature of the DNA lesions are achieved by the recent inclusion of DNA repair enzymes to treat nucleoids after cell lysis. We here report that varying the lysis conditions (duration and pH) gives additional information on DNA adducts induced by certain mutagens. Acrylamide (AA) is a genotoxic industrial compound, but it is also formed in starch-rich foods upon heating hence representing a genotoxic load to the general population (1). The estimated dietary daily intake of AA is 0.4–1 µg/kg bodyweight (2–4). In humans, ~12% of the urinary metabolites of AA arise via the formation of glycidamide (GA) (5) which is the ultimate DNA-reactive metabolite. Using chromatographic analysis, GA has been shown to form DNA adducts, i.e. N7-(2-carbamoyl-2-hydroxyethyl)-deoxyguanosine (N7-GA-dG), and to a lesser extent N3-(2-carbamoyl-2-hydroxyethyl)-deoxyadenosine (N3-GA-dA), both in vivo and in vitro (6–8). In addition, the N1-adenine adducts of GA (analysed after conversion to N⁶-GA-deoxyadenosine-5′-monophosphate) were detected in DNA reacted with GA and in DNA from cells exposed to GA but not in DNA from mice treated with AA (9). In reactions with pure nucleosides, GA forms adducts not only with the purines but also with cytidine and thymidine (10,11). GA induces DNA damage as single-strand breaks (SSB) and alkali-labile sites (ALS), detected in the comet assay in various human and rodent cell types after in vitro exposure (12–15), and in vivo (16,17). The GA-induced SSB and ALS were detected at moderate to high doses and concentrations (in the millimolar range). Recently, chronic exposure of mice to very low AA concentrations (1 µg/ml in drinking water) gave increased levels of DNA damage in spermatozoa (18). In addition to the GA-modified bases which are likely to lead to base loss and subsequent formation of apurinic/apyrimidinic sites (AP sites) (6), modification of DNA could arise via alkylation of the DNA phosphate backbone (19). High sensitivity of detection of AA-related genotoxicity in cells and tissues is essential for evaluation of a potential health risk. In general, the comet assay allows measurement of DNA damage in single cells at biologically relevant levels of damage. The sensitivity of this assay depends on whether the initial DNA modification leads to the formation of a strand break. Single-strand DNA breaks may occur either directly or during endogenous DNA repair, or they may represent chemically unstable lesions such as AP sites leading to DNA strand breakage during the alkaline treatment conditions (pH 13) normally used in the assay. A major advance in the comet assay was the introduction of lesion-specific enzymes with the ability to cleave agarose-embedded lysed and histone-depleted DNA, resulting in a substantially increased sensitivity and specificity (20,21). DNA glycosylases in base excision repair (BER) pathways thus recognise various oxidised or alkylated base modifications (20,22–24). In particular, formamidopyrimidine-DNA glycosylase/AP-lyase (Fpg) was found to detect oxidised guanines (8-oxo-7,8-dihydroguanine, 8-oxoG) more reproducibly than various analytical methods (25). For a variety of other DNA adducts, methods based on liquid chromatographic (LC) separation and mass spectrometric (MS) detection are considered more sensitive and specific for the detection of modified DNA bases [reviewed in (26)]. This has in the past also been the case for GA; in the in vivo study by Maniere et al. (17), significant formation of DNA adducts was detected by LC-MS analysis for the lowest GA dose used, in contrast to insignificant DNA damage detected by the traditional comet assay at the same dose. Thielen and co-workers and also our group, using the comet assay, reported that Fpg markedly increases the detection of GA-induced DNA damage (13,27). In general, this bifunctional enzyme is known to recognise and cleave not only oxidised purines such as 8-oxoG, but also substituted and unsubstituted imidazole ring-opened guanines (Fapy) (28–32). We previously reported that the human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is more specific towards oxidised DNA lesions than Fpg in the comet assay (33), does not detect GA-induced DNA lesions (13). We therefore suggested that the Fpg-sensitive GA-induced lesions result from N7-GA-dG adducts being ring-opened during the lysis step (pH 10), ultimately resembling 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-FapyG). Such adducts and oxidised purines may have different genotoxic outcomes, and knowledge of the exact nature of the DNA lesions induced by GA—and their repair—is highly important for risk assessment of GA. In this paper, we have investigated the ability of the comet assay to detect these adducts. We explored various parameters of the lysis step in the comet assay (incubation time, pH and temperature) and their importance for Fpg-sensitive lesion detection. Parallel experiments were carried out with GA and also methyl methane sulfonate (MMS; giving mainly methylation of N-purines), X-rays (inducing SSB, ALS, oxidised pyrimidines and purines and double strand breaks) and a phototoxic compound primarily inducing 8-oxoG. Furthermore, we studied the nature of the GA-induced lesions by subjecting GA-treated naked DNA to Fpg in a cell-free system and analysing the ‘fall-off’ products by means of LC-MS. Overall, the results strongly suggest that the high efficiency of detection of GA-induced lesions with Fpg is due to N7-GA-dG adducts which are ring-opened during the alkaline lysis step (pH 10). The data demonstrate a novel use of the comet assay not only to detect DNA damage but also to partially analyse their identity. Material and methods Induction of GA adducts in calf thymus DNA treated with Fpg Five milligrams of double-stranded calf thymus DNA (Sigma, St. Louis, MO) were dissolved in 0.1 M phosphate buffer, pH 7.6 overnight (ON, 16–18 h) at 4°C under circulation and light protection as described in the manufacturer’s protocol. The final concentration was determined spectrophotometrically (NanoDrop® ND-1000 spectrophotometer, Saveen & Werner A/S) and adjusted to 2 mg/ml. GA dissolved in sterile H2O at 0.5 M was added to 1 ml DNA solutions to give a final GA concentration of 21 mM, followed by incubation of samples in the dark at 37°C for 18 h (pH 7.6). Subsequently, enzyme buffer [1 mM EDTA, 0.1 M KCl, 20 mM Tris–HCl, pH 7.6 and 2 mg/ml BSA (final concentrations)] with or without 10 or 100 µg/ml Fpg was added to DNA aliquots with or without GA (stoichiometric ratio for GA versus Fpg approximately the same as in the comet assay). All samples were further incubated at 37°C for 1 h. NaCl was then added to a final concentration of 1 M, and DNA was precipitated with ethanol at −20°C for 3 days. Supernatants were collected after two centrifugations at 1150×g for 10 min; samples were stored at −20°C until chromatographic analysis. DNA samples incubated without GA and Fpg were included in the analysis for comparison. Chromatographic methods The LC separations were performed on Agilent 1100 and 1200 systems, both consisting of a binary pump, a vacuum degasser, an autosampler and a thermostated column oven. The 1100 system was connected to a triple quadrupole mass spectrometer (MS) and the 1200 system was hyphenated with the Q-ToF MS. The compounds in the DNA supernatants were separated on a reversed-phase C18 analytical column (5 µm, 4 × 125 mm, Hypersil BDS-C18). The column was eluted with a gradient consisting of 0.01 M aqueous ammonium acetate and acetonitrile starting from 1% acetonitrile and ending after 20 min at 30% acetonitrile at the flow rate 0.5 ml/min and with the injection volume 20 µl. A Micro Triple-Quadrupole MS (Micromass, Manchester, UK) equipped with an electrospray interface was used in the multiple reaction monitoring (MRM) mode. The source block temperature was 120°C and the desolvation temperature was 325°C. Nitrogen was used as the desolvation gas (616 l/h) and the cone gas (33 l/h), while argon was applied as the collision gas, at a collision cell pressure of 5.8 × 10−3 mbar. Positive ions were acquired with a dwell time of 0.25 s and inter-channel delay of 0.05 s. A microTOF-Q (Bruker Daltonics, Bremen, Germany) was used for screening of adducts in the DNA supernatants and for recording the exact mass of adducts. Ionisation was performed with electrospray ionisation in the positive mode and at a capillary voltage of 4500 V. Nitrogen was used as the nebulizer gas (pressure 1.4 bar) and drying gas (8.0 l/min, temperature 200°C). Masses of m/z 50–600 were recorded. The spectrometer was calibrated daily prior to the analyses using a sodium formate solution. A post-run internal mass scale calibration was performed at the end of each chromatographic run calibration. The instrument was operated at a resolution of about 10 000 (FWHM). Chemical treatment of human lymphocytes Human peripheral blood lymphocytes (hPBL) were isolated from one (the same in all experiments) healthy male non-smoking volunteer (in compliance with Norwegian legislation and with ethical permission) using LymphoprepTM tubes as described in the manufacturer’s protocol (Axis Shield, Oslo, Norway). Cells were finally suspended in ice-cold cell medium (BioWhittaker®RPMI 1640 medium with 25 mM HEPES and L-glutamine, Lonza Verviers, Belgium). Cell membrane integrity was measured with trypan blue exclusion (>95%) indicating high cell viability. Stock solutions of GA (Toronto Research Chemicals Inc., Canada, CAS no. 5694-00-8; the dry compound was stored under argon at −20°C) were made fresh for each experiment; GA was dissolved in phosphate-buffered saline (PBS). MMS (Sigma-Aldrich, Oslo, Norway) was diluted in dimethyl sulfoxide (DMSO). GA and MMS dilutions or their solvents were added to hPBLs (1–2 × 106 cells/ml). These cell samples were then incubated in Eppendorf tubes for 2 h at 37°C in the dark. The phototoxic compound Ro 12-9786 (a gift from Dr Elmar Gocke, Roche Ltd, Basel, Switzerland) was dissolved in DMSO and stored at −20°C; this stock solution was added at a final concentration of Ro 12-9786 of 3 µM (0.5% DMSO final) to cell suspensions on ice. After 1 min, the latter samples were exposed to visible light (18 000 lux) at 10 cm distance from a fibre-optic light source, for different time periods. The cell treatments were terminated by adding ice-cold cell medium plus 10% foetal bovine serum (PAA laboratories GMbH, Austria) followed by centrifugation (200×g, 4°C, 5 min). Cell pellets were gently resuspended in cell medium for comet analysis. X-ray exposure (260 kV, filtered through 0.5 mm Cu, dose rate 10 Gy/min (13) took place while cell samples were on ice, at doses 0, 6 and 10 Gy. Comet analysis with Fpg and various lysis conditions DNA SSB plus ALS and Fpg-sensitive sites were measured with a modified alkaline comet assay, as previously described (13). In brief, cells were mixed 1:10 with 0.75% low melting point agarose and added as 12–48 gels per GelBond film (Cambrex, Rockland, ME). Samples were placed for specified time periods in lysis solution at 4°C [2.5 M sodium chloride, 0.1 M Na2EDTA, 10 mM Trizma base, 1% Lauroylsarcosine sodium salt (≥94%; Sigma-Aldrich, Oslo, Norway), with 1% Triton X-100 (Sigma-Aldrich, Oslo, Norway) and 10% DMSO freshly added]. The normal pH of this standard lysing solution is pH 10; in some experiments it was adjusted to pH 7.5. After lysis films were transferred to an enzyme buffer [40 mM HEPES, 0.5 mM Na2EDTA, 0.1 M KCl and 0.2 mg/ml BSA, pH 7.6] for 1 h at 4°C, followed by incubation at 37°C for 1 h in fresh enzyme buffer with or without 1 µg/ml Fpg (prepared from an E. coli extract (13,24,34). After unwinding (40 min, 4°C) in electrophoresis solution (pH ≥13.2), electrophoresis was performed at 0.8 V/cm, 8–10°C for 20 min as described (35). Films were further neutralised and fixed in ethanol. SybrGold (Invitrogen, Oslo, Norway) stained gels were microscopically examined and comet tails were scored using the Comet assay IV software (Perceptive Instruments Ltd., Suffolk, UK). DNA damage was defined as %Tail DNA, i.e. the fluorescence intensity in the tail relative to the total intensity of the comet, as described (35). Samples were scored in triplicate each of 30 cells, and medians were calculated. Each experiment was repeated up to three times. The net increase in DNA damage measured with Fpg is denoted as Fpg-sensitive lesions. The activity of the Fpg prep used in this study has been tested using an Oligo Repair Chip (LXRepair SAS, Grenoble) showing high 8-oxoG and AP-site activity in vitro (data not shown, personal communication from Dr Sylvie Sauvaigo). Statistics Concentration–effect relationships of GA and each lysis condition were explored using linear regression analyses with goodness to fit; the %Tail DNA measurements were square root transformed to obtain normal distribution of the dependent variables and residuals. The slopes for the regression lines for GA were compared using the one hour lysis at pH 10 (1 h pH10) group as the reference value. Statistically significant differences between slopes are indicated in the result section and in figures and their legends. JMP Pro (JMPPRO WINDOWS 13, SAS) was used for the statistical analyses. Results Reaction of naked DNA with GA Chromatographic analyses with LC-MS were carried out to confirm the formation of GA-purine adducts, and to investigate whether Fpg treatment has any effect on the pattern of bases released from GA-treated DNA at neutral pH. The Fpg treatment step (pH 7.6) in the comet assay is in general carried out after an alkaline lysis step (pH 10) included to remove cellular proteins and histones from DNA. Assuming that the Fpg enzyme should be similarly active on naked double-stranded DNA in solution, reactions were set up in which similar relative amounts of GA, Fpg enzyme and calf thymus DNA were used. GA (21 mM) was mixed with calf thymus DNA and the reaction mixtures were thereafter incubated at 37°C with Fpg (pH 7.6) to allow enzymatic release of DNA adducts. Possible reaction products (DNA bases) released from ethanol precipitated DNA were subjected to LC-MS chromatographic analysis. The identification of the GA adducts was based on exact mass and the isotopic pattern determinations provided by the Q-ToF mass analyser and on mass spectra generated by MRM transitions in the triple quadrupole MS. The N7-GA-Gua and N3-GA-Ade nucleobase derivatives were detected in the supernatants from samples in which DNA was treated with GA. In the case of N7-GA-Gua, the chromatographic retention matched the retention time of pure N7-GA-Gua aglycon (prepared by reacting GA with dG and hydrolysis of the sugar unit). The MRM transition 239–152 and 223–136 for N7-GA-Gua and N3-GA-Ade, respectively (Figure 1), corresponded to the loss of the GA unit from the nucleobase (i.e. the aglycon). The masses of both adducts measured by the Q-ToF analyser differed from the calculated masses by less than 5 ppm corresponding to a mass error of only 0.7 mDa. LC-Q-ToF MS was applied for screening for the occurrence of additional adduct-identities, but no other adducts than N7-GA-Gua and N3-GA-Ade were observed. The MRM transitions generated a peak area three times higher for N7-GA-Gua than for N3-GA-Ade, in all samples analysed. Due to the lack of N3-GA-Ade standard, it was not possible to conclude from these experiments that N7-GA-Gua was the dominating adduct. However, the ratio of formed adducts was the same in all samples. Furthermore, the peak intensities were approximately the same in all samples. In conclusion, the data suggest that the addition of the Fpg enzyme did not affect the total adduct yield in the supernatant, and new products were not identified. No adducts could be detected in the supernatant from untreated DNA (data not shown). Fig. 1. View largeDownload slide Multiple reaction monitoring (MRM) chromatograms with retention times (minutes) for N7-GA-Gua and N3-GA-Ade from the analysis of the supernatant obtained by precipitation of calf thymus DNA incubated with (A) GA (21 mM) and the Fpg enzyme (10 µg/ml); and (B) GA in the absence of Fpg. Fig. 1. View largeDownload slide Multiple reaction monitoring (MRM) chromatograms with retention times (minutes) for N7-GA-Gua and N3-GA-Ade from the analysis of the supernatant obtained by precipitation of calf thymus DNA incubated with (A) GA (21 mM) and the Fpg enzyme (10 µg/ml); and (B) GA in the absence of Fpg. DNA damage detection in the comet assay and the effect of lysis time, temperature and pH A higher sensitivity for detection of DNA lesions induced by GA in cells is obtained when the comet assay includes Fpg treatment of nucleoids after cell lysis (often 1 h at pH 10). However, this increase was markedly higher after over-night lysis at pH 10 prior to the Fpg treatment (Figures 2 and 3A). Using this protocol, effects of approximately 20–50 times lower concentrations of GA can be measured, in whole blood or primary cell cultures from mice and human lymphocytes (13,27). In the present study, the level of GA-induced DNA lesions in human lymphocytes detected without Fpg was low (0–7% Tail DNA) and is subtracted so that Figures 2 and 3A show net levels of Fpg-sensitive lesions. The standard lysis condition used in our comet assay protocol is over-night (16–18 h) at 4°C in lysis buffer (pH 10). Figures 2 and 3A show that the level of GA-induced Fpg-sensitive lesions was significantly different (Figure 3A), approximately twice as high, when lysis lasted over-night rather than for 1 h. One hour lysis time at pH 10 is in accordance with the traditional protocol by Tice et al. (36). When we increased the lysis treatment to 40 h or 6 days, no further increase in the level of GA-induced DNA-damage was observed beyond that obtained with over-night lysis (Figure 2). The temperature did not seem to be important since lysis at room temperature for 6 days (144 h) gave similar levels of Fpg-sensitive sites as lysis at 4°C (Figure 2). The data in Figure 3A show that, in addition to the duration of the lysis step, the pH of the lysis solution was highly important for the detection of the GA-induced Fpg-sensitive sites. At pH 7.5, no Fpg-sensitive GA-induced lesions were detected. Fig. 2. View largeDownload slide Induction of Fpg-sensitive sites in human peripheral blood lymphocytes by glycidamide (GA), as a function of duration of alkaline lysis (hours, pH 10) in the comet assay. The shortest lysis time is 1 h. Open symbols: lysis performed at 4°C; median %Tail DNA values of all comets from three technical replicates for each experiment (n = 1 or 2). Closed symbols: Lysis at room temperature for 6 days (144 h) (n = 1). GA concentrations during incubation of lymphocytes are indicated in µM. Fig. 2. View largeDownload slide Induction of Fpg-sensitive sites in human peripheral blood lymphocytes by glycidamide (GA), as a function of duration of alkaline lysis (hours, pH 10) in the comet assay. The shortest lysis time is 1 h. Open symbols: lysis performed at 4°C; median %Tail DNA values of all comets from three technical replicates for each experiment (n = 1 or 2). Closed symbols: Lysis at room temperature for 6 days (144 h) (n = 1). GA concentrations during incubation of lymphocytes are indicated in µM. Fig. 3. View largeDownload slide Influence of lysis time (1 h or overnight (ON)) and pH in the comet assay on the detection of Fpg-sensitive sites and SSB/ALS, in human peripheral blood lymphocytes, exposed to either (A) glycidamide (GA, µM); (B) methyl methane sulfonate (MMS, µM); (C) 3 µM Ro 12–9786 + light exposure (1.5 or 3 min) or (D) X-rays (Gy). Data points represent various lysis conditions (pH and duration) as indicated in the figures. Net Fpg-sensitive sites are shown in (A–C), whereas in (D) the X-ray-induced lesions (SSB/ALS) were assayed without Fpg. (A) shows dose-response curves (versus GA concentration; ±SEM n = 3); stars denote lysis dependent regression lines significantly different from lysis at 1 h pH 10 (P < 0.001). (B–D) show mean %Tail DNA, calculated from medians of technical replicates, from at least two independent experiments. The concentration-response relationship in (B) (for MMS) was not tested by statistical methods, since n = 2. For (C) and (D), there were no significant effects of lysis conditions. Fig. 3. View largeDownload slide Influence of lysis time (1 h or overnight (ON)) and pH in the comet assay on the detection of Fpg-sensitive sites and SSB/ALS, in human peripheral blood lymphocytes, exposed to either (A) glycidamide (GA, µM); (B) methyl methane sulfonate (MMS, µM); (C) 3 µM Ro 12–9786 + light exposure (1.5 or 3 min) or (D) X-rays (Gy). Data points represent various lysis conditions (pH and duration) as indicated in the figures. Net Fpg-sensitive sites are shown in (A–C), whereas in (D) the X-ray-induced lesions (SSB/ALS) were assayed without Fpg. (A) shows dose-response curves (versus GA concentration; ±SEM n = 3); stars denote lysis dependent regression lines significantly different from lysis at 1 h pH 10 (P < 0.001). (B–D) show mean %Tail DNA, calculated from medians of technical replicates, from at least two independent experiments. The concentration-response relationship in (B) (for MMS) was not tested by statistical methods, since n = 2. For (C) and (D), there were no significant effects of lysis conditions. In order to examine how different types of lesions respond to the lysis conditions (37), cells were exposed to other genotoxic treatments. The detection of Fpg-sensitive sites induced by the methylating agent MMS was influenced by duration of lysis and pH in a similar way as for GA (Figure 3). In the case of treatment with Ro 12-9786 plus light which in analogy with similar compounds leads to the formation of 8-oxoG (25,38,39), alkaline lysis conditions had no significant effect on the total yield of Fpg-sensitive sites (Figure 3C). For X-rays, inducing SSB and ALS, the lesions were as expected detectable in the comet assay without Fpg; the different lysis conditions gave similar levels of damage (Figure 3D). X-rays are known to also induce frequent oxidative lesions at the doses applied (6 and 10 Gy), but sequestering of Fpg due to its high affinity to SSBs, and damage clustering of such lesions with DNA strand breaks (40), prevent their efficient cleavage by Fpg until the strand breaks have been largely repaired, as we have discussed previously (41). Accordingly, we observed very low levels of Fpg-sensitive lesions in the comet assay after X-rays (data not shown). In some of the experiments illustrated in Figure 3, the over-night lysis was carried out not at pH 10 but at pH 7.5, except for 1 h initial treatment at pH 10. Figure 3A–D show that no major increase in lesion detection was associated with extending the lysis time beyond 1 h at pH 7.5. Discussion When calf thymus DNA was incubated with GA at neutral conditions, N7-GA-Gua and N3-GA-Ade adducts were detected in the supernatant by LC-MS analysis implying that the modified bases had been released from DNA. These adducts are the same major GA-induced DNA-adducts as those detected in previous in vivo rodent studies (6–8,17,42). Incubation with Fpg prior to DNA precipitation did not change the occurrence of the adducts, nor were new types of adducts/modified bases detected in the supernatant. This strongly suggests that the N7-GA-dG and N3-GA-dA adducts per se were not major substrates for Fpg. The N7-GA-Gua and N3-GA-Ade adducts detected are likely to have been released from the sugar phosphates via spontaneous depurination. N3-alkylated adenosines are not known to be ring-opened or recognised by Fpg; the predominant reaction for labile N7-alkylated guanosines at physiological pH is depurination and not imidazole ring opening (reviewed in (43)). Ring opening of N7-alkylated guanosines is facilitated by strongly electron-withdrawing substituents (44); in some cases ring-opening rates may be comparable to depurination rates under physiologically relevant conditions (43). GA does possess electron-withdrawing groups; however, the N7-GA-adducts formed in the non-cellular reaction (Figure 1) seemed not to be ring-opened for recognition and cleavage by Fpg to detectable levels at these (neutral pH) conditions. Purified or crude extracts of the bacterial DNA glycosylase Fpg have in recent years been much used for detection of oxidised purines, i.e. 8-oxoG and unsubstituted FapyG (29,31), in the comet assay (23–25,45) and also with alkaline elution (34,46). This bifunctional enzyme has strong substrate affinity also to me-FapyG (28,30,47), and was previously shown to detect DNA damage induced by alkylating agents in the comet assay (33,48,49). Fpg is known to have high substrate affinity also to imidazole ring-opened guanosines containing larger substituents than methyl at the N7-position of the guanine, such as ethyl (50), aminofluorene (51), hydroxyethyl-thioethyl (52) and aminoethyl (53). The substituent at the N7-position in the imidazole ring-opened guanosine seems to be a determinant for the enzyme activity, with me-FapyG being more efficiently excised than ethyl-FapyG (50) but at a similar rate as the larger substituent hydroxyethyl-thioethyl (52). This implies that Fpg has broad substrate specificity towards various substituted N7-guanosines if they become imidazole ring-opened. It is hence likely that the enzyme would be able to recognise and cleave imidazole ring-opened N7-GA-dG. We hypothesise that this mechanism explains the Fpg sensitivity of DNA from GA-treated cells and also the characteristic dependence of the comet lysis conditions (Figures 2 and 3A). Fpg has previously been shown also to be able to cleave substrates from naked DNA such as oligonucleotides and double-stranded DNA (47,54). Fpg did not change the pattern of GA-induced DNA lesions in the chromatographic analysis of the supernatant from the non-cellular reaction of GA and naked DNA (Figure 1). Initially, we found this to be in contrast to the apparent high efficiency of Fpg in transforming GA-induced DNA lesions into SSB/ALS detectable in the comet assay (13,27,55). However, this is now explained by the pH dependence of the comet lysis step, during which the N7-GA-dG adduct is imidazole ring-opened producing a structure which can be recognised and cleaved by Fpg. This ring-opening is likely to be pH- and time-dependent, in accordance with the results presented in Figures 2 and 3A. Alkaline lysis (pH 10) for 1 h is apparently only partially sufficient for complete imidazole ring-opening of the N7-GA-dG adduct. Similar patterns were seen for MMS-induced Fpg-sensitive sites (Figure 3B). The t1/2 for imidazole ring-opening of N7-methylated deoxyguanosine (N7-me-dG) in double-stranded DNA is reported to be more than 54 h at pH 8.9 and 37°C (reviewed in (43); for the free methylated nucleoside, t1/2 = 4 h at pH 10 and 24°C (56). N7-me-dG and N3-me-dA constitute 84 and 8%, respectively, of the methylated adducts induced by in vitro exposure of mammalian cells to MMS (reviewed in (57)). The reaction scheme which we propose for the conversion of GA-induced DNA lesions into the ring-opened structure is illustrated in Figure 4. From the comet analysis it seemed that 1 h, pH 10 and 4°C gave only partial ring-opening (Figure 2), whereas the reaction seemed complete after over-night lysis at pH 10 at 4°C since no more lesions were detected by Fpg by extending the alkaline lysis period to a maximum of 6 days. Without Fpg, very little increase in GA-induced lesions were detected even after 6 days of alkaline lysis (data not shown), suggesting that spontaneous N3-GA-dA and N7-GA-dG depurination must be very slow even at pH 10; such depurination would have produced ALS detectable in the comet assay without Fpg treatment. Some spontaneous depurination does apparently occur also at neutral pH, not seen in the comet assay but reflected by adducts more sensitively detected with LC-MS in the supernatant from GA-treated naked DNA (Figure 1). Fig. 4. View largeDownload slide A possible mechanism for ring-opening of the N7-GA-dG adduct under alkaline conditions (pH 10), yielding a formamidopyrimidine dG adduct. Fig. 4. View largeDownload slide A possible mechanism for ring-opening of the N7-GA-dG adduct under alkaline conditions (pH 10), yielding a formamidopyrimidine dG adduct. Using the comet assay to study specific adducts such as N7-GA-dG and N3-GA-dA formed in vivo may be particularly relevant for evaluation of the human health risk of AA. The N7-position of guanine is not involved in the hydrogen bonds for base pairing; similar to the N7-me-dG (58), the N7-GA-dG adduct is not expected to block DNA replication or lead to miscoding during replication. However, slow depurination of N7-alkylated guanosines under physiological conditions results in apurinic sites which may block DNA replication if not repaired (58,59). The t1/2 of the N7-GA-dG adduct is 40–80 h in rodents (6,17). If N7-GA-dG is ring-opened over time in vivo, a process which has been observed for N7-me-dG (60,61), this secondary form of the adduct may also cause DNA replication block (31,62). As discussed above, ring-opening of N7-GA-dG would be expected to be slow at physiological pH. The N3-GA-dA is formed at 50–100 times lower rates than N7-GA-dG but the former adduct is more labile and depurinates at a higher rate (6,17). The N3-GA-dA adduct probably blocks DNA replication similar to N3-me-dA (58) since the N3-purine position is important for DNA polymerase function (reviewed in (63)). In fact, we have observed a clear accumulation in S-phase of lymphoblastoid cells following GA-exposure (E. Ansok and K.B. Gutzkow, personal communication). N-methylpurines, if not successfully repaired by a methylpurine DNA glycosylase, may cause strand breaks and chromosomal aberrations as a consequence of DNA replication blockage and potentially damaging BER intermediates (reviewed in (63,64)). Indeed, AA exposure of rodents through drinking water for 30 days resulted in GA-induced structural DNA damage (in mice, not in rats) including micronuclei, but no somatic mutations as measured using the Pig-A mutation assay (65). This all adds to the conclusion that AA (and GA) is clastogenic and carcinogenic (66–68), with dominant lethality and reproductive toxicity as well as carcinogenicity as likely outcomes (69–71). In this study it was apparent that the alkaline lysis conditions did not influence the detection of lesions induced by the phototoxic compound Ro 12–9786 inducing mostly 8-oxoG lesions (25,38,39) which are excellent substrates for Fpg (31) (Figure 3C). Lesions induced by X-rays, i.e. SSB and ALS, and detected without Fpg, were as expected not affected by the lysis conditions (Figure 3D). Taken together, the different types of DNA lesions (GA-induced and MMS-induced (alkylations), vs 8-oxoG and SSB/ALS), show strikingly different patterns of comet alkaline lysis dependence (Figure 3A–D). In general, this can give information on the nature of the DNA damage, thus extending the applicability of the comet assay. Most importantly, it highlights the importance of using relevant comet assay protocols when testing new chemicals for genotoxicity. Conclusions This study shows that the comet assay may give specific information on genotoxic DNA adducts using DNA repair enzymes and specific assay protocols. The chemical modification of adducts during the alkaline lysis step represents a novel application of the assay. These results also emphasise the need for strict control of steps in the comet assay protocol, which may affect the stability of DNA lesions and lead to erroneous conclusions on genotoxicity. Funding This work was financed by the Research Council of Norway in project 159572/I20, and through its Centres of Excellence funding scheme, project number 223268/F50 CERAD. Further financial support was from the European Commission, Priority 5 on Food Quality and Safety, ‘Heat-generated food toxicants—identification, characterisation, and risk minimisation’, [FOOD-CT-2003–506820 Specific Targeted Project]. This publication reflects the authors’ views and not necessarily those of the EC. The information in this document is provided as is with no guarantee or warranty that the information is fit for any particular purpose. The user, therefore, uses the information at their sole risk and liability. Acknowledgements We thank Fredrik N. Fritzøe, Minh Hoang, Solveig M. B. Lakså and Anne Graupner for valuable technical assistance, and Dr Dag Markus Eide for assistance with statistical tests. The contribution of Dr Nur Duale in preparing the Fpg enzyme is gratefully acknowledged. 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MutagenesisOxford University Press

Published: Jan 1, 2018

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