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Chem. Res. Toxicol. 1999, 12, 106-111
Detection of Concomitant Formation of and O6-Methyl-2′-deoxyguanosine in DNA Exposed to Nitrosated Glycine Derivatives Using a Combined Immunoaffinity/HPLC Method O6-Carboxymethyl-
Kathryn L. Harrison,† Rebekah Jukes,† Donald P. Cooper,‡ and David E. G. Shuker*,† MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, U.K., and CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester M20 9BX, U.K. Received March 26, 1998
A previous observation that an N-nitroso-N-carboxymethyl derivative reacts with DNA to give both O6-carboxymethyl-2′-deoxyguanosine (O6-CMdGuo) and O6-methyl-2′-deoxyguanosine (O6-MedGuo) [Shuker, D. E. G., and Margison, G. P. (1997) Cancer Res. 57, 366-369] has been confirmed using a range of nitrosated glycine derivatives [N-acetyl-N′-nitroso-N′prolylglycine (APNG), azaserine (AS), and potassium diazoacetate (KDA)]. In addition, mesyloxyacetic acid (MAA) was also found to give both O6-adducts in DNA. O6-CMdGuo and O6-MedGuo were assessed in enzymatic hydrolysates of treated calf thymus DNA using a combined immunoaffinity/HPLC/fluorescence procedure. The ratio of O6-CMdGuo to O6-MedGuo varied somewhat between the different compounds with APNG giving the most methylation (O6-CM:O6-Me ratio of 10) and AS the least (39), with KDA and MAA giving intermediate amounts (16 and 18, respectively). The formation of O6-MedGuo by the four compounds probably arises through decarboxylation at various stages in the decomposition pathways, but the exact mechanisms remain to be clarified. The formation of O6-MedGuo from reactions of nitrosated glycine derivatives with DNA in vitro may explain the frequent detection of this adduct in human gastrointestinal DNA, as nitrosation of dietary glycine may occur. O6-CMdGuo is likely to be a useful biomarker of this pathway in vivo and has been detected in human tissues.
Introduction Alkylation of DNA is considered to be a key step in the induction of cancer by many different chemicals (1). For many compounds, including N-alkyl-N-nitroso compounds, alkylation at the O6 atom of 2′-deoxyguanosine appears to be the major mutagenic lesion, although O4alkylthymidines may also be mutagenic (2). In both prokarytic and eukaryotic cells, efficient specific repair mechanisms exist for the removal of O6-methyl-2′-deoxyguanosine (O6-MedGuo)1 residues as well as some higher homologues, albeit more slowly (3). Among the many N-alkyl-N-nitroso compounds that are known to be carcinogenic, there are a number which are derived from glycine, the simplest R-amino acid (Scheme 1). N-Nitrosoglycocholic acid (NOGC) is a carcinogenic and mutagenic derivative of the naturally occurring bile acid conjugate, glycocholic acid (4-6). Incubation of NOGC with calf thymus DNA in vitro gave * To whom correspondence should be addressed. † University of Leicester. ‡ Christie Hospital (NHS) Trust. 1 Abbreviations: NOGC, N-nitrosoglycocholic acid; 7-CMGua, 7-carboxymethylguanine; 3-CMAde, 3-carboxymethyladenine; O6-CMGua, O6-carboxymethylguanine; APNG, N-(N′-acetyl-L-prolyl)-N-nitrosoglycine; AS, azaserine; O6-CMdGuo, O6-carboxymethylguanine-2′-deoxyguanosine; PBS, phosphate-buffered normal saline; TFA, trifluoroacetic acid; O6-MedGuo, O6-methyl-2′-deoxyguanosine; O6-MeGua, O6methylguanine; KDA, potassium diazoacetate; HFBA, heptafluorobutyric acid; MAA, mesyloxyacetic acid.
Scheme 1. Structures of Nitrosated Glycine Derivatives and Mesyloxyacetic Acid Used in This Study
rise to 7-carboxymethylguanine (7-CMGua), 3-carboxymethyladenine (3-CMAde), and O6-carboxymethylguanine (O6-CMGua) (7). Furthermore, administration of [14C]NOGC to rats gave rise to dose-dependent excretion of 7-CMGua in urine (7). N-Nitroso peptides, which are C-terminal in glycine, such as N-(N′-acetyl-L-prolyl)N-nitrosoglycine (APNG), are mutagenic (8) and carcinogenic (9) and would be expected to be carboxymethy-
10.1021/tx980057n CCC: $18.00 © 1999 American Chemical Society Published on Web 12/16/1998
O6-CMG and O6-MeG from Nitrosated Glycines
lating agents by analogy with NOGC. Similarly, N-nitrosoN-carboxymethylurea is a gastrointestinal carcinogen (10, 11). Azaserine (AS), a pancreatic carcinogen, is also known to carboxymethylate DNA with [14C]-7-CMGua being detected in DNA extracted from acinar cells treated in vitro with [14C]AS (12). Recently, we have shown that O6-CMdGuo is formed in DNA treated with several nitrosated glycine derivatives (13). In studies on the formation of DNA adducts by NOGC, it was also observed that methylation (O6methyl- and 7-methylguanine) of DNA occurred at the same time as carboxymethylation (O6-CMGua and 7-CMGua) (14). This potential source of methylating agents is particularly interesting in view of the number of observations of detectable levels of O6-MedGuo in DNA extracted from human gastrointestinal tissues (15-19). It was therefore of interest to determine if DNA methylation was a common property of a range of carboxymethylating agents. This required the development of an assay for quantitating the two adducts in the same DNA sample, and available antibodies to O6-CMdGuo and O6MedGuo were used to affinity purify each deoxynucleoside adduct sequentially from DNA hydrolysates prior to determination of each adduct as bases in separate HPLC/fluorescence analyses.
Materials and Methods Warning: Reagents that generate carboxymethyldiazonium ions are alkylating agents and should be treated with extreme caution. Unused solutions of N-carboxymethyl-N-nitroso compounds should be decomposed in 0.1 M sodium hydroxide solutions overnight in a fume cupboard. Unused solutions of diazoacetic acid derivatives should be decomposed by treatment with 1 M aqueous acetic acid. AS was purchased from Sigma (Poole, Dorset, U.K.). APNG (20) and mesyloxyacetic acid (MAA; 21) were synthesized according to published procedures. Potassium diazoacetate (KDA; 22) was prepared by alkaline hydrolysis of ethyl diazoacetate according to the method of Kreevoy (23), and suitable dilutions of the resulting solution were used without further purification for reactions with DNA. Attempts to isolate the salt resulted in decomposition and polymerization (23). Immunoaffinity column kits (polystyrene columns and frits) and dimethylpimelimidate were purchased from Pierce [Pierce and Warriner (UK) Ltd., Chester, U.K.]. [3H]dThd labeled on the 5′-methylene was obtained from Amersham Life Sciences (Amersham, Bucks, U.K.). HPLC columns were purchased from Shandon HPLC (Runcorn, Cheshire, U.K.). Immunoaffinity Purification of O6-CMdGuo. Immunopurification of O6-CMdGuo from DNA hydrolysates was carried out as described by Harrison et al. (13). Briefly, an immunoaffinity matrix was prepared by covalently binding the total immunogloblin fraction from a rabbit antiserum for O6-CMdGuo. Bound O6-CMdGuo was eluted from immunoaffinity columns with 1 M trifluoroacetic acid (TFA) and converted to O6-CMGua which was detected and quantified by RP-HPLC with fluorescence detection (see below). Immunoaffinity Purification of O6-MedGuo. (1) Preparation of Immunoaffinity Columns. O6-MedGuo monoclonal antibody (24), which had been freeze-dried, was reconstituted in 1 mL of water to give a protein concentration of 1.7 mg/mL. This solution was then placed in a small beaker (25 mL) to which cold saturated ammonium sulfate was added to a final concentration of 40% and stirred for 5 min. After centrifugation, the supernatant was discarded and the precipitate washed twice with cold 50% saturated ammonium sulfate. The pellet was then resuspended in PBS (5 mL) and dialyzed overnight against PBS (3 L). The dialysate was centrifuged at 3000g to remove suspended matter and the supernatant used directly in the next
Chem. Res. Toxicol., Vol. 12, No. 1, 1999 107 step to make immunoaffinity columns. The antibody was bound to Protein A-Sepharose as described by Harrison et al. (13) using 2 mL of gel. The resulting affinity gel was mixed with 3 mL of Sepharose CL4B to enable 5 × 1 mL columns to be made. (2) Determination of Optimal Elution Conditions for O6-MedGuo. [3H]-O6-MedGuo [prepared from O6-methylguanine (O6-MeGua) and [3H]dThd (25), 48 pmol, 900 dpm) in 2 mL of PBS/azide (0.02%) was applied to immunoaffinity columns, followed by an additional 3 mL of PBS/azide (0.02%). The columns were then washed with 10 mL of HPLC grade water. Fractions (1 mL) of the column eluates were collected directly into scintillation vials, and 3 mL of Hydrofluor scintillation cocktail (National Diagnostics, Atlanta, GA) was added prior to scintillation counting. Quantitative elution of the [3H]-O6MedGuo was obtained with a range of aqueous methanol solutions (60-80% v/v). However, the 80% methanol resulted in the sharpest elution peak in the smallest volume (3 mL). Columns were regenerated with 15 mL of PBS/azide (0.02%). (3) Determination of the Column Capacity for O6MedGuo. The determination of the capacity of the columns for O6-MedGuo was achieved using a saturation assay. [3H]-O6MedGuo (48 pmol, 900 dpm) and O6-MedGuo (0-1500 ng) were applied to the immunoaffinity columns which were then washed with PBS/azide (0.02%, 3 mL) and water (10 mL). Elution with 80% methanol (4 mL) was then carried out. The eluate was collected directly into scintillation vials. Liquid scintillation fluid (Hydrofluor, 3 mL) was added to each vial, and the amount of radioactivity was determined by scintillation counting. Immunoaffinity Purification of O6-CMdGuo and O6MedGuo from DNA Hydrolysates in a Combined Protocol. Preliminary experiments established that both types of immunoaffinity columns could be loaded with a solution resulting from enzymatic hydrolysis of 2.5 mg of calf thymus DNA without affecting column performance. The ability of the immunoaffinity columns to be used in a combined protocol to purify both O6CMdGuo and O6-MedGuo was examined as follows. Aliquots of calf thymus DNA (2.5 mg) spiked with (a) O6-CMdGuo (0-50 pmol), (b) O6-MedGuo (0-50 pmol), or (c) O6-CMdGuo (0-50 pmol) and O6-MedGuo (0-50 pmol) were digested to deoxynucleoside using a method outlined by Beranek et al. (26). DNA samples were hydrolyzed in 50 mM BisTris/1 mM MgCl2 (pH 6.5) at 50 °C for 8 h, using 24 units of nuclease P1, 2.4 units of bacterial alkaline phosphatase, and 0.3 unit of wheat germ acid phosphatase per milligram of DNA (to a final concentration of 1 mg of DNA/mL). The reactions were stopped by heating at 100 °C for 5 min, and the mixtures were then centrifuged to remove the denatured enzymes. To each digest was added 0.02% PBS/azide (2 mL), and the sample was briefly vortexed before being loaded onto the O6-CMdGuo immunoaffinity column which eluted directly onto a O6-MedGuo immunoaffinity column washed with a further 3 mL of 0.02% PBS/ azide. The columns were then separated for an independent wash of water (10 mL) and elution with either 4 mL of 1 M TFA (O6-CMdGuo) or 4 mL of 80% methanol (O6-MedGuo), which were collected in 15 mL tubes (Scheme 2). The solutions containing O6-CMdGuo and O6-MedGuo were prepared for HPLC/fluorescence analysis as described below. The dGuo content of each DNA hydrolysate was determined in a separate analysis as follows. A 10 µL aliquot was analyzed by HPLC [RPBDS C18 3 µm Hypersil reversed-phase column, 10 cm × 2 mm, with an isocratic flow rate of 0.2 mL/min of 0.1 M triethylammonium acetate (pH 5) with 4% methanol] with UV detection at 260 nm. The amount of dGuo was calculated by comparison to an authentic standard. (1) O6-CMdGuo. The eluates from the O6-CMdGuo immunoaffinity columns were heated at 50 °C for 60 min to completely hydrolyze O6-CMdGuo to O6-CMGua. The solution was then freeze-dried, redissolved in water (1 mL), and finally concentrated to dryness in a centrifugal vacuum evaporator. Just prior to HPLC analysis, the residue was dissolved in 0.1% aqueous heptafluorobutyric acid (HFBA, 20 µL). Analytical RP-HPLC was carried out using a RP-BDS C18 3 µm Hypersil (10 cm ×
108 Chem. Res. Toxicol., Vol. 12, No. 1, 1999 Scheme 2. Diagrammatic Representation of the Combined Immunoaffinity Cleanup Used To Separate O6-CMdGuo (Hatched Shape) and 6 O -MedGuo (Black Shape) from Hydrolyzed DNA
Harrison et al. and left gently stirring at 37 °C in the dark overnight. Reactions were carried out in triplicate for each compound and treatment concentration. After treatment, DNA was precipitated from the reaction medium with sodium acetate (0.1 volume, 2.5 M) and cold ethanol (2 volumes) and centrifuged gently (3000g for 5 min), and the DNA was washed several times with ethanol. The DNA pellet was recovered, dried, and resuspended in water to the original volume. O6-CMdGuo and O6-MedGuo were quantitated as described above, and the results were expressed as picomoles of adduct per micromole of dGuo.
Results and Discussion
2.0 mm) column with a prefilter, with an isocratic flow rate of 0.2 mL/min of 0.1% HFBA with 10% methanol. Peaks were detected by fluorescence (excitation wavelength of 286 nm and emission wavelength of 378 nm) and quantified by comparison with standard curves obtained with standard O6-CMGua (13). (2) O6-MedGuo. The eluates from the O6-MedGuo columns were evaporated to dryness in a centrifugal vacuum evaporator, redissolved in 0.1 M HCl (10 µL), and heated at 50 °C for 30 min to hydrolyze O6-MedGuo to O6-MeGua. The samples were re-dried and dissolved in 0.1% aqueous HFBA (20 µL). Analytical RP-HPLC was carried out using a RP-BDS C18 3 µm Hypersil (10 cm × 2.0 mm) column with a prefilter, with an isocratic flow rate of 0.2 mL/min of 0.1% HFBA with 15% methanol. Peaks were detected by fluorescence (excitation wavelength of 286 nm and emission wavelength of 378 nm) and quantified by comparison with standard O6-MeGua (14). An identical overall procedure for detection and quantification of O6-CMGua and O6-MeGua was used to analyze calf thymus DNA treated in vitro with various carboxymethylating agents (see below). Treatment of Calf Thymus DNA with Carboxymethylating Agents in Vitro. Aliquots of calf thymus DNA (2.5 mg) from a stock solution (5 mg/mL in PBS) were treated with either APNG or KDA (0.5, 1, 2.5, and 5 mM solution), azaserine (1, 2.5, 5, and 10 mM solutions), or mesyloxyacetic acid (5 mM)
Immunoaffinity columns were prepared which selectively purified and concentrated O6-CMdGuo (13) and O6MedGuo prior to quantitation by HPLC/fluorescence. Enzymatic hydrolysates of treated DNA were applied, first of all, to an O6-CMdGuo immunoaffinity column, and the eluate from that column was passed directly onto the O6-MedGuo immunoaffinity column. The two columns were then washed and eluted separately. O6-CMdGuo was eluted from immunoaffinity columns using 1 M trifluoroacetic acid. The eluate was heated at 50 °C for 60 min to completely hydrolyze the adduct to O6-CMGua (13). Under these conditions, there was no conversion of O6-CMGua to O6-MeGua. O6-MedGuo was eluted from immunoaffinity columns using 80% aqueous methanol. Eluates were evaporated to dryness, redissolved in a small volume of 0.1 M HCl, and heated at 50 °C for 30 min which resulted in complete hydrolysis of the adduct to O6-MeG. The O6-CMGua- and O6-MeGua-containing extracts were analyzed using different HPLC conditions. The overall analytical protocol is summarized in Scheme 2. Typical RP-HPLC/fluorescence chromatograms for both adducts are shown in Figure 1. Note that the HPLC chromatograms in Figure 1 were obtained using different solvent compositions and that O6-CMGua and O6-MeGua do not coelute (see the legend of Figure 1). The overall recoveries of O6-MedGuo (Figure 2) and O6-CMdGuo (13) through the combined procedure were found to be 79 and 59%, respectively, and linear calibration lines were obtained over the range of 0-15 pmol/2.5 mg of DNA and 0.2 pmol/2.5 mg of DNA, respectively. The results for each adduct were corrected for these recovery values. A number of methods for quantification of O6-MedGuo have been developed (27), many of which involve immunochemical steps, and most recently, a combined immunoaffinity/32P-postlabeling method has been developed (28). The sensitivity of each of these methods is a function of both the absolute detection limit and the amount of DNA that can be used per sample. The most sensitive methods typically can detect approximately five adducts per 108-109 bases. The limits of quantitation for each of the adducts using the immunoaffinity/HPLC/fluorescence assay described above were 0.1 pmol of O6-CMGua/ injection and 0.05 pmol of O6-MeGua/injection. If the equivalent of 1 mg of DNA was used per injection, the overall limits of detection of the combined immunoaffinity/HPLC/fluorescence assay corresponded to 3.7 O6CMdGuo adducts per 108 normal bases and 1.85 O6MedGuo adducts per 108 normal bases. Thus, the assays described in this paper have a sensitivity comparable to those of existing methods for O6-alkyldGuo and have the advantage of not requiring the use of radiolabeled compounds. When the O6-CMdGuo and O6-MedGuo immunoaffinity columns were combined in a sequential procedure, both adducts could be quantitated in the same DNA hydrolysate (Scheme 2). As there was no detectable
O6-CMG and O6-MeG from Nitrosated Glycines
Chem. Res. Toxicol., Vol. 12, No. 1, 1999 109
Figure 2. Calibration lines for the determination of the amount of O6-MeGua in calf thymus DNA by immunoaffinity purification followed by HPLC/fluorescence detection. Details are given in Materials and Methods. The upper line was obtained from standards of O6-MeGua injected directly onto the HPLC apparatus, and the lower line was obtained from standards that had gone through the entire immunoaffinity procedure. Table 1. Ratios of Levels of O6-CMdGuo to O6-MedGuo in Calf Thymus DNA following Treatment with APNG, AS, KDA, and MAA at pH 7.4 compound
O6-CMdG:O6-MedG
APNG KDA MAA AS
10a 16a 18b 38a
a The ratios were determined from the slopes of the doseresponse lines in Figure 3. b This ratio was obtained at a single concentration of MAA (5 mM).
Figure 1. RP-HPLC/fluorescence chromatograms of O6-MeGua (10 min, A-C) and O6-CMGua (10 min, D-F) in DNA hydrolysates following immunoaffinity purification. Traces obtained from reactions of 5 mM KDA [(A) O6-MeGua and (D) O6-CMGua from injections of 25 or 50 µg of DNA hydrolysate, respectively], 5 mM AS [(B) O6-MeGua and (E) O6-CMGua using 1 mg of DNA hydrolysate], and untreated DNA [1 mg of hydrolysate injected, (C) O6-MeGua and (F) O6-CMGua] are illustrated. The vertical axes correspond to the signal intensity of fluorescence emission at 378 nm (excitation at 286 nm) and are normalized to the largest peak in each trace. The elution conditions for traces A-C and D-F were different as described in Materials and Methods.
cross-reactivity of the O6-CMdGuo antibody with O6MedGuo, no retention of O6-MedGuo was observed in the first column (13). In principle, a simpler method of analysis is possible via acid hydrolysis of DNA followed by HPLC/fluorescence determination of the amount of O6alkyl guanines. However, while this method gave acceptable results with high levels of adducts in DNA treated in vitro (14), analysis of DNA with low levels of modification or from animal tissues was problematic, and immunoaffinity cleanup prior to HPLC/fluorescence analysis led to vastly superior results (13). The combined analytical method was used to quantitate the levels of O6MedGuo and O6-CMdGuo in calf thymus DNA treated in vitro with a range of nitrosated glycine derivatives as well as MAA. Aliquots of calf thymus DNA were treated with a range of concentrations of KDA (0.5-5 mM), APNG (0.5-5 mM), and AS (1-10 mM), and in all cases, dose-dependent increases in the levels of O6-CMdGuo and O6-MedGuo were seen (Figure 3A-C). DNA was treated with a single
Figure 3. Yields of O6-CMdGuo (b) and O6-MedGuo (9) in calf thymus DNA treated with (A) KDA (0-5 mM), (B) APNG (0-5 mM), and (C) azaserine (0-10 mM). Reaction conditions and the analytical procedures are described in Materials and Methods.
concentration of MAA and gave levels of O6-CMdGuo and O6-MedGuo of 11.45 ( 0.88 and 0.631 ( 0.010 µmol/mol of dG, respectively. The ratios of O6-CMdGuo to O6MedGuo were determined for each compound, and the results are summarized in Table 1. The results presented in this paper show that concomitant methylation and carboxymethylation at O6 of guanine in DNA appears to be a general property of N-carboxymethyl-N-nitroso compounds (APNG) as well as of diazoacetic acid derivatives (KDA and AS) (Figure 3). The formation of O6-MeGua does not appear to be an artifact of the analytical method. It seems that all the compounds studied undergo decarboxylation at some point during decomposition. However, the fact that the
110 Chem. Res. Toxicol., Vol. 12, No. 1, 1999
Harrison et al.
Scheme 3. Generation of Carboxymethylating and Methylating Agents from the Compounds Studieda
a As indicated in the text, it is not clear to what extent each of the illustrated intermediates contributes to the overall yields of O6MedGuo and O6-CMdGuo. Intermediates in square brackets are proposed structures only and have not been formally characterized.
different compounds yield quite different amounts of methyl and carboxymethyl adducts on reaction with DNA appears to rule out a single common intermediate such as the carboxymethyl diazonium ion (diazonium acetate) that gives rise to both products. The possibility exists that several intermediates that are not in rapid equilibrium, and that arise to varying degrees from the various precursors, may partition between the carboxymethylation and methylation reactions to different extents. This is summarized in Scheme 3. Indeed, there is evidence suggesting that under some conditions the diazoacetate and diazonium acetate might not be in equilibrium (23). The possibility that these and other intermediates related to Scheme 3 give rise to different extents of methylation and carboxymethylation in DNA is currently under investigation. Interestingly, a decarboxylation pathway is not unique to nitrosated glycine derivatives since MAA also gives rise to both O6-MedGuo and O6-CMdGuo adducts. MAA was studied as an example of a supposed SN2-type alkylating agent which, if anything, was expected to result only in the formation of low levels of O6CMdGuo. In the event, the relatively high levels of O6alkylation by 5 mM MAA (similar to that formed by 1 mM APNG) are consistent with its potent mutagenicity (29). There have been several indications that N-nitrosoN-carboxymethyl compounds can give rise to methylating agents, but this evidence has so far been indirect and was derived from mutagenicity studies of N-nitroso-Ncarboxymethylurea derivatives (30, 31). The finding that nitrosated glycine derivatives give rise to DNA methylation may be very significant in our understanding of the etiology of cancers of the gastrointestinal (GI) tract. A number of methylating agents have been used to induce experimental GI tract tumors, such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG)
and N-nitroso-N-methylurea (MNU) for gastric cancers (32) and 1,2-dimethylhydrazine (DMH) for colorectal cancer (33). All of these agents either spontaneously generate methylating agents or do so after metabolic activation and result in the formation of DNA methyl adducts in the target organs of treated animals (32, 34). In combination with this experimental data, there are a number of reports that O6-MedGuo levels are raised in human subjects with an elevated risk of GI tract tumors (15-19) which suggests that methylating agents may be involved in the etiology of these cancers. Furthermore, several studies have indicated that intragastric nitrosation (35) and intraintestinal nitrosation (36) of dietary precursors may be significant sources of alkylating agents. However, it is unlikely that nitroso compounds such as MNNG or MNU occur naturally in the human GI tract, and in fact, the endogenous nitrosation of dietary amino acids and peptides is a more likely reaction (37, 38), although the bile acid conjugates also constitute a large endogenous source of nitrosatable substrates (46, 39, 40). As glycine is one of the most abundant amino acids in nature, it would seem likely that its nitrosation products would constitute a major source of alkylating agents (41). Thus, our findings that nitrosated glycine derivatives, either N-nitrosopeptides or diazoacetic acid derivatives, decompose to give DNA-methylating agents lend considerable support to this hypothesis. Of additional interest is the finding that the major O6-guanine adduct of nitrosated glycine derivatives, O6-CMdGuo, is not repaired by O6-alkylguanine alkyl transferees (14) and is thus likely to accumulate in the DNA of GI tract 2 K. L. Harrison and D. E. G. Shuker, Detection of O6-carboxymethylguanine adducts in DNA from animal and human tissues by immunoslotblot assay (manuscript in preparation).
O6-CMG and O6-MeG from Nitrosated Glycines
tissues and be a promutagenic lesion. Using a very sensitive immunoslot blot assay, O6-CMdGuo has been detected in DNA extracted from human gastric biopsies and white blood cells,2 suggesting that formation of carboxymethylating agents does indeed occur in humans.
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Acknowledgment. This study was supported in part by a contract from the U.K. Ministry of Agriculture, Fisheries and Food (Contract FS1617). We gratefully acknowledge the critical comments of Professor James Fishbein (Wake Forest University, Winston-Salem, NC) on an earlier draft of the manuscript.
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