Combined High-Performance Liquid Chromatography/32P
Transcript of Combined High-Performance Liquid Chromatography/32P
[CANCER RESEARCH 50. 6580-6584. October 15. 1990]
Combined High-Performance Liquid Chromatography/32P-Postlabeling Assay of
yV7-Methyldeoxyguanosine
P. G. Shields, A. C. Povey,1 V. L. Wilson, A. Weston, and C. C. Harris2
Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892 ¡P.G. S., A. C. P., A. W., C. C H.], and Molecular GeneticsLaboratory, The Children's Hospital, Denver, Colorado 80218-1088 [V. L. H'.J
ABSTRACT
A highly sensitive and specific assay for the detection of A/7-methyl-2'-deoxyguanosine (NTmethyldG) has been developed by combininghigh-performance liquid Chromatograph), "P-postlabeling, and nucleo-
tide chromatography. Separation of normal nucleotides and adducts byhigh-performance liquid chromatography and then combining a portionof 2'-deoxyguanosine to the N'TmethyldG allows for quantitation using
an internal standard. The directly determined molar ratio is not subjectto errors in digestion, variable ATP-specifìcactivity, or assumptions inrelative adduct-labeling efficiency. The detection limit was oneN7methyldG adduct in IO7 unmodified 2'-deoxyguanosine bases.
N7methyIdG adducts have been detected in 5 human lung samples inwhich O6-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of NTmethyldG to O6-methyl-2'-deoxyguanosine
was determined to be approximately 10. The current assay complementsthe high-performance liquid chromatography/'"P-postlabeling assay forO6-methyI-2'-deoxyguanosine and increases the detection sensitivity of
DNA methylated by exogenous alkylating agents.
INTRODUCTION
Molecular epidemiologists seek markers of carcinogen exposure and cancer risk in individuals or groups (1). Biologicalmarkers to carcinogenic /V-nitrosamines in DNA would behelpful in exposure assessments. Humans are frequently exposed to /V-nitrosamines through a variety of sources such astobacco use, diet, cancer chemotherapy, and occupation (2-4).
On a molecular level, alkylation of DNA may be promuta-genic and associated with activation of protooncogenes (3, 5-9); O^methyldG1 is one such lesion that is also associated withbrain and liver cancer in laboratory animals (3, 5-9). Thisadduct has been detected in humans using HPLC and 32P-postlabeling (10) or immunoassay (11). O6methyldG is associated with cancer of the esophagus in people from Lin-Xiang,People's Republic of China, when compared with people inFrance (11). O^methyldG has also been detected in 16 of 17lung specimens of smokers and nonsmokers (12). The half-lifeof O^methyldG is dependent upon the repair of this lesion, anddifferences in half-lives vary among cells, tissues, and individuals (13, 14).
Methylation of NTmethyldG also occurs, but NTmethyldGis not promutagenic. However, NTmethyldG occurs in higherlevels than O6methyldG and does not undergo significant en-
Received4/9/90;accepted7/17/90.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Present address: Carcinogenesis Department. Christie Hospital and Holt
Radium Institute, Manchester. M20 9B.X. England.! To whom requests for reprints should be addressed, at Laboratory of Human
Carcinogenesis. Division of Cancer Etiology. National Cancer Institute. Building37. Room 2C05. 9000 Rockville Pike. Bethesda. M D 20892.
3The abbreviations used are: O6methyldG, O*-methyl-2'-deoxyguanosine:dCp. 2'-deoxycytosine 3'-monophosphate; dGp. 2'-deoxyguanosine 3'-mono-phosphate; pdGp. 2'-deoxyguanosine _V.5'-bisphosphate: HPLC. high-performance liquid chromatography; NTmethyldG. A7-methyl-2'-deoxyguanosine;N7methyldGp. A7-methyl-2'-deoxyguanosine 3'-monophosphate; pN7-methyldGp, A7-methyl-2'-deoxyguanosine 3'.5'-bisphosphate; NMU, A'-nitroso-A'-mcthylurea; RO. ring-opened; TLC, thin-layer chromatography.
zymatic repair. It may serve, therefore, as a surrogate markerfor the promutagenic O^methyldG (and others) (15, 16). Nu
merous assays have been developed to detect N7methyldGincluding mass spectroscopy (17), 12P-postlabeling and nucleo-
tide chromatography (18), immunochemical detection (19),HPLC/fluorescence (20), and HPLC/electrochemical detection(21). However, only HPLC/fluorescence has been used on alimited basis to detect NTmethyldG in human tissues becauseof its low sensitivity (22).
12P-Postlabeling of DNA adducts, as originally described by
Randerath et al. (23), has become widely utilized because of itssensitivity and can serve as a useful tool for detecting bulkyaromatic adducts. Workers have utilized HPLC with 32P-
postlabeling for adduct detection (24, 25), but this has not beenapplied to DNA from humans or animal tissues exposed tocomplex carcinogen mixtures. We have recently combinedHPLC and the 12P-postlabeling assay for detection and quantitation of O6methyldG (10). The use of chemically synthesized
adducts, micropreparative techniques such as HPLC, and concomitant labeling of dGp as an internal standard are importantelements that increased the assay's specificity and accuracy.This report describes a method for combining HPLC with "P-
postlabeling of NTmethyldG that is equally specific and sufficiently sensitive for examining human tissues.
MATERIALS AND METHODS
Chemicals. Nucleotide monophosphates, methyl iodide, and /V-ni-troso-AAmethylurea were purchased from Sigma (St. Louis, MO). Ra-diolabeled [7-':P]ATP (>5000 Ci/mmol) and ['H]-A/-nitroso-A/-meth
ylurea (514 Ci/mmol) were obtained from Amersham (ArlingtonHeights, IL). Calf thymus DNA and bisphosphate nucleotides (pdGpand 2'-deoxycytosine 3',5'-bisphosphate) were purchased from Phar
macia (Piscataway, NJ).Enzymes. Micrococcal nuclease and nuclease P, were obtained from
Sigma (St. Louis, MO), calf spleen phosphodiesterase was purchasedfrom Boehringer Mannheim (Indianapolis, IN), and T4 polynucleotidekinase, lacking 3'-phosphatase activity, was obtained from New Eng
land Nuclear (Boston, MA).Preparation of N7Methyldeoxyguanosine Monophosphate and Bis-
phosphate. Synthetic standard N7methyldGp was purchased from Kar-kinos Biochemical (Phoenix. AZ) and then subsequently synthesized inthis laboratory by treatment of 2'-deoxyguanosine mono- or bisphos
phate (17 mg) with methyl iodide (20 p\) in dimethyl sulfoxide (4 ml).The reaction mixture was stirred at 20-24°Covernight and then puri
fied by ion-pair reverse-phase HPLC as described below. The structurewas confirmed by both UV scan (neutral and alkaline pH) and TLCanalysis (silica and cellulose plates) following alkaline phosphatase oracid treatment. The concentration of adduct was determined by UVabsorbance (257 nm; <= 8500).
Methylation of DNA. Calf thymus DNA (2 mg) in dimethyl sulfoxide(3 ml) was treated in vitro with methyl iodide (0-20 ^1) for 4 h at 22°C
and precipitated with 3 volumes of chloroform. Calf thymus DNA (3.5mg) was also treated with NMU (0-10 mg) in Tris buffer (66 mM, pH8.0) for l h at 37°Cand precipitated with 2 volumes of ethanol and
NaCl (5 M; 1/50, v/v).HPLC Separation of Normal and Methylated Nucleotides. HPLC was
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iV'-METHYLDEOXYGUANOSINE IN HUMAN LUNG
performed using a Hewlett-Packard 1050 quaternary pump and Hewlett-Packard 1040A Diode Array UV detector or 1050 variable wavelength detector. Nucleotides were separated using a Beckman AltexUltrasphere ion-pair column (50 ¿im;4.6 mm x 25 cm) fitted with anUltrasphere ion-pair guard column. Triethylamine acetate (0.1 M, pH7.0; Applied Biosystems, Foster City, CA) and 1% acetonitrile weremixed isocratically for 20 min. The acetonitrile was increased to 5%for 10 min, held isocratically for 10 min, and then increased to 10%over the next 5 min. Flow rate was 1 ml/min and UV absorbance wasmonitored at 254 nm. Fractions (1 ml) were collected (Redirac 2112;LKB Instruments, Inc., Gaithersburg, MD) and either assayed forradioactivity in an LKB 1216 Rackbeta II liquid scintillation counteror pooled and lyophilized for -"P-postlabeling.
HPLC/"P-Postlabeling of DNA (Fig. 1). DNA (10-100 Mg) wasdigested to nucleoside 3'-monophosphates as described previously (26).
The entire digestion mixture was fractionated by HPLC and simultaneously scanned by UV. Three 1-min collections corresponding tofractions containing normal or adducted nucleotides were pooled andlyophilized. Of the fractions containing dGp, 0.001 was added back tothe pooled fractions containing N7methyldGp. The mixture was dissolved in H2O (5 M'), dithiothreitol (1 jtl; 0.1 M), and buffer [1.2 /a(bicine, 0.1 M, spermidine. 10 HIM,MgCl2, 0.1 M), pH 7.0). ATP (0.8ill; 0.5 mM) and [7-"P]ATP (2 n\; >5000 Ci/mmol) were added in thepresence of T4-polynucleotide kinase (2 »1:10 units) resulting in atransfer of the 7-phosphate group from ATP to the 5'-position of thenucleotide. The mixture was incubated at 37°Cfor l h and then an
additional aliquot of T4 polynucleotide kinase (2 nI) was added. After1 h, a portion (5-10 ^') of the mixture was spotted on 20 x 20 cmpolyethyleneimine cellulose plates (Merck. Germany). These plateswere prewashed for 5 min in methanol and prespotted with unlabeled3',5'-pdGp and synthetic 3',5'-pN7methyldGp so that they could be
visualized by UV light (254 nm). The plates were developed in the firstdimension with 0.75 M lithium chloride and 5% butanol. After washingfor 15 min in methanol, the plates were developed in the seconddimension with saturated ammonium sulfate/isopropyl alcohol/1.0 Msodium acetate (80/2/18, v/v). Normal nucleotides and adducts werelocalized by autoradiography (10-30 min and 3 h, respectively) usingKodak XAR5 film and MCI Optonix enhancer screens (Cedar Knolls,NJ) at -70°C.Only a short exposure was required to localize the high-
level radioactivity associated with dGp and dCp, but a second andlonger exposure was necessary to localize the lower amount ofN7methyldGp adduct. Location of nucleotides was additionally confirmed by coelution with the UV markers. Radiographically and UV
Genomic DNA
Micrococcal NucleaseCalf-SpleenPhosphodiesterase
3' -Monophosphate Nucleotide Digest
HPLCSeparation
dGp
dilute 1/1000
N7methyldGp
1T4 Polynucteotide KinaseATP
pdGp + pN7methyldGp
I2DTLC
AutoradiographyScintillation Counting
Fig. 1. HPLC/32P-postlabeling schema.
localized nucleotides were scraped with a razor, mixed with HC1 (1 ml;0.1 M), and counted for radioactivity. A direct ratio of pN7methyldGpto pdGp was determined.
Preparation and HPLC/J2P-Postlabeling of Human Lung Samples.
DNA was extracted from human lung samples obtained from freshautopsy samples of trauma victims with known occupational and smoking histories as reported previously (12). O'methyldG levels in these
samples were determined previously (12).
RESULTSKnown mixtures of standard 3'-monophosphate nucleotides
and 3'-monophosphate N7methyldG were separated by HPLCand were 12P-postlabeled. Fig. 2 shows a representative auto-
radiograph for the chemically synthesized standard nucleotides.Labeling efficiency was maximized by determining optimalATP/dGp ratio and reaction time (data not shown). A one-to-one labeling efficiency for pN7methyldGp and pdGp over 3 logratios was identified (Fig. 3). The limit of detection forN7methyldGp was approximately 1 in IO7 normal dGp using
100 fig of DNA (or 7 fmol in 70 nmol of dGp). It was notedthat the HPLC elution of N7methylguanine coincided withN7methyldGp so that this compound can be conveniently usedas an UV marker for locating the monophosphate adduct (Fig.4). Furthermore, the HPLC retention time for N7-methylgu-anosine-3'-monophosphate is 10 min slower than
N7methyldGp so that RNA contamination does not falselyelevate quantitative levels (data not shown).
HPLC/"P-postlabeling assay was applied to methyl iodide-and NMU-treated calf thymus DNA. A typical HPLC profileand location of the N7methyldGp adduct is shown in Fig. 4.A lesser peak (approximately 10% of the N7methyldGppeak) resulting from NMU treatment occurs at 40-42 min,corresponding to the O6-methyl-2'-deoxyguanosine 3'-mono-
phosphate adduct. An earlier peak at 4 min, accounting for3.4% of total radioactivity, has UV spectral characteristics ofRO-N7methyldG. The autoradiography following "P-postlabeling of NMU-treated DNA is shown in Fig. 2. Significantcontamination with dCp is shown to occur despite a 2-4-minseparation. However, the dCp did not interfere with detectionor quantitation. Fig. 5 shows a dose-response curve for calfthymus DNA treated with NMU over 3 logs and for methyliodide-treated DNA over 2 logs. A wider variability and lowerslope were found at higher doses for NMU versus methyl-iodide. This assay was further validated, in addition to UVquantitation, by assaying adduct levels in calf thymus DNAtreated with ['H]-NMU and liquid scintillation counting. One-to-one labeling efficiency was again demonstrated (Fig. 5).
The utility of this assay for use in biological tissues is demonstrated by the detection of N7methyldGp in human lungsamples. Five samples were analyzed as part of a larger groupobtained from autopsy donors (12). Fig. 6 shows the HPLCprofile and autoradiographs for one sample. N7methyldGp wasdetected in all 5 samples. Levels are indicated in Table 1 alongwith the corresponding levels of O('-methyl-2'-deoxyguanosine3'-monophosphate as reported previously (12). The
N7methyldG adduct occurred in approximately 10-fold higherlevels than O6methyldG (range. 0.57-30). Age, occupational
history, and tobacco consumption are presented. Dietary histories are unknown.
DISCUSSIONN7methyldGp can be measured by the HPLC/32P-postlabel-
ing assay with sufficient sensitivity and specificity to be detected6581
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/V7-METHYLDEOXYGUANOSINE IN HUMAN LUNG
Fig. 2. Autoradiography following HPLC/"P-postlabeling. In A, NT- methyldGp wasmixed with dGp (ratio = 1 adduci in IO4 dGp)and assayed as described, fi, NMU-treatedDNA. Level of modification was determinedto be 1.6 x IO4. Other observed spots include2 '-deoxycytosine-3 '.5'-bisphosphate (pdCp),
which results from insufficient HPLC separation of adducts from dCp and B spots resultingfrom presumed labeled ATP products (alsoseen in controls without the 3'-monophos-
phate nucleotide). Circles, visualized positionsof UV markers. First dimension (Ili) and second dimension (2D) are shown.
pN7mdGp
pdGp
Origin Origin
in human samples. The limit of detection is 1 adduci in IO7
dGp, which is equal to or better than other assays but utilizesa smaller quantity of DNA (11, 18,19,21, 27-29). The accuracyof quantitation was compared with UV spectroscopy and radi-olabeled chemically synthesized standards. This method reliesupon an internal standard for quantitation, resulting in thedetermination of a direct molar ratio, while eliminating someof the potential variables in classic "P-postlabeling methods,
e.g., enzymatic digestion, DNA quantitation, kinase activity(lot-to-lot or adduct-specific), ATP specific activity, and pipet-ing of labeled products onto the TLC plate. Chemical specificityof this assay lies in the use of 3 Chromatographie separationsand UV localization of adducts, which distinguishes it fromclassical postlabeling as developed by Randerath et al. (23).Comparisons and correlations of the classical i:P-postlabeling
assay to other methods, for example, immunoassays or withenvironmental monitoring, may be confusing due to a nonspecific pattern of labeling and failure to adequately characterizethe labeled product (30-33). Thus, the use of chemically syn-
ffl•DÇ
II
1.0
0.1
0.01
0.001
0.0001
r 0970
[1 .OE-7 1.0E-6 1.0E-5
N7methyldGp/dGp Ratio
1.0E-4
Known
Fig. 3. Determination of pN7methyldGp and pdGp levels in a standardmixture by HPLC/"P-postlabeling. Experimentally determined ratio (ordinate)is correlated with the known adduct-nucleotide ratio at levels as low as 1 adductin IO7 dGp. Determined ratios differ from known ratios by a factor of 0.001 dueto the dilution of dGp. Points, means of 4-6 determinations; bars. ±2 SD.
400
_ 300
200
100
100
50 o
20 30
Fig. 4. HPLC elulion profiles of methyl iodide-treated DNA. The DNA wasspiked with ¿V7-methylguanineto serve as a marker for the adduci (2). Radioactivity profile for DNA treated with ['H]NMU resulted in a major peak (/)coeluting with the NVniethyldGp and .W-methylguanine. a lesser peak at 40 mincoeluting with O*methyldGp standard, and an earlier peak at 4 min that isindicative of ring-opened N7methyldGp.
thesized standards, micropreparative techniques such as HPLC,and direct quantitation of adducts to normal nucleotides shouldimprove upon previous adduct assessments in complex humansamples, facilitate interlaboratory comparisons, and validatedifferent types of assays.
Levels of N7methyldGp were detected in all 5 human lungsamples at approximately one adduct in IO6 or IO7 2'-deoxy-
guanosine residues. Herrón and Shank (22) previously reporteda level of one adduct in 10' 2'-deoxyguanosine residues in the
liver of an individual poisoned with /V-nitrosodimethylamine.They also calculated a ratio of N7methyldG to O6methyldG of4.5. Based upon ':P-postlabeling for both N7methyldG andO6methyldG, we have identified a ratio of approximately 10with a range of 0.57 to 30 (Table 1). Different /V-nitrosocompounds can produce ratios ranging from 9 to 250 (4). Thewide range in this pilot study likely reflects interindividualvariability in exposure and repair (14). The number of samples
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/V'-METHYLDEOXYGUANOSINE IN HUMAN LUNG
a 1.0E-2
a- LOE'3
1.OE'5
LOE-«
Methyl Iodide
NMU
.001 .01 .10
mmole Ilog I1.0
1.0EJ
LOE'5
1.0E-6
1.0E-7LOE'' LOE'6 1.0E5
N7methyldGp/dGp - Tritium (log)
LOE-1
Fig. 5. Dose-response curves for DNA treated with methyl iodide or NMUtreatment (A). B, [3H]NMU. Levels of modification were determined by measurement of 3H-specific activity or 32P-postlabeling. Points, means of 3-4 determina
tions ±2 SD.
analyzed is too small to draw any conclusions regarding adductlevels and associations with tobacco consumption, age or occupational history. Dietary habits are unknown in this group.
32P-Postlabeling of NTmethyldG has been previously re
ported by Reddy et al. (18), but this method did not involveeither direct quantitation or HPLC. Thus, application of theirmethod to human samples with complex exposures would belimited. A method for HPLC/32P-postlabeling of alkali-treatedDNA, in vitro, that results in RO-N7methyldG has recentlybeen reported, but this technique did not utilize 2-dimensionalTLC or an internal standard, and reported a 10-fold lowerlabeling efficiency (34, 35).
The N7methyldG adduct can spontaneously depurinate orundergo ring fission due to an unstable quaternary structure(36, 37). The HPLC/32P-postlabeling method would not be able
to identify these products, and so loss from biological samplescan occur. However, this was not a problem in the samplesanalyzed here as demonstrated through the use of radiolabeledmaterial. The fortuitous HPLC coelution of the free base andintact N7methyldGp showed that '2P-postlabeling quantita
tively accounted for all of the tritium in those fractions. TheRO-N7methyldGp élûtesfaster than the N7methyldGp as identified by UV spectral determinations of alkali-treated DNA(data not shown). Using tritiated NMU without alkali showsthat the respective peak accounts for less than 3.4% of radioactivity compared with N7methyldGp, suggesting that loss atneutral pH due to ring fission is not significant.
In conclusion, a combined HPLC/32P-postlabeling assay for
the detection of N7methyldG has been developed that is chemically specific and sufficiently sensitive to be used in humantissues. This assay, in conjunction with detection of O6-alkyl-2'-deoxyguanosine by a similar assay, should enhance exposure
assessments for alkylating agents and provide new informationregarding DNA repair.
ACKNOWLEDGMENTS
The authors wish to thank Dr. A. Dipple for his careful review ofthis manuscript. We also thank Bob Julia for his skillful editorialassistance in the preparation in this manuscript.
I
a.sg
uuuuJ_
5 10 15 20 2530354046
C
opN7methyldGp
Exp. = 10'
Origin
pN7methyldGp
pdC
Exp. = 3°
i Origin
Fig. 6. Representative analysis of human lung DNA. A, HPLC elution profilewithout AT-methylguanine marker. The position of NTmethyldGp is shown.Bottom, autoradiogram of the 2-dimensional TLC separation of the 32P-postla-
beled mixtures after separation by HPLC and combining fractions containingNTmethyldGp and 0.001 of dGp as described in the text. Autoradiography wasfor 10 min to localize pdGp and 2'-deoxycytosine-3',5'-bisphosphate (B). Auto-radiography for 3 h at —70'Cwith intensifying screens to localize NVmethyldGp;2'-deoxycytosine 5'-monophosphate pdC is also seen (C). Circles, visualized
position of UV markers.
Table 1 Alkyl adduci levels in human lung
Age(yr)7157613935GenderMMMFFRace"BWBWWTC*304030830OccupationCookPainterStock
handlerAccountantSalespersonO'methyl-
dGpfJ11147I24145
3N7methyl-
dGpc-'3372
±19144137
±8°B, black, W, white.* TC, tobacco consumption expressed as pack-years (number of packs/day
smoked x years smoking).c Adduct levels per IO7dGp.d O'methyldGp values reported previously (12).' Levels represent 1-2 determinations (limited availability of DNA).
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1990;50:6580-6584. Cancer Res P. G. Shields, A. C. Povey, V. L. Wilson, et al.
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