[CANCER RESEARCH 50. 6580-6584. October 15. 1990]
Combined High-Performance
yV7-Methyldeoxyguanosine
Liquid Chromatography/32P-Postlabeling
Assay of
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 Genetics
Laboratory, 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-methyl2'-deoxyguanosine (NTmethyldG) has been developed by combining
high-performance liquid Chromatograph), "P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by
high-performance liquid chromatography and then combining a portion
of 2'-deoxyguanosine to the N'TmethyldG allows for quantitation using
an internal standard. The directly determined molar ratio is not subject
to errors in digestion, variable ATP-specifìcactivity, or assumptions in
relative adduct-labeling efficiency. The detection limit was one
N7methyldG adduct in IO7 unmodified 2'-deoxyguanosine bases.
N7methyIdG adducts have been detected in 5 human lung samples in
which O6-methyl-2'-deoxyguanosine adducts had been previously deter
mined. The mean ratio of NTmethyldG to O6-methyl-2'-deoxyguanosine
was determined to be approximately 10. The current assay complements
the high-performance liquid chromatography/'"P-postlabeling assay for
O6-methyI-2'-deoxyguanosine and increases the detection sensitivity of
DNA methylated by exogenous alkylating agents.
INTRODUCTION
Molecular epidemiologists seek markers of carcinogen ex
posure and cancer risk in individuals or groups (1). Biological
markers to carcinogenic /V-nitrosamines in DNA would be
helpful in exposure assessments. Humans are frequently ex
posed to /V-nitrosamines through a variety of sources such as
tobacco use, diet, cancer chemotherapy, and occupation (2-4).
On a molecular level, alkylation of DNA may be promutagenic and associated with activation of protooncogenes (3, 59); O^methyldG1 is one such lesion that is also associated with
brain and liver cancer in laboratory animals (3, 5-9). This
adduct has been detected in humans using HPLC and 32Ppostlabeling (10) or immunoassay (11). O6methyldG is associ
ated with cancer of the esophagus in people from Lin-Xiang,
People's Republic of China, when compared with people in
France (11). O^methyldG has also been detected in 16 of 17
lung specimens of smokers and nonsmokers (12). The half-life
of O^methyldG is dependent upon the repair of this lesion, and
differences in half-lives vary among cells, tissues, and individ
uals (13, 14).
Methylation of NTmethyldG also occurs, but NTmethyldG
is not promutagenic. However, NTmethyldG occurs in higher
levels than O6methyldG and does not undergo significant enReceived4/9/90;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 in
accordance 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. Building
37. 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'-monophosphate; pdGp. 2'-deoxyguanosine _V.5'-bisphosphate: HPLC. high-perform
ance liquid chromatography;
NTmethyldG. A7-methyl-2'-deoxyguanosine;
N7methyldGp.
A7-methyl-2'-deoxyguanosine
3'-monophosphate;
pN7methyldGp, A7-methyl-2'-deoxyguanosine 3'.5'-bisphosphate; NMU, A'-nitrosoA'-mcthylurea; RO. ring-opened; TLC, thin-layer chromatography.
zymatic repair. It may serve, therefore, as a surrogate marker
for the promutagenic O^methyldG (and others) (15, 16). Nu
merous assays have been developed to detect N7methyldG
including mass spectroscopy (17), 12P-postlabeling and nucleotide chromatography (18), immunochemical detection (19),
HPLC/fluorescence (20), and HPLC/electrochemical detection
(21). However, only HPLC/fluorescence has been used on a
limited basis to detect NTmethyldG in human tissues because
of its low sensitivity (22).
12P-Postlabeling of DNA adducts, as originally described by
Randerath et al. (23), has become widely utilized because of its
sensitivity and can serve as a useful tool for detecting bulky
aromatic adducts. Workers have utilized HPLC with 32Ppostlabeling for adduct detection (24, 25), but this has not been
applied to DNA from humans or animal tissues exposed to
complex carcinogen mixtures. We have recently combined
HPLC and the 12P-postlabeling assay for detection and quan
titation of O6methyldG (10). The use of chemically synthesized
adducts, micropreparative techniques such as HPLC, and con
comitant labeling of dGp as an internal standard are important
elements that increased the assay's specificity and accuracy.
This report describes a method for combining HPLC with "Ppostlabeling of NTmethyldG that is equally specific and suffi
ciently sensitive for examining human tissues.
MATERIALS
AND METHODS
Chemicals. Nucleotide monophosphates, methyl iodide, and /V-nitroso-AAmethylurea were purchased from Sigma (St. Louis, MO). Radiolabeled [7-':P]ATP (>5000 Ci/mmol) and ['H]-A/-nitroso-A/-meth
ylurea (514 Ci/mmol) were obtained from Amersham (Arlington
Heights, IL). Calf thymus DNA and bisphosphate nucleotides (pdGp
and 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 purchased
from Boehringer Mannheim (Indianapolis, IN), and T4 polynucleotide
kinase, lacking 3'-phosphatase activity, was obtained from New Eng
land Nuclear (Boston, MA).
Preparation of N7Methyldeoxyguanosine Monophosphate and Bisphosphate. Synthetic standard N7methyldGp was purchased from Karkinos Biochemical (Phoenix. AZ) and then subsequently synthesized in
this 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 structure
was confirmed by both UV scan (neutral and alkaline pH) and TLC
analysis (silica and cellulose plates) following alkaline phosphatase or
acid treatment. The concentration of adduct was determined by UV
absorbance (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 (3.5
mg) was also treated with NMU (0-10 mg) in Tris buffer (66 mM, pH
8.0) 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
6580
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iV'-METHYLDEOXYGUANOSINE
performed using a Hewlett-Packard 1050 quaternary pump and Hew
lett-Packard 1040A Diode Array UV detector or 1050 variable wave
length detector. Nucleotides were separated using a Beckman Altex
Ultrasphere ion-pair column (50 ¿im;4.6 mm x 25 cm) fitted with an
Ultrasphere ion-pair guard column. Triethylamine acetate (0.1 M, pH
7.0; Applied Biosystems, Foster City, CA) and 1% acetonitrile were
mixed 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 was
monitored at 254 nm. Fractions (1 ml) were collected (Redirac 2112;
LKB Instruments, Inc., Gaithersburg, MD) and either assayed for
radioactivity in an LKB 1216 Rackbeta II liquid scintillation counter
or pooled and lyophilized for -"P-postlabeling.
HPLC/"P-Postlabeling of DNA (Fig. 1). DNA (10-100 Mg) was
digested to nucleoside 3'-monophosphates as described previously (26).
The entire digestion mixture was fractionated by HPLC and simulta
neously scanned by UV. Three 1-min collections corresponding to
fractions containing normal or adducted nucleotides were pooled and
lyophilized. Of the fractions containing dGp, 0.001 was added back to
the pooled fractions containing N7methyldGp. The mixture was dis
solved 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 (0.8
ill; 0.5 mM) and [7-"P]ATP (2 n\; >5000 Ci/mmol) were added in the
presence of T4-polynucleotide kinase (2 »1:10 units) resulting in a
transfer of the 7-phosphate group from ATP to the 5'-position of the
nucleotide. The mixture was incubated at 37°Cfor l h and then an
additional aliquot of T4 polynucleotide kinase (2 nI) was added. After
1 h, a portion (5-10 ^') of the mixture was spotted on 20 x 20 cm
polyethyleneimine cellulose plates (Merck. Germany). These plates
were prewashed for 5 min in methanol and prespotted with unlabeled
3',5'-pdGp and synthetic 3',5'-pN7methyldGp so that they could be
visualized by UV light (254 nm). The plates were developed in the first
dimension with 0.75 M lithium chloride and 5% butanol. After washing
for 15 min in methanol, the plates were developed in the second
dimension with saturated ammonium sulfate/isopropyl alcohol/1.0 M
sodium acetate (80/2/18, v/v). Normal nucleotides and adducts were
localized by autoradiography (10-30 min and 3 h, respectively) using
Kodak XAR5 film and MCI Optonix enhancer screens (Cedar Knolls,
NJ) at -70°C.Only a short exposure was required to localize the highlevel radioactivity associated with dGp and dCp, but a second and
longer exposure was necessary to localize the lower amount of
N7methyldGp adduct. Location of nucleotides was additionally con
firmed by coelution with the UV markers. Radiographically and UV
Genomic DNA
Micrococcal Nuclease
Calf-SpleenPhosphodiesterase
3' -Monophosphate Nucleotide Digest
HPLCSeparation
dGp
N7methyldGp
dilute 1/1000
1
T4 Polynucteotide Kinase
ATP
pdGp + pN7methyldGp
I
2DTLC
Autoradiography
Scintillation Counting
Fig. 1. HPLC/32P-postlabeling schema.
IN HUMAN LUNG
localized nucleotides were scraped with a razor, mixed with HC1 (1 ml;
0.1 M), and counted for radioactivity. A direct ratio of pN7methyldGp
to pdGp was determined.
Preparation and HPLC/J2P-Postlabeling of Human Lung Samples.
DNA was extracted from human lung samples obtained from fresh
autopsy samples of trauma victims with known occupational and smok
ing histories as reported previously (12). O'methyldG levels in these
samples were determined previously (12).
RESULTS
Known mixtures of standard 3'-monophosphate nucleotides
and 3'-monophosphate N7methyldG were separated by HPLC
and were 12P-postlabeled. Fig. 2 shows a representative autoradiograph for the chemically synthesized standard nucleotides.
Labeling efficiency was maximized by determining optimal
ATP/dGp ratio and reaction time (data not shown). A one-toone labeling efficiency for pN7methyldGp and pdGp over 3 log
ratios was identified (Fig. 3). The limit of detection for
N7methyldGp was approximately 1 in IO7 normal dGp using
100 fig of DNA (or 7 fmol in 70 nmol of dGp). It was noted
that the HPLC elution of N7methylguanine coincided with
N7methyldGp so that this compound can be conveniently used
as an UV marker for locating the monophosphate adduct (Fig.
4). Furthermore, the HPLC retention time for N7-methylguanosine-3'-monophosphate
is
10 min
slower
than
N7methyldGp so that RNA contamination does not falsely
elevate quantitative levels (data not shown).
HPLC/"P-postlabeling
assay was applied to methyl iodideand NMU-treated calf thymus DNA. A typical HPLC profile
and location of the N7methyldGp adduct is shown in Fig. 4.
A lesser peak (approximately
10% of the N7methyldGp
peak) resulting from NMU treatment occurs at 40-42 min,
corresponding to the O6-methyl-2'-deoxyguanosine
3'-monophosphate adduct. An earlier peak at 4 min, accounting for
3.4% of total radioactivity, has UV spectral characteristics of
RO-N7methyldG. The autoradiography following "P-postla
beling of NMU-treated DNA is shown in Fig. 2. Significant
contamination with dCp is shown to occur despite a 2-4-min
separation. However, the dCp did not interfere with detection
or quantitation. Fig. 5 shows a dose-response curve for calf
thymus DNA treated with NMU over 3 logs and for methyl
iodide-treated DNA over 2 logs. A wider variability and lower
slope were found at higher doses for NMU versus methyliodide. This assay was further validated, in addition to UV
quantitation, by assaying adduct levels in calf thymus DNA
treated with ['H]-NMU and liquid scintillation counting. Oneto-one labeling efficiency was again demonstrated (Fig. 5).
The utility of this assay for use in biological tissues is dem
onstrated by the detection of N7methyldGp in human lung
samples. Five samples were analyzed as part of a larger group
obtained from autopsy donors (12). Fig. 6 shows the HPLC
profile and autoradiographs for one sample. N7methyldGp was
detected in all 5 samples. Levels are indicated in Table 1 along
with the corresponding levels of O('-methyl-2'-deoxyguanosine
3'-monophosphate
as reported
previously
(12). The
N7methyldG adduct occurred in approximately 10-fold higher
levels than O6methyldG (range. 0.57-30). Age, occupational
history, and tobacco consumption
tories are unknown.
are presented. Dietary his
DISCUSSION
N7methyldGp can be measured by the HPLC/32P-postlabeling assay with sufficient sensitivity and specificity to be detected
6581
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/V7-METHYLDEOXYGUANOSINE
IN HUMAN LUNG
pN7mdGp
Fig. 2. Autoradiography following HPLC/
"P-postlabeling. In A, NT- methyldGp was
mixed with dGp (ratio = 1 adduci in IO4 dGp)
and assayed as described, fi, NMU-treated
DNA. Level of modification was determined
to be 1.6 x IO4. Other observed spots include
2 '-deoxycytosine-3 '.5'-bisphosphate (pdCp),
pdGp
which results from insufficient HPLC separa
tion of adducts from dCp and B spots resulting
from presumed labeled ATP products (also
seen in controls without the 3'-monophosphate nucleotide). Circles, visualized positions
of UV markers. First dimension (Ili) and sec
ond dimension (2D) are shown.
Origin
in human samples. The limit of detection is 1 adduci in IO7
dGp, which is equal to or better than other assays but utilizes
a smaller quantity of DNA (11, 18,19,21, 27-29). The accuracy
of quantitation was compared with UV spectroscopy and radiolabeled chemically synthesized standards. This method relies
upon an internal standard for quantitation, resulting in the
determination of a direct molar ratio, while eliminating some
of 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 pipeting of labeled products onto the TLC plate. Chemical specificity
of this assay lies in the use of 3 Chromatographie separations
and UV localization of adducts, which distinguishes it from
classical 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 with
environmental monitoring, may be confusing due to a nonspe
cific pattern of labeling and failure to adequately characterize
the labeled product (30-33). Thus, the use of chemically syn1.0
r 0970
0.1
•DÇ
ffl
II
0.01
Origin
100
400
_
300
50 o
200
100
20
30
Fig. 4. HPLC elulion profiles of methyl iodide-treated DNA. The DNA was
spiked with ¿V7-methylguanineto serve as a marker for the adduci (2). Radioac
tivity profile for DNA treated with ['H]NMU
resulted in a major peak (/)
coeluting with the NVniethyldGp and .W-methylguanine. a lesser peak at 40 min
coeluting with O*methyldGp standard, and an earlier peak at 4 min that is
indicative of ring-opened N7methyldGp.
thesized standards, micropreparative techniques such as HPLC,
and direct quantitation of adducts to normal nucleotides should
improve upon previous adduct assessments in complex human
samples, facilitate interlaboratory comparisons, and validate
different types of assays.
Levels of N7methyldGp were detected in all 5 human lung
samples at approximately one adduct in IO6 or IO7 2'-deoxy-
0.001
guanosine residues. Herrón and Shank (22) previously reported
a 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 of
[
1.0E-4
1.0E-6
1.0E-5
1 .OE-7
4.5. Based upon ':P-postlabeling for both N7methyldG and
N7methyldGp/dGp
Ratio
O6methyldG, we have identified a ratio of approximately 10
Known
with a range of 0.57 to 30 (Table 1). Different /V-nitroso
Fig. 3. Determination of pN7methyldGp and pdGp levels in a standard
mixture by HPLC/"P-postlabeling.
Experimentally determined ratio (ordinate)
compounds can produce ratios ranging from 9 to 250 (4). The
is correlated with the known adduct-nucleotide ratio at levels as low as 1 adduct
in IO7 dGp. Determined ratios differ from known ratios by a factor of 0.001 due
wide range in this pilot study likely reflects interindividual
to the dilution of dGp. Points, means of 4-6 determinations; bars. ±2 SD.
variability in exposure and repair (14). The number of samples
6582
0.0001
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
/V'-METHYLDEOXYGUANOSINE
a
1.0E-2
a-
LOE'3
IN HUMAN LUNG
I
a.
sg
Methyl Iodide
1.OE'5
NMU
LOE-«
.01
.001
.10
1.0
mmole Ilog I
1.0EJ
uuuu
LOE'5
J_
5
10
15
20
2530354046
C
1.0E-6
o
1.0E-7
LOE''
LOE'6
N7methyldGp/dGp
1.0E5
-
LOE-1
Tritium (log)
pN7methyldGp
pN7methyldGp
Fig. 5. Dose-response curves for DNA treated with methyl iodide or NMU
treatment (A). B, [3H]NMU. Levels of modification were determined by measure
ment of 3H-specific activity or 32P-postlabeling. Points, means of 3-4 determina
tions ±2 SD.
pdC
analyzed is too small to draw any conclusions regarding adduct
levels and associations with tobacco consumption, age or oc
cupational 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 involve
either direct quantitation or HPLC. Thus, application of their
method to human samples with complex exposures would be
limited. A method for HPLC/32P-postlabeling of alkali-treated
DNA, in vitro, that results in RO-N7methyldG has recently
been reported, but this technique did not utilize 2-dimensional
TLC or an internal standard, and reported a 10-fold lower
labeling efficiency (34, 35).
The N7methyldG adduct can spontaneously depurinate or
undergo 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 samples
can occur. However, this was not a problem in the samples
analyzed here as demonstrated through the use of radiolabeled
material. The fortuitous HPLC coelution of the free base and
intact N7methyldGp showed that '2P-postlabeling quantita
tively accounted for all of the tritium in those fractions. The
RO-N7methyldGp élûtes
faster than the N7methyldGp as iden
tified by UV spectral determinations of alkali-treated DNA
(data not shown). Using tritiated NMU without alkali shows
that the respective peak accounts for less than 3.4% of radio
activity compared with N7methyldGp, suggesting that loss at
neutral 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 chem
ically specific and sufficiently sensitive to be used in human
tissues. This assay, in conjunction with detection of O6-alkyl2'-deoxyguanosine by a similar assay, should enhance exposure
Exp. = 3°
Exp. = 10'
Origin
i Origin
Fig. 6. Representative analysis of human lung DNA. A, HPLC elution profile
without AT-methylguanine marker. The position of NTmethyldGp is shown.
Bottom, autoradiogram of the 2-dimensional TLC separation of the 32P-postlabeled mixtures after separation by HPLC and combining fractions containing
NTmethyldGp and 0.001 of dGp as described in the text. Autoradiography was
for 10 min to localize pdGp and 2'-deoxycytosine-3',5'-bisphosphate
(B). Autoradiography 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
dGpfJ11147I24145
dGpc-'3372
19144137
±
handlerAccountantSalespersonO'methyl3N7methyl- ±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).
REFERENCES
assessments for alkylating agents and provide new information
regarding DNA repair.
ACKNOWLEDGMENTS
The authors wish to thank Dr. A. Dipple for his careful review of
this manuscript. We also thank Bob Julia for his skillful editorial
assistance in the preparation in this manuscript.
6583
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
Combined High-Performance Liquid Chromatography/32
P-Postlabeling Assay of N7-Methyldeoxyguanosine
P. G. Shields, A. C. Povey, V. L. Wilson, et al.
Cancer Res 1990;50:6580-6584.
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