Preparation of a methylated DNA standard, and its stability on storage

Article abstract of DOI:10.1021/tx990182e

Several methods are available for the determination of low levels of O⁶-methylguanine in DNA; this base arises after methylation by environmental carcinogens. The reliability of these assays is much improved by the use of a standard. We have prepared such a standard by treating calf thymus DNA with [³H-methyl]-N-methyl-N-nitrosourea. We characterized the methylated bases by hydrolysis of a sample to nucleosides, followed by liquid chromatography and liquid scintillation counting of the tritium content. The level of O⁶- methylguanine was 0.6 per one million nucleotides. This base is stable, and its level was unchanged after storage for 5 years at -20 °C. The methylated DNA also contained 7-methylguanine and 3-methyladenine, which are slowly cleaved from the DNA on standing. The half-life for loss of 7-methylguanine at neutral pH was estimated to be 70 h at 39 °C, 460 h at 22 °C, 3800 h at 10 °C, and about 4 years at -20 °C.

Full text of DOI:10.1021/tx990182e

Chem. Res. Toxicol. 2000, 13, 257-261
257
P r ep a r a tion of a Meth yla ted DNA Sta n d a r d , a n d Its
Sta bility on Stor a ge
Martin R. Osborne* and David H. Phillips
Molecular Carcinogenesis Section, Institute of Cancer Research, Haddow Laboratories,
Cotswold Road, Sutton, Surrey SM2 5NG, U.K.
Received October 28, 1999
Several methods are available for the determination of low levels of O6-methylguanine in
DNA; this base arises after methylation by environmental carcinogens. The reliability of these
assays is much improved by the use of a standard. We have prepared such a standard by
treating calf thymus DNA with [3H-methyl]-N-methyl-N-nitrosourea. We characterized the
methylated bases by hydrolysis of a sample to nucleosides, followed by liquid chromatography
and liquid scintillation counting of the tritium content. The level of O6-methylguanine was 0.6
per one million nucleotides. This base is stable, and its level was unchanged after storage for
5 years at -20 °C. The methylated DNA also contained 7-methylguanine and 3-methyladenine,
which are slowly cleaved from the DNA on standing. The half-life for loss of 7-methylguanine
at neutral pH was estimated to be 70 h at 39 °C, 460 h at 22 °C, 3800 h at 10 °C, and about
4 years at -20 °C.
methyl-6-phenylimidazo[4,5-b]pyridine, PhIP), and by a
methylating agent (N-methyl-N-nitrosourea). The prepa-
ration and characterization of the 4-aminobiphenyl stan-
dard have been reported (9). Here we report the prepa-
ration, characterization, and stability of the methylated
DNA standard.
In tr od u ction
With the advent of several sensitive methods for their
determination, carcinogen-DNA adducts have become
useful biomarkers of human exposure to genotoxic agents
(1). These can be analyzed by immunological methods (for
example, enzyme-linked immunosorbent assay, ELISA),
biochemical methods (for example, 32P-postlabeling), and
physicochemical methods (for example, fluorescence spec-
troscopy and mass spectrometry) (1-4). However, tech-
niques vary widely between laboratories, and there have
been relatively few attempts to conduct interlaboratory
comparisons to verify results (5-7).
Exp er im en ta l P r oced u r es
P r ep a r a tion of Meth yla ted DNA. Calf thymus DNA, which
had been purified by treating an aqueous solution with RNase
and proteinase K (10), was supplied by F. Kadlubar (National
Center for Toxicological Research, J effersonville, AR). Its purity
was checked by its ultraviolet absorbance ratios: A230/A260
)
What the field has lacked, and needs, is standards.
Without these, it can be difficult to determine the
efficiency with which adducts are detected by a particular
analytical method in a particular laboratory. This has
also made comparisons between results obtained in
different laboratories difficult and uncertain. For DNA
adduct measurements to be able to be applied to safety
assessment and possibly for regulatory purposes, it is
essential that research-based methods become more
standardized and subjected to the rigors of quality
assurance. To this end, the availability of validated
standards can be of significant assistance. For a large
interlaboratory trial of postlabeling methods, a number
of such standards were prepared and analyzed in the
participating laboratories and initially used to assist in
the process of developing standardized protocols (8). In
a subsequent stage of the trials, the standards were used
as positive controls for the analysis of unknown samples,
and for the normalized quantitation of adduct levels in
the unknowns. The four standards that were prepared
consisted of DNA modified by a polycyclic aromatic
hydrocarbon (benzo[a]pyrene), by an aromatic amine (4-
aminobiphenyl), by a heterocyclic amine (2-amino-1-
0.42 and A260/A280 ) 1.87. 3- and 7-methyladenine and -guanine
were from Sigma-Aldrich (Poole, U.K.); O6-methyldeoxygua-
nosine was provided by P. D. Lawley. [3H-methyl]-N-Methyl-N-
nitrosourea (1.5 Ci/mmol) was supplied by NEN-Du Pont
(Stevenage, U.K.). Ca u tion : This substance is probably a
human carcinogen, and should be handled with due care.
To 1.4 µg (20 µCi) of tritiated methylnitrosourea was added
80 µg of unlabeled methylnitrosourea in 20 mL of ethanol. This
was added to 200 mg of DNA dissolved in 100 mL of 0.08 M
sodium acetate (pH 6), and the mixture was kept at 37 °C for
1.5 h. A 50 µL sample of the mixture gave 7572 tritium counts/
min, a counting efficiency of 34%. The DNA was precipitated
with 250 mL of ethanol containing 3% ammonium acetate,
washed, dried, and redissolved in 60 mL of water. Ten milliliters
of 14% sodium acetate and 180 mL of ethanol were added to
reprecipitate the DNA; it was washed twice with ethanol and
twice with ether and dried in air to yield 160 mg of methylated
DNA. This sample contained about 20 methyl groups per one
million bases; DNA with different levels of modification was
obtained by varying the amount of unlabeled methylnitrosourea
that was added, or occasionally by adding an unmodified DNA
solution before the final precipitation. The modification level
was determined as described below (Analysis of DNA).
Most of the methylated DNA was stored at -80 °C as the
solid or as a frozen solution (samples of 1 mg in 1 mL) in 5 mM
BisTris + 0.1 mM EDTA buffer, pH 7. Other 1 mL samples were
stored at room temperature, at 10 °C, -20 °C or under liquid
nitrogen, to determine the long-term stability at various tem-
* To whom correspondence should be addressed. E-mail: martino@
icr.ac.uk and davidp@icr.ac.uk.
10.1021/tx990182e CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/18/2000

258 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Osborne and Phillips
peratures. Samples were taken by weight or by volume of
solution for analysis as described below.
Loss of Meth ylp u r in es. Methylated DNA (20 mg) was
prepared as described above, containing one methyl group per
2000 bases, with a tritium activity of 1.4 µCi/g of DNA. It was
dissolved in aqueous buffer solution [50 mM sodium phosphate
(pH 7) and 4 mM sodium azide], and 50 µL each of 7-meth-
ylguanine and 3-methyladenine solutions (1 mg/mL) added as
markers. The mixture was incubated at 10, 22, or 39 °C.
Samples (0.5 mL) were taken, and DNA was precipitated by
adding 1.3 mL of ethanol containing 3% ammonium acetate. The
DNA was washed with ethanol and ether, dried, and analyzed
as described below. The supernatant was evaporated at low
pressure to about 0.2 mL, and then 0.4 mL of 0.05 M ammonium
formate was added and the mixture filtered if necessary. It was
analyzed on a Nucleosil ODS column (5 µm, 250 mm × 4.6 mm)
eluted with 15% methanol in 0.04 M ammonium formate (pH
∼6.5), at a rate of 0.8 mL/min. Fractions (1.5 min) were assayed
for ultraviolet absorbance and tritium activity, to determine the
amount of tritiated 7-methylguanine (eluted at ca. 10 min) and
3-methyladenine (ca. 20 min).
F igu r e 1. Release of 7-methylguanine from methylated DNA,
as measured by its tritium radioactivity. The upper two curves
are negative exponential curves with the data superimposed.
Ta ble 1. Ha lf-Lives of Meth ylp u r in es in DNA
An a lysis of DNA. Methylated DNA was hydrolyzed by
treatment with enzymes: for 1 mL of solution (containing 1-2
mg of DNA), 20 µL of DNase 1 (Boehringer; 5 mg/mL) and 20
µL of buffer [0.5 M Tris (pH 7) and 0.5 M MgCl2] for 17 h, 10 µL
of snake venom phosphodiesterase (Sigma type VII, 3.3 units/
mL) and 0.1 mL of buffer [0.5 M Tris (pH 9)] for 7 h, and 10 µL
of alkaline phosphatase (Sigma type III, 300 units/mL) for 17
h, all at 37 °C. The resulting mixture was analyzed by liquid
chromatography on an ODS column as described above, but
eluted with a water/methanol gradient [0.05 M ammonium
formate (pH 6.5) for 20 min, and then to 40% methanol over
the course of 40 min, at a rate of 0.8 mL/min]. Fractions (2 min)
were collected and assayed for ultraviolet absorbance and
tritium activity.
The column separated deoxycytidine (17 min), 5-methyldeoxy-
cytidine (37 min), deoxyguanosine and thymidine (together at
40 min), and deoxyadenosine (51 min); these were quantified
by their ultraviolet absorption at 260 nm (extinction coefficients,
by our estimates: dCyd, 7000; 5-Me-dCyd, 5000; dGuo and Thd,
10 000; and dAdo, 15 000). The molar ratios were close to the
expected values (22:50:28 C:G/T:A). The methylpurines were
released as the bases during hydrolysis, except for O6-meth-
yldeoxyguanosine; any other remaining methylpurine nucleo-
sides, being positively charged, should be eluted by the aqueous
starting solvent, but only 5-10% of the total tritium activity
was recovered in this region. The retention times of 3-meth-
ylguanine, 7-methylguanine, 7-methyladenine, 3-methylade-
nine, and O6-methyldeoxyguanosine were about 30, 40, 43, 46,
and 58 min, respectively. They were quantified by their tritium
activity, by adding scintillation fluid (Optiphase Safe, Fisher,
Loughborough, U.K.) and counting in a Packard scintillation
counter.
Qu a n tita tion of Meth yl Gr ou p s in DNA. The efficiency
of scintillation counting was estimated as follows. 7-Methylgua-
nine with a lower specific activity was prepared by treating 1
mg of DNA with 2 mg (5 µCi) of methylnitrosourea, and then
heating it (100 °C, 30 min). The resulting free 7-methylguanine
was chromatographically isolated and quantified by both ultra-
violet absorption (molar absorption coefficient of 7400 at 283
nm; 11) and scintillation counting, and hence, the counting
efficiency for this base was determined to be 35%. The occur-
rence of adducts in other samples of methylated DNA could then
be estimated per one million bases as (counts per minute in
product × millimoles of methylnitrosourea used)/[0.35(efficiency)
× 2.22 × millicurie of methylnitrosourea used × millimoles of
DNA].
10 °C
22 °C
461
39 °C
70
half-life for release of
7Me-guanine (h)
half-life for release of
3Me-adenine (h)
3800
not determined
266
30
with N-methyl-N-nitrosourea at pH 6-7, at a sufficient
concentration to produce about seven methylated bases
per one million nucleotides, and with tritium in the
methyl groups to the minimum level needed for assay
by radioactivity detection (0.5 µCi/g). Other samples were
also prepared with higher levels of methylation. The
7-methylguanine:3-methyladenine:O6-methylguanine:7-
methyladenine ratio in the DNA was estimated to be 79:
8:9:3. This is similar to that reported by Lawley and Shah
(78:12:7.7:2.5; 12).
Ra te of Relea se of Meth ylp u r in es fr om DNA.
Methylated DNA (ca. one methyl group per 2000 bases)
was incubated in aqueous buffer at neutral pH at 10, 22,
or 39 °C, and samples were taken at various times. The
DNA was precipitated, and the supernatant analyzed by
liquid chromatography for 7-methylguanine and 3-meth-
yladenine. These were identified by their coelution with
added marker compounds, and quantified by their tritium
radioactivity. The results are shown in Figure 1. The
curves at 22 and 39 °C exhibit first-order kinetics, and
that at 10 °C would probably exhibit the same if followed
to completion. The half-lives were estimated using a
programmable calculator procedure (13) and are given
in Table 1.
The 7-methylguanine peak may also contain 7-methyl-
adenine, but this should amount to only 2-3% of the
7-methylguanine and is released quickly, and would not
affect the results.
The precipitated DNA was hydrolyzed to nucleosides
with enzymes, and separated by liquid chromatography
on a reverse-phase column, using a water/methanol
gradient. A typical elution pattern is shown in Figure 2.
Labile methylated nucleosides still remaining in the DNA
lost deoxyribose during hydrolysis, and were eluted from
the column as 7-methylguanine and 3-methyladenine at
about 40 and 46 min. O6-Methyldeoxyguanosine eluted
at about 58 min.
Resu lts
The results of the stability study at 39 °C are shown
in Figure 3. The amount of 7-methylguanine fell with
time, as expected; the initial rate of fall was consistent
P r ep a r a tion of Meth yla ted DNA. Standard meth-
ylated DNA was prepared by treating calf thymus DNA

Methylated DNA Standard
Chem. Res. Toxicol., Vol. 13, No. 4, 2000 259
F igu r e 4. Loss of methylguanines from DNA on storage: (O)
at -20 °C and (b) at -80 °C. The total methyl content (top
curves) decreased from 7.5 to 4.0 per one million DNA bases in
5 years and the 7-methylguanine content (middle curves) from
4.7 to 2.4, while the O6-methyldeoxyguanosine content did not
change significantly (mean value of 0.59 ( 0.09 per one million
bases, bottom curves). Analysis of DNA stored as a frozen
solution at -190 °C for 0.6 or 2.3 years, or as a solid at -80 °C
for 0.6 or 1 years, gave the same results as the frozen solution
at -80 °C (data not shown).
F igu r e 2. Elution of methylated DNA components from ODS,
using an ammonium formate/methanol gradient. The meth-
ylated bases and nucleosides were located by reference to UV-
absorbing markers, and quantified by their tritium radioactivity.
The dotted line shows the elution of deoxyguanosine + thymi-
dine and deoxyadenosine, and added 7-methylguanine, 3-meth-
yladenine, and O6-methyldeoxyguanosine markers; the 7-meth-
ylguanine was coincident with 5-methyldeoxycytidine from the
DNA. The histogram shows tritium activity eluted per minute.
The example that is shown is from methylated DNA after
storage for 5 years at -20 °C.
in 5 mL of 0.05 M phosphate buffer (pH 7) and heated in
a water bath at 89 °C. Samples (1 mL) were taken after
12, 30, 77, 160, and 230 min, and the DNA was isolated
from each by adding 2.5 mL of ethanol containing 3%
ammonium acetate, cooling, and centrifugation; it was
no longer fibrous because of extensive strand breakage.
Hydrolysis to nucleosides, followed by chromatographic
separation, gave the normal nucleosides and tritium-
labeled O6-methyldeoxyguanosine. The 12 min sample
yielded a small amount 7-methylguanine, but the other
samples gave none of the labile methylated purines, as
they are lost from the DNA within a few minutes. The
amount of O6-methyldeoxyguanosine remained constant,
showing that this base is stably attached to the DNA,
and that its tritium content is not easily exchanged with
the solvent at neutral pH.
Sta bility of F r ozen Meth yla ted DNA. Methylated
DNA was stored as a frozen solution (1 mg/mL) in
BisTris/EDTA buffer, and kept at various temperatures
for extended periods of time. Samples were precipitated
to remove labile material, then hydrolyzed to nucleosides
with enzymes, and analyzed on the reverse-phase column
as described above. Some results are given in Figure 4.
The results show a slow loss of radioactivity from the
DNA, which is largely accounted for by loss of 7-meth-
ylguanine. The loss of 7-methylguanine proceeded with
a half-life of about 4 years at -20 °C; it was only slightly
slower (ca. 6 years) at -80 °C, which was somewhat
surprising. The yield of O6-methyldeoxyguanosine did not
change significantly with time (95% confidence limits for
the half-life at -20 °C were 12 years and infinity). The
observed variations were probably due to difficulties in
keeping the assay consistent over time, rather than any
intrinsic variability.
F igu r e 3. Change in content of methylated DNA during
incubation at 39 °C and neutral pH.
with the half-life of 70 h estimated above, but the last
four values were higher than expected. This discrepancy
from first-order kinetics was not observed in the amount
of 7-methylguanine that was released (Figure 1), sug-
gesting a defect in the measurement of the amount of
7-methylguanine in the precipitated DNA. The sizes of
the 3-methyladenine and O6-methyldeoxyguanosine peaks
were initially each about 15% of that of 7-methylguanine;
they both remained constant with time. This was as
expected for O6-methyldeoxyguanosine, which is stable,
but unexpected for 3-methyladenine, which should have
disappeared within the first 5 days; the reason for its
apparent persistence was unclear. It could have been due
to a stable adduct which coeluted with 3-methyladenine,
but the other known adducts are not eluted here.
In a separate experiment, DNA was precipitated from
solutions containing 2 mg/mL DNA and 2-20 mg/L
7-methylguanine or 3-methyladenine, to see whether the
DNA would carry the purine down with it. In each case,
more than 90% of the methylpurine remained in solution.
Sta bility of O6-Meth ylgu a n in e in DNA. Another
sample (10 mg) of this methylated DNA was dissolved
Discu ssion
There are many agents which methylate DNA. To
create a standard methylated DNA, we wanted an agent
which would give a relatively high yield of O6-methylgua-
nine residues, as these are of the greatest biological

260 Chem. Res. Toxicol., Vol. 13, No. 4, 2000
Osborne and Phillips
interest. We chose N-methyl-N-nitrosourea, where the
proportion of methylation at O6-guanine was known to
be about 7%, and which was readily available as a
tritium-labeled species. The range and proportion of
products with this agent are closely similar to those seen
in DNA isolated from the liver of rats treated with the
environmental carcinogen N,N-dinitrosodimethylamine
(14).
There are several good methods for analyzing meth-
ylated DNA (reviewed by Lawley in ref 15). We chose
hydrolysis by enzymes to nucleosides, followed by liquid
chromatography, as this gave acceptable results for both
the labile products (which were recovered as methylpu-
rines) and the chief stable adduct, O6-methyldeoxygua-
nosine. For the present purposes, we ignored the minor
products 1-methyladenine, 3-methylguanine, methylated
deoxycytidine and thymidine, and methyl phosphotri-
esters. The amounts of these can be estimated by other
methods (15).
F igu r e 5. Effect of temperature on the loss of 7-methylguanine
from DNA. Arrhenius plot of log(rate constant) (s-1) vs reciprocal
temperature (K). The rate in frozen solution at -20 °C is
included as a black square.
have been lost during the preparation of the standard
DNA samples.
Since O6-methylguanine residues in DNA appear to be
stable even at 89 °C, and there is no known mechanism
for their loss from DNA in vitro, it may be assumed that
the frozen methylated DNA standard will retain a
constant level of O6-methylguanine for many years. The
other methylated bases are slowly cleaved from the DNA,
and analysis of the 7-methylguanine or 3-methyladenine
content must include the solution in which it is stored;
otherwise, the estimates will slowly change with time.
The methylated standard was stable on storage, except
for the radioactive decay of the tritium label, and the
hydrolytic cleavage of methylpurines from the DNA. The
methylpurines remain in solution and can still be found
by analysis, unless the DNA is precipitated before the
assay, when they are lost in the supernatant. It appeared
to make little difference whether the DNA was stored as
a solid at -80 °C or as a frozen solution at -20, -80, or
-190 °C.
The methylated standard was distributed to partici-
pants in an international interlaboratory trial of 32P-
postlabeling (8). A published method for determining the
level of O6-methylguanine in DNA (20) was tested on the
DNA standard in participating laboratories without
investigators knowing the exact level of modification. In
the first trial, their mean value for the level of O6-
methylguanine was very close to the value given in this
paper, provided that the results from those participants
who failed to detect any adducts were excluded from the
analysis (8). In a subsequent trial, an unknown sample
was analyzed and the results were normalized against
our DNA standard, the level of modification of the latter
by now known to the investigators. The mean level
calculated for the unknown sample by this procedure was
In principle, the tritium label in the methyl groups may
be subject to exchange with water. However, the high
rate of recovery of tritiated O6-methyldeoxyguanosine
after 5 years, and the high rate of recovery of 7-meth-
ylguanine if the supernatant was included, indicate that
less than 10% of the observed loss of methylated base
was due to tritium exchange.
The rates of loss from DNA of the two major products,
7-methylguanine and 3-methyladenine, were best mea-
sured by precipitating the DNA, and estimating the
quantity of these purines remaining in solution. In
principle, it should be possible to measure the rates
equally well by analyzing the precipitated DNA, but we
found that this gave less accurate results, possibly
because of interference by other products with unknown
compositions. The rates given in Table 1 are therefore
based on analysis of the purines released from DNA. The
half-life for 7-methylguanine loss has been determined
by other authors, as follows: 105 h at pH 7 and 37 °C
(16), about 4.3 h at pH 8 and 65 °C (17), and 4.1 min at
pH 7 and 100 °C (18); the rate decreases only slightly
with pH (19). The corresponding rate constants are
plotted in Figure 5, along with those obtained at lower
temperatures in the work presented here. They fit the
Arrhenius equation ln k (s-1) ) 27-12507/T (K), corre-
sponding to an activation energy of 25 kcal/mol, and a
trebling of the rate for each 10° rise in temperature.
Extrapolation of the line would predict a half-life of about
80 years at -20 °C, but the data of Figure 4 indicate a
half-life of 4 years in frozen solution. Storage in a liquid
solution (e.g., a water/glycerol mixture at -20 °C) may
therefore be a viable alternative to storage as the frozen
solution.
3
found to be 50% higher than that calculated from the H
incorporation method. With the use of the methylated
standard and also with the use of standards for DNA
adducts of bulky carcinogens, the trials demonstrated
that interlaboratory variability is reduced when results
are normalized to values for validated standards that are
analyzed concomitantly (9).
For investigators wishing to use a standard in their
assays for methylated DNA adducts, the standard de-
scribed here is available in 1 mg aliquots on application
to the authors, while stocks last.
Ack n ow led gm en t. This work was supported by the
Cancer Research Campaign, U.K., and by a grant from
the European Union (Project EV5V-CT94-0449).
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