Modified immunoenriched 32P-HPLC assay for the detection of O4-ethylthymidine in human biomonitoring studies

Article abstract of DOI:10.1021/tx015582s

Increased excretion of ethylated DNA bases has been reported in the urine of cigarette smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched ³²P-postlabeling assay for O⁴-ethylthymidine (O⁴-etT) was developed. O⁴-etT-3′-monophosphate (O⁴-etT-3′P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA was enzymatically digested to 2′-deoxynucleoside-3′-monophosphate followed by immunoprecipitation of O⁴-etT-3′P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O⁴-etT-3′P was recovered by ethanol treatment. The enriched O⁴-etT-3′P was labeled with [γ-³²P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5′labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online detection of radioactivity. Adduct recovery was > 80%, and the detection limit was approximately 500 amol. To further validate the method, O⁴-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O⁴-etT was observed. Furthermore, O⁴-etT was found to be present in the cells obtained from the lower respiratory tract by sputum induction of two out of four smokers but not in three nonsmokers. O⁴-etT is a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and specific to detect O⁴-etT in surrogate cells from cigarette smoke exposed humans.

Full text of DOI:10.1021/tx015582s

Chem. Res. Toxicol. 2002, 15, 433-437
433
Mod ified Im m u n oen r ich ed 32P -HP LC Assa y for th e
Detection of O4-Eth ylth ym id in e in Hu m a n Biom on itor in g
Stu d ies
Roger Godschalk,† J agadeesan Nair,*,† Hans-Christian Kliem,‡
Manfred Wiessler,‡ Guy Bouvier,§ and Helmut Bartsch†
Divisions of Toxicology and Cancer Risk Factors and Molecular Toxicology,
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Received November 7, 2001
Increased excretion of ethylated DNA bases has been reported in the urine of cigarette
smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched 32P-
postlabeling assay for O4-ethylthymidine (O4-etT) was developed. O4-etT-3′-monophosphate
(O4-etT-3′P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA
was enzymatically digested to 2′-deoxynucleoside-3′-monophosphate followed by immunopre-
cipitation of O4-etT-3′P using specific monoclonal antibodies. The immunoconjugate was washed
by filtration, and O4-etT-3′P was recovered by ethanol treatment. The enriched O4-etT-3′P was
labeled with [γ-32P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5′-
labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online
detection of radioactivity. Adduct recovery was >80%, and the detection limit was approximately
500 amol. To further validate the method, O4-etT levels were determined in calf thymus DNA
treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O4-etT was observed.
Furthermore, O4-etT was found to be present in the cells obtained from the lower respiratory
tract by sputum induction of two out of four smokers but not in three nonsmokers. O4-etT is
a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring
smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and
specific to detect O4-etT in surrogate cells from cigarette smoke exposed humans.
of O4-etT in tobacco smoke-induced carcinogenesis,
whereby the amount of DNA available from target organs
is often limited. To date, the 32P-postlabeling assay is
widely applied to measure structurally diverse DNA
modifications, including alkylation products (9, 10). A 32P-
postlabeling method was previously developed to assess
O4-etT levels in human samples (10), but this assay
required relatively large amounts of DNA (up to 100 µg)
to reach a reasonable limit of detection and was time-
consuming. Therefore, we developed a modified immu-
noenriched 32P-postlabeling procedure with which sub-
femtomole levels of O4-etT could be reliably detected. This
method was further validated by applying it to DNA
treated with the direct-acting ethylating carcinogen
N-ethyl-N-nitrosourea (ENU) and to DNA obtained from
cells of the lower respiratory tract of smokers and
nonsmokers.
In tr od u ction
Alkylating agents react extensively with DNA to
produce several types of promutagenic structural base
modifications (1), which represent the biologically effec-
tive dose that can be used in exposure and possibly in
risk assessment (2). Tobacco smoke contains many alkyl-
ating compounds, and their mutagenic potential mainly
depends on the position of binding within the four DNA
bases (3, 4). O-Substitution by alkylating agents results
in relevant DNA adducts in terms of mutagenesis and
carcinogenesis, including O6-alkylguanine and O4-alkyl-
thymine. Animal studies indicated that, after exposure
to ethylating compounds, O4-ethylthymidine (O4-etT)1 is
initially formed in very low levels but is poorly repaired
(5) and thus accumulates to biologically relevant levels.
Also, cigarette smoke contains hitherto uncharacterized
ethylating agents, because increased levels of N-ethylated
DNA bases have been reported in the urine of smokers
as compared to nonsmokers (6-8). Highly sensitive
methods for the detection of O4-etT are required for
human biomonitoring and to further investigate the role
Exp er im en ta l P r oced u r es
Ma ter ia ls a n d Equ ip m en t. (1) Ma ter ia ls. 3′-Monophos-
phate of O4-etT was synthesized using 5′-dimethoxytrityl-3′-
phosporamidite thymidine (Amersham/Pharmacia Biotech,
Freiburg, Germany) (11). Calf thymus DNA (sodium salt) and
micrococcal endonuclease were purchased from Sigma-Aldrich
(Taufkirchen, Germany). Spleen phosphodiesterase was ob-
tained from Worthington Biochemical Corp. (Lakewood, NJ ) and
T4-polynucleotide kinase (T4-PNK) from Amersham/Pharmacia
Biotech (Freiburg, Germany). [γ-32P]ATP with a specific activity
of >220 TBq/mmol was purchased from Hartmann Analytic
(Braunschweig, Germany). (Ca u tion : [γ-32P]ATP is a hazard-
ous radioactive compound and should only be handled with
* Corresponding author. E-mail: J .Nair@dkfz.de. Phone: +49 6221
42 3306/3301. Fax:+49 6221 42 3359.
† Division of Toxicology and Cancer Risk Factors, German Cancer
Research Center.
‡ Division of Molecular Toxicology, German Cancer Research Center.
§ Current address: Aventis Crop Science, Regulatory Toxicology
Insecticide/Fungicide, 355 rue Dosto¨ıevski, BP 15306903 Sophia An-
tipolis, France.
Abbreviations: O4-etT, O4-ethylthymidine; ꢀdA, 1,N6-ethenode-
1
oxyadenosine; I.S., internal standard; ENU, N-ethyl-N-nitrosourea.
10.1021/tx015582s CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/22/2002

434 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Godschalk et al.
of an aqueous solution of ammonium acetate (1 M) and extracted
with 80 mL of chloroform. The organic solvent was dried over
Na2SO4, filtered, and removed in a vacuum. For deprotection
of the 3′-phosphate group, the residue was incubated with 10
mL of ammonia for 3 h. Ammonia was removed in vacuo, and
the solution was then lyophilized. Reversed-phase chromatog-
raphy (0-50% acetonitrile/water in 30 min) yielded pure 4 (4.1
mg, 35%). ESI-MS (m/z): 351.2 ([M + H+], 45%), 373.9 ([M +
Na+], 17%), 701.1 ([2M + H+], 100%), 723.3 ([2M + Na+], 30%);
349.1 ([M - H]-, 100%). 31P NMR (101 MHz, D2O, δ): 0.228
(brs, P). 13C NMR (62 MHz, D2O, δ): 174.3 (C-4), 160.5 (C-2),
143.2 (C-6), 110.5 (C-5), 89.2 (C-4′), 88.9 (C-1′), 77.1 (C-3′), 67.1
(C-5′), 63.9 (O4-C), 441.4 (C-2′), 16.2 (CH3), 14.1 (CH3). 1H NMR
(250 MHz, D2O, δ): 7.88 (q, 1H, H-6), 6.30 (dd, 1H, H-1′), 4.40
(q, 2H, O4-CH2), 4.20 (m, 1H, H-4′), 3.88 (dd, 1H, H-5′a), 3.80
(dd, 1H, H-5′b), 2.64 (ddd, 1H, H-2′a), 2.35 (ddd, 1H, H-2′b), 1.98
(d, 3H, C5-CH3), 1.38 (t, 3H, O4-C-CH3) (J 1′,2′a ) J 1′,2′b ) 6.5
Hz, J 2′a,2′b ) 13.9 Hz, J 2′a,3′ ) 4.2 Hz, J 2′b,3′ ) 6.7 Hz, J 4′,5′a ) 3.3
Hz, J 4′,5′b ) 4.9 Hz, J 5′a,5′b ) 12.5 Hz, J 5,CH ) 1.1 Hz, J CH
)
2,CH3
3
7.1 Hz; H-3 is covered by the signal of D2O; q ) quadruplet).
LC-MS: the mole peak [M - H]- and a dimer peak [2M - H]-
were observed at m/z: 349 and 699, respectively, with a major
fragment ion at m/z: 195 representing the base-free 2′-deox-
yribose-3′-monophosphate.
F igu r e 1. Scheme for the synthesis of O4-ethylthymidine-3′-
monophosphate (4): DMTr, 4,4′-dimethoxytrityl.
In Vitr o Tr ea tm en t of Ca lf Th ym u s DNA w ith ENU. Calf
thymus DNA was dissolved in water at a concentration of 0.4
mg/mL, and N-ethyl-N-nitrosourea (ENU) dissolved in DMSO
was added to reach final concentrations of 20, 2.0, 0.2, 0.02, and
0.002 µmol of ENU/incubation of 200 µL (Ca u tion : N-ethyl-
N-nitrosourea is a hazardous carcinogenic compound and should
be handled carefully). After incubation for 1 h at 37 °C, DNA
was precipitated by the addition of 1/30 volume of sodium acetate
(3 M, pH 5.2) and 2 volumes of ice-cold ethanol. DNA was
washed twice with 70% ethanol, dried in vacuo, and redissolved
in distilled water at a concentration of 1 µg/µL.
sufficient protection and shielding.) Polyethyleneimine (PEI) was
obtained from J .T. Baker, Inc. (Phillipsburg, NJ ). Antibody ER-
01 (12), which specifically recognizes O4-etT-3′- and 5′-mono-
phosphates, was provided by Dr. P. Lorenz (Uni-Klinikum,
University of Essen, Germany) and was purified before use by
an antibody purification kit (Immunopure (A/G) IgG purification
kit, Pierce, Rockford, IL). Antibodies were subsequently con-
centrated on centrifugation filters (MY-50, Millipore, Bedford,
MA).
(2) Equ ip m en t. An HPLC-model 1090 from Hewlett-Packard
(Waldbronn, Germany) with
a
250 × 4 mm C18 column
Sp u tu m In d u ction a n d DNA Isola tion fr om Sm ok er s
a n d Non sm ok er s. After clearance by the medical ethical
committee of the Maastricht University (The Netherlands), four
smokers and three nonsmokers volunteered to undergo sputum
induction to obtain cells from the lower respiratory tract (mostly
bronchoalveolar macrophages). Sputum induction was per-
formed by inhalation of ultrasonically nebulized 4.5% saline
delivered from an Ultra-Neb 2000 (De Vilbiss, Somerset, PA).
The subjects all produced expectorate into a 50-mL tube, and
the induced sputum was processed within 2.5 h of sampling, as
previously described by Besarati-Nia et al. (13). Cells were lysed
with 0.5% SDS in 100 mM NaCl, 20 mM EDTA, and 50 mM
Tris (pH 8.0) at 37 °C overnight. The resulting suspension was
treated with RNase for 3 h at 37 °C, followed by treatment with
proteinase K. DNA was isolated by extraction with phenol/
chloroform/isoamyl alcohol (25/24/1, by vol) and chloroform/
(Bischoff, Leonberg, Germany) was connected to an EG&G
Berthold (Bad Wildbad, Germany) radioactivity detector with
cell-model Z200-4.
Syn th esis of O4-etT-3′-Mon op h osp h a te Sta n d a r d . (1) 5′-
Dim eth oxytr itylth ym id in e-3′-(biscya n oeth yl)m on op h os-
p h a te 2 (see F igu r e 1). The solution of commercially available
thymidine-3′-phosphite 1 (500 mg, 0.67 mmol) in 10 mL of
acetonitrile was incubated with 0.5 mL of hydroxypropionitrile
and 2.3 mL of tetrazole (1 mM solution in acetonitrile) at
ambient temperature. After 90 min, 10 mL of a solution of 3%
cumene-hydroperoxide in acetonitrile was added to oxidize the
phosphite diester to phosphate 2. The reaction was stopped after
2 h by adding 3 mL of ethanol, then diluted with 50 mL of
chloroform, and washed with a concentrated aqueous solution
of NaHCO3 (2 × 30 mL) and water (2 × 30 mL). The organic
layer was separated, dried with Na2SO4, and filtered, and the
solvent was removed in a vacuum. The resulting product was
purified by flash chromotography (silica gel, chloroform/
methanol (95/5, v/v)) to yield 2 (187 mg, 38%). ESI-MS (m/z):
753.0 ([M + Na+], 100%).
isoamyl alcohol (24/1), respectively, and precipitated with 2
1
volumes of 100% cold ethanol and
/
volume of 3 M sodium
30
acetate (pH 5.3). Precipitated DNA was rinsed with 70% ethanol
and dissolved in 2 mM Tris (pH 7.4). Purity and concentration
of the DNA were determined spectrophotometrically by absor-
bance at 230, 260, and 280 nm, and the concentration was
adjusted to 2 mg/mL.
(2) O4-Eth yl-5′-d im eth oxytr itylth ym id in e-3′-(biscya n o-
eth yl)m on op h osp h a te 3. A freshly prepared solution of dia-
zoethane (approximately 8 mmol) in diethyl ether (10 mL) was
poured into a solution of 2 (185 mg, 0.25 mmol) in 5 mL of
methanol to form the O4-ethylated adduct 3. (Ca u tion : Dia-
zoethane is a hazardous alkylating compound and should be
handled carefully.) After 30 min, the solvents were removed in
a vacuum, and the residue was purified by flash chromatogra-
phy (chloroform/ethanol (98/2, v/v)) to yield compound 3 (26 mg,
13%). ESI-MS (m/z): 781.2 ([M + Na+], 40%), 1539.6 ([2M +
Na+], 100%).
(3) O4-Eth ylth ym id in e-3′-m on op h osp h a te 4. For 5′-depro-
tection, 3 (25 mg, 33 mmol) was dissolved in 2 mL of dry
nitromethane, and 2 mL of a saturated solution of ZnBr2 in
nitromethane was added. The color of the reaction solution
turned bright orange. The reaction was quenched with 40 mL
Digestion of DNA a n d Im m u n oen r ich m en t. DNA dried
in vacuo (25 µg) was reconstituted in 27.5 µL of a Tris-buffer
(80 mM Tris and 20 mM CaCl2 (pH 6.8)) and subsequently
digested to 3′-monophosphate nucleosides by the addition of 12.5
µL of micrococcal endonuclease (0.2 units/µL) and 10 µL of
spleen phosphodiesterase (0.025 units/µL) followed by incubation
at 37 °C for 4 h (14). Immunoprecipitation was performed
according to a modified procedure of Kang et al. (10). Fifty
microliters of immunobuffer (10 mM Tris (pH 7.5), 140 mM
NaCl, 3 mM NaN3, 1% BSA, and 0.1% rabbit IgG) and 50 µL of
purified monoclonal antibody were added and incubated for 1
h at room temperature with continuous mixing. Saturated
ammonium sulfate was added (1.3 mL) and placed on ice for 30

Biomonitoring for O4-Ethylthymidine
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 435
F igu r e 3. Mean recovery of O4-etT ((SD) using different
concentrations of the specific antibody ER-01 in three indepen-
dent experiments in which 25 µg of calf thymus DNA was spiked
with 1 fmol of O4-etT.
F igu r e 2. Profiles for (A) O4-ethylthymidine by HPLC with
online detection of radioactivity: (I) calf thymus DNA exposed
to ENU, (II) DNA of a smoker’s induced sputum, and (III) DNA
spiked with O4-etT standard. (B) Analysis of normal nucleotides
by HPLC-UV: I.S., internal standard.
min. The antibody-adduct interaction product was collected by
centrifugation and was redissolved in 400 µL of water (the
supernatant was used for the quantitation of normals; see the
next section). This solution was transferred into a microcon
centrifugation filter (MY-50; molecular weight cutoff, 50 000
Da), which retains the antibody-adduct complex but not
residual normal nucleotides and excess of ammonium salt.
Filters were washed 5 times with water (450 µL), and the
retentates were recollected by inversion of the filter and short
centrifugation (10 000 rpm for 5 min). Proteins were precipitated
by the addition of 500 µL of cold ethanol, and the supernatants
were dried and stored at -80 °C. Recovery of O4-etT was
determined by using purified O4-etT-5′-32P-monophosphate (for
labeling conditions, see the following discussion) and by the
comparison of peak areas obtained from spiked calf thymus DNA
with those of adduct standards.
F igu r e 4. Linear relationship between the peak area of O4-
etT and the peak area of a fixed amount of internal standard
(800 amol of 1,N6-ethenodeoxyadenosine, ꢀdA).
Resu lts
An a lysis of O4-Eth ylth ym id in e. Numerous attempts
to prepare immunoaffinity columns for O4-etT were
unsuccessful. Therefore, O4-etT was enriched by immu-
noprecipitation and subsequent desalting of the adduct-
antibody conjugate on membrane filters, which enabled
the analysis of O4-etT-3′P levels without inhibition of
labeling due to excessively high salt concentrations.
Typical HPLC chromatograms for the detection of O4-
etT are shown in Figure 2A. Adduct levels were related
to the amount of normal thymidine present in each
sample as determined by HPLC-UV (Figure 2B). The
recovery of adducts depended on the antibody concentra-
tion; the use of unconcentrated antibodies (13 µg/mL)
resulted in a maximal recovery of O4-etT of less than 50%,
whereas 5-fold concentrated antibodies (65 µg/mL) showed
a reproducible recovery of >80% (Figure 3), and this
antibody concentration was therefore used for the rest
of the experiments.
Qu a n tita tion of Am ou n t of DNA in th e An a lysis. After
precipitation of antibodies with ammonium sulfate, the super-
natant contained all unbound material, including normal nucle-
otides. To determine the amount of DNA in the analysis, 10 µL
was analyzed by reversed-phase HPLC, isocratically eluted with
100 mM ammonium formate (pH 7.5). The normals were
detected by UV absorption at 260 nm and quantitated by
comparison with standard solutions with known amounts of
nucleotides (200 pmol of each nucleotide). A typical chromato-
gram is shown in Figure 2B.
La belin g a n d HP LC On lin e An a lysis of O4-etT. Internal
standard (800 attomol 1,N6-ethenodeoxyadenosine, ꢀdA; see ref
14 for details) and 4 µL of a kinase buffer (125 mM Tris-HCl,
25 mM MgCl2, and 25 mM DTT (pH 6.8)) were added to the
dried enriched samples. After the addition of 2 µL of [γ-32P]-
ATP (20 µCi), 10 units of T4-PNK were added, and the mixture
was incubated for 2 h at 37 °C. Another 10 units of T4-PNK
were added after 1 h of incubation. Polyethyleneimine (PEI)
minicolumns were prepared by filling 2.5 mg of PEI into a 200
µL pipet tip closed with a small fritt. The labeled samples were
pipetted on top of the PEI and centrifuged for 5 min at 10 000
rpm at 4 °C. The labeled adducts were eluted with 5 × 25 µL of
1 M acetic acid, whereas excess ATP bound to PEI. The eluent
was injected into a C18-HPLC with online detection of radio-
activity. The mobile phase consisted of 100 mM ammonium
formate (pH 7.5) with 5% MeOH during the first 12 min at a
flow rate of 1 mL/min. Then, the methanol concentration was
increased to 20% over an 8 min time period (from 12 to 20 min)
and further increased to 50% over a period of 10 min (from 20
to 30 min). The internal standard eluted at 17 min and O4-etT-
5′32P at 22 min, whereas residual normal nucleotides eluted
before 12 min (Figure 2A). In the case of high background
radioactivity by tailing of residual normal nucleotides, appropri-
ate fractions were collected, dried, and reinjected.
1,N6-Ethenodeoxyadenosine (ꢀdA; 800 amol) was added
to all samples as an internal standard for quantitation
purposes. A linear relationship between the amount of
O4-etT and ꢀdA was observed (Figure 4). The labeling
efficiency of O4-etT was found to be 70%, but in the
presence of internal standard (800 amol) and normal
nucleotides (10 fmol of each), the labeling efficiency
decreased to ca. 20%. Increasing the amount of ATP or
labeling by the method as originally described by Kang
et al. (10) did not improve the labeling efficiency, which
appears to be a major limitation for the detection of O4-
etT. Optimal conditions were obtained by labeling at pH
6.8 and two additions of 10 units of T4-PNK.
DNA Ad d u ct Levels in Ca lf Th ym u s DNA Tr ea ted
w ith ENU. To further validate this method, calf thymus
DNA was treated with increasing concentrations of ENU

436 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Godschalk et al.
O4-etT-3′P levels without inhibition of 5′-labeling. The
specificity of antibodies is combined with the high
sensitivity of 32P-postlabeling, and this method can also
be used in conjunction with other enrichment procedures,
for example, HPLC preseparation as described previously
for O4-etT (10). Over 15 years ago, an immunoslot-blot
(ISB) methodology for the detection of O4-etT had been
described (17). Although this ISB method has a higher
absolute sensitivity for O4-etT (100 amol) as compared
to our method (500 amol), it may not reach the sensitivity
required for human DNA samples because of the limited
amount of DNA that can be used in the assay (e3 µg).
The lowest molar ratio which can be detected by ISB was
reported to be 100 amol of O4-etT in 3 µg of DNA (17)
(i.e., 4 adducts/108 normal thymidines). Thus, the major
advantage of our method is that more DNA can be used,
which permits the measurement of relative DNA adduct
levels as low as 2-4 adducts/108 thymidines, as observed
in human sputum samples.
F igu r e 5. Sigmoidal dose-response relationship of O4-etT in
ENU treated calf thymus DNA.
for 60 min and analyzed for the presence of O4-etT in
triplicate (sample I in Figure 2A). A clear dose-response
was observed (Figure 5) with adduct levels ranging from
44.5 ( 12.4 (mean ( SD) to 2913.8 ( 428.1 O4-etT/108
normal thymidines. The overall assay variation was
found to be approximately 20%.
Although the active removal of O4-etT from human
DNA was reported (18), the repair mechanisms have not
yet been elucidated. It is suggested that nucleotide
excision repair is involved (19), and the current evidence
suggests that O4-etT is only poorly repaired by O6-
alkylguanine-DNA alkyltransferase (5). O4-etT was found
to be highly persistent in animal tissues (16). Thus, even
small amounts of O4-etT in genomic DNA may have
biological consequences, and a method to quantify this
type of adduct requires high sensitivity. The relative
detection limit of ∼2 adducts/108 normal thymidines (500
amol of O4-etT in 25 µg of DNA) was found to be
sufficiently low to detect O4-etT in human lung samples
of smokers, in which the mean adduct levels were found
to be approximately 4-8 adducts/108 thymidines (20).
Exposure of calf thymus DNA to increasing concentra-
tions of ENU resulted in a dose-dependent formation of
O4-etT, with an overall assay variation of less than 20%.
Ethylating compounds are rarely present as environ-
mental contaminants. Therefore, the measurement of O4-
etT may offer a highly specific DNA modification to study
exposure to tobacco smoke, although the nature of the
DNA-reactive agent(s) remains to be determined. Until
now, other types of tobacco-derived carcinogen-DNA
adducts have been used for this purpose, but in some
cases, because of their ubiquitous presence in the envi-
ronment, it could not be unequivocally established from
what source these carcinogens originate. For example,
polycyclic aromatic hydrocarbons occur in cigarette smoke,
but can also be found in large quantities as food byprod-
ucts. Oral and inhalatory exposure to these hydrocarbons
may both lead to detectable DNA adducts (21) and are
thus unspecific indicators for cigarette smoke exposure.
In addition to pyridyloxobutyl-DNA adducts, which
provided a promising approach for human dosimetry of
major tobacco specific N-nitrosamines (22), O4-etT may
represent another useful specific biomarker for cumula-
tive exposure to cigarette smoke, because (i) urinary
excretion of ethylated DNA-bases proved to be indepen-
dent from diet (6) and (ii) in our feasibility study, O4-etT
was detected in human cells obtained from the lower
respiratory tract of smokers but not in those of nonsmok-
ers. Moreover, smoking related O4-etT was previously
detected in human lung tissue, with adduct levels being
2-fold higher in smokers than in ex-smokers, whereas,
in the same samples, mean levels of O6-methylguanine
and O4-methylthymine did not differ (20). Thus, mea-
DNA Ad d u ct F or m a tion in In d u ced Sp u tu m fr om
Sm ok er s a n d Non sm ok er s. To test the applicability
of this newly developed assay for human samples, cells
from the lower respiratory tract were obtained by sputum
induction and analyzed for the presence of O4-etT. O4-
etT was found in low levels in two out of four smokers
(sample II in Figure 2A) and in none of the nonsmokers.
O4-etT levels in the two smokers with detectable levels
were found to be 2.4 and 4.0 adducts/108 thymidines. In
all other samples, adduct levels were e2.0 adducts/108
thymidines (detection limit).
Discu ssion
O4-etT-3′P was synthesized and characterized by vari-
ous spectrometric data, which enabled us to systemati-
cally investigate stability, recovery, and labeling effi-
ciency of this biologically relevant DNA-alkylation product.
Among about a dozen different alkylation products
induced in genomic DNA by carcinogenic N-nitroso
compounds, O4-etT can be considered as one of the major
promutational lesions causing TA f CG transitions (15).
Although O4-etT is initially formed at far lower amounts
in cellular DNA than are other alkylation products, it
accumulates because of its inefficient repair and may,
thus, play an important role in human tobacco-induced
cancers (5, 16). The possible involvement of ethylating
agents in cigarette smoke has been demonstrated in
humans by studies on urinary excreted N-3-ethylated
adenine (6-8). To further investigate the role of O4-etT
in tobacco smoke-induced malignancies, a sensitive and
specific assay with which subfemtomole levels of O4-etT
can be detected was developed. The assay described in
this study is capable of detecting 500 amol of O4-etT,
which is ca. 2-fold more sensitive than the methodology
developed previously (10). It thus offers the possibility
to use smaller amounts of DNA (approximately 25 µg) to
reach a similar relative detection limit of 1-2 adducts/
108 parent nucleotides. Furthermore, this new enrich-
ment procedure for DNA adducts is quick and, in
principle, applicable to all types of DNA adducts for
which antibodies are available. In this modified method,
repeated washings of the adduct-antibody conjugate on
membrane filters effectively removed excessive am-
monium salts (because of the precipitation with am-
monium sulfate) and enabled the subsequent analysis of

Biomonitoring for O4-Ethylthymidine
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 437
surements of O4-etT levels in target or surrogate cells
(e.g., obtained by sputum induction) may identify those
individuals among smokers which are highly exposed or
are unable to repair this type of DNA damage. As those
subjects are likely to be at an increased risk for develop-
ing smoking-related malignancies, once identified, they
could be subjected to preventive measures.
(10) Kang, H. I., Konishi, C., Eberle, G., Rajewsky, M. F., Kuroki, T.,
and Huh, N. H. (1992) Highly sensitive, specific detection of O6-
methylguanine, O4-methylthymine, and O4-ethylthymine by the
combination of high-performance liquid chromatography prefrac-
tionation, 32P postlabeling, and immunoprecipitation. Cancer Res.
52, 5307-5312.
(11) Worth, C. C., Schmitz, O. J ., Kliem, H. C., and Wiessler, M. (2000)
Synthesis of fluorescently labeled alkylated DNA adduct stan-
dards and separation by capillary electrophoresis. Electrophoresis
21, 2086-2091.
Ack n ow led gm en t. Dr. Roger Godschalk was a re-
cipient of a visiting scientist fellowship awarded by the
DKFZ in 1999. The authors would like to thank Dr. H.
Besarati-Nia and Dr. F. J . Van Schooten (Maastricht
University, The Netherlands) for providing induced
sputum samples and Dr. W. Hull (German Cancer
Research Center, Heidelberg, Germany) for NMR analy-
ses. Dr. R. Owen (German Cancer Research Center,
Heidelberg, Germany) is acknowledged for helpful sci-
entific discussions.
(12) Adamkiewics, J ., Drosdziok, W., Eberhardt, W., Langenberg, U.,
and Rajewsky, M. F. (1982) High-affinity monoclonal antibodies
specific for DNA components structurally modified by alkylating
agents. In Indicators of genotoxic exposure (Bridges, B. A.,
Butterworth, B. E., and Weinstein I. B., Eds.) pp 265-276, Cold
Spring Harbor Laboratory, Plainview, NY, (Banbury, report 13).
(13) Besarati-Nia, A. B., Maas, L. M., Van Breda, S. G., Curfs, D. M.,
Kleinjans, J . C., Wouters, E. F., and Van Schooten, F. J . (2000)
Applicability of induced sputum for molecular dosimetry of
exposure to inhalatory carcinogens: 32P-postlabeling of lipophilic
DNA adducts in smokers and nonsmokers. Cancer Epidemiol.,
Biomarkers Prev. 9, 367-372.
(14) Nair, J ., Barbin, A., Guichard, Y., and Bartsch, H. (1995) 1,N6-
ethenodeoxyadenosine and 3,N4-ethenodeoxycytine in liver DNA
from humans and untreated rodents detected by immunoaffinity/
32P-postlabeling. Carcinogenesis 16, 613-617.
(15) Singer, B., Spengler, S. J ., Fraenkel-Conrat, H., and Kusmierek,
J . T. (1986) O4-methyl, ethyl, or isopropyl substituents on thy-
midine in poly(dA-dT) all lead to transitions upon replication.
Proc. Natl. Acad. Sci. U.S.A. 83, 28-32.
Refer en ces
(1) Shuker, D. E., and Bartsch, H. (1994) DNA adducts of
nitrosamines. IARC Sci. Publ. 125, 73-89.
(2) Vineis, P., and Perera, F. (2000) DNA adducts as markers of
exposure to carcinogens and risk of cancer. Int. J . Cancer 88, 325-
8.
(3) Barbin, A., and Bartsch, H. (1989) Nucleophilic selectivity as a
determinant of carcinogenic potency (TD50) in rodents: a com-
parison of mono- and bifunctional alkylating agents and vinyl
chloride metabolites. Mutat. Res. 215, 95-106.
(16) Scherer, E., Timmer, A. P., and Emmelot, P. (1980) Formation
by diethylnitrosamine and persistence of O4-ethylthymidine in
rat liver DNA in vivo. Cancer Lett. 10, 1-6.
(4) Vogel, E. W., Barbin, A., Nivard, M. J ., and Bartsch, H. (1990)
Nucleophilic selectivity of alkylating agents and their hypermut-
ability in Drosophila as predictors of carcinogenic potency in
rodents. Carcinogenesis 11, 2211-2217.
(17) Nehls, P., Adamkiewicz, J ., and Rajewsky, M. F. (1984) Immuno-
slot-blot: A highly sensitive immunoassay for the quantitation
of carcinogen-modified nucleosides in DNA. J . Cancer Res. Clin.
Oncol. 108, 23-29.
(5) Bronstein, S. M., Skopek, T. R., and Swenberg, J . A. (1992)
Efficient repair of O6-ethylguanine, but not O4-ethylthymine or
O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyl-
transferase and nucleotide excision repair activities in human
cells. Cancer Res. 52, 2008-2011.
(6) Prevost, V., and Shuker, D. E. (1996) Cigarette smoking and
urinary 3-alkyladenine excretion in man. Chem. Res. Toxicol. 9,
439-444.
(7) Prevost, V., Shuker, D. E., Friesen, M. D., Eberle, G., Rajewsky,
M. F., and Bartsch, H. (1993) Immunoaffinity purification and
gas chromatography-mass spectrometric quantification of 3-alkyl-
adenines in urine: metabolism studies and basal excretion levels
in man. Carcinogenesis 14, 199-204.
(8) Eberle, G., Glu¨senkamp, K. H., Drosdziok, W., and Rajewsky, M.
F. (1990) Monoclonal antibodies for the specific detection of
3-alkyladenines in nucleic acids and body fluids. Carcinogenesis
11, 1753-1759.
(18) Wani, A. A., Wani, G., and D’Ambrosio, S. M. (1990) A human
DNA repair activity specific for O4-ethylthymine: identification
and partial characterization. Carcinogenesis 11, 1419-1424.
(19) Klein, J . C., Bleeker, M. J ., Roelen, H. C., Rafferty, J . A.,
Margison, G. P., Brugghe, H. F., van den Elst, H., van der Marel,
G. A., van Boom, J . H., and Kriek, E., et al. (1994) Role of
nucleotide excision repair in processing of O4-alkylthymines in
human cells. J . Biol. Chem. 269, 25521-25528.
(20) Kang, H., Konishi, C., Bartsch, H., Kuroki, T., and Huh, N. (1995)
Detection of smoking related O4-ethylthymine in human lung
tissues. Proc. Am. Assoc. Cancer Res., Abstr. 810.
(21) Godschalk, R. W., Moonen, E. J ., Schilderman, P. A., Broekmans,
W. M., Kleinjans, J . C., and Van Schooten, F. J . (2000) Exposure-
route-dependent DNA adduct formation by polycyclic aromatic
hydrocarbons. Carcinogenesis 21, 87-92.
(22) Hecht, SS. (1999) DNA adduct formation from tobacco-specific
(9) Povey, A. C., and Cooper, D. P. (1995) The development, validation
and application of a 32P-postlabeling assay to quantify O6-
methylguanine in human DNA. Carcinogenesis 16, 1665-1669.
N-nitrosamines. Mutat. Res. 424, 127-142.
TX015582S