Lung DNA Adducts Detected in Human Smokers Are Unrelated to
Typical Polyaromatic Carcinogens
Jamal M. Arif,†,#Carolyn Dresler,|,⊥Margie L. Clapper,|C. Gary Gairola,¶
Cidambi Srinivasan,¶Ronald A. Lubet,§and Ramesh C. Gupta*,‡,†
Department of Pharmacology and Toxicology/Brown Cancer Center, UniVersity of LouisVille, LouisVille,
Kentucky 40202, Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh
11211, Saudi Arabia, Department of Statistics, UniVersity of Kentucky, Lexington, Kentucky 40506, DiVision of
Population Science, Fox Chase Cancer Center, Philadelphia, PennsylVania 19111, and DiVision of Cancer
PreVention & Control, National Cancer Institute, Bethesda, Maryland 20892
ReceiVed September 7, 2005
Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other
polyaromatics by32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues
of human smokers. However, our experimental studies in rodent models, which used modified
chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic
DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines.
In the present study, we have performed similar analysis of human lung tissues to study the chemical
nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active,
ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and
n-butanol enrichment-mediated32P-postlabeling assay. All DNA samples yielded low to highly intense
adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents.
Adduct burden ranged from 6.6 to 2930 per 1010nucleotides. However, when adducts were resolved in
a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide
in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms,
this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs
mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic
amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated
DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known
to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by32P-postlabeling
resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-
containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was
used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms
similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-
associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they
are not related to PAHs and aromatic amines.
Lung cancer is the leading cause of cancer-related deaths in
the United States, and the lung cancer deaths in the new
millennium are estimated at 25% for females and 32% for males
of the total cancer mortality (1, 2). Epidemiological studies
strongly implicate cigarette smoking as a major etiological factor
in the incidence of lung cancer (3, 4). Despite improvement in
smoking cessation programs and antitobacco smoking aware-
ness, about 25% of the U.S. adult population continues to smoke
cigarettes, a percentage which has remained largely constant
since 1990 (2). Epidemiological studies have also indicated
possible gender-specific susceptibility to lung cancer (5, 6). In
contrast to men, the incidence of lung cancer in women over
the past decade has increased despite lower overall consumption
of cigarettes by women (2, 6).
Cigarette smoke is a complex mixture of chemicals and
contains more than 50 known or probable human carcinogens,
including polycyclic aromatic hydrocarbons (PAHs),1aromatic
amines (AAs), and tobacco-specific nitrosamines (3, 7). Many
* Corresponding author: R. Gupta, Department of Pharmacology &
Toxicology/Brown Cancer Center, University of Louisville, Louisville,
KY 40202. Tel, (502) 852-3682; fax, (502) 852-3662; e-mail, rcgupta@
†Prior affiliation of J. Arif and R. Gupta where large part of this work
was performed: Department of Preventive Medicine and Environmental
Health, and Graduate Center for Toxicology, University of Kentucky,
Lexington, KY 40536.
#King Faisal Specialist Hospital and Research Center.
|Fox Chase Cancer Center.
⊥Present address: Tobacco and Cancer Group, International Agency
for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon CEDEX
‡University of Louisville.
¶University of Kentucky.
§National Cancer Institute.
1Abbreviations: AAs, aromatic amines; 4-ABP, 4-aminobiphenyl; ABZ,
N′-acetylbenzidine; AF, 2-aminofluorene; anti-BADE, benz[a]anthracene-
3,4-dihydrodiol-1,2-epoxide (anti); anti-BFDE, benzo[k]fluoranthene-8,9-
dihydrodiol-10,11-epoxide (anti); BP, benzo(a)pyrene; anti-BPDE, benzo-
(a)pyrene-7,8-dihydrodiol-9,10-epoxide (anti); anti-DBADE, dibenz(a,h)-
anthracene-1,2-dihydrodiol-3,4-epoxide (anti); DRZs, diagonal radioactive
zones; ETS, environmental tobacco smoke; NNK, 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone; PAH, polycyclic aromatic hydrocarbons; PEI,
polyethyleneimine; RAL, relative adduct labeling; TLC, thin-layer chro-
Chem. Res. Toxicol. 2006, 19, 295-299
10.1021/tx0502443 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/25/2006
of these compounds possess mutagenic activity, cause DNA
damage, and form DNA adducts (8). Covalent interaction of
DNA with the electrophilic metabolites of carcinogens is thought
to be an essential early step in the initiation of cancer (8).
Despite the diversity of chemical carcinogens in tobacco smoke,
an analysis by32P-postlabeling, fluorescence and immunoassays,
and so forth has suggested the presence of PAH- and AA-
derived DNA adducts in various human tissues of smokers (7,
9-12). However, our experimental studies have failed to detect
benzo(a)pyrene (BP)- and 4-aminobiphenyl (4-ABP)-derived
DNA adducts in the respiratory (lung, trachea) and nonrespi-
ratory (bladder, heart) tissues of smoke-exposed rodents (13-
15). Analysis of environmental tobacco smoke (ETS)-induced
lung tumor multiplicity in the A/J mice have indicated that the
vapor phase of ETS is as tumorigenic as whole ETS, suggesting
that the tobacco-specific nitrosamines such as 4-(methyl-
nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or BP may not
be involved in tumor induction in that particular A/J mouse
Improvements in the resolution of DNA adducts produced
by various carcinogens, including PAHs, nitrated PAHs, and
AAs, using an ammonium hydroxide-based chromatographic
solvent system (17), has prompted us to re-examine lung tissues
from human smokers for the presence of PAH- and AA-derived
DNA adducts. We postulate that DNA adducts produced in the
tissues of human smokers, as diagonal radioactive zones (DRZs)
in standard urea-based solvent systems, may not represent the
typical PAH- and AA-derived DNA adducts. In the present
study, absence or presence of BP- and 4-ABP-derived DNA
adducts were analyzed in lung tissues from human lung cancer
Materials and Methods
Subjects and Sampling. Lung tumor tissue samples were
obtained from 50 (26 male and 24 female) lung cancer patients
(42-83 years of age), with current (n ) 25), ex- (n ) 22), or never-
smoking (n ) 3) status, who were undergoing surgical resection
of their tumors at Fox Chase Cancer Center, Philadelphia, PA. The
ex-smokers included in the study were those who had quit smoking
for at least 1 year prior to the surgery. The Institutional Review
Boards of Fox Chase Cancer Center and the University of Kentucky
approved the protocols. A detailed questionnaire regarding the
smoking history (e.g., age of initiation, age at the time of quitting,
and pack-year history, etc.) was administered at the time of their
scheduled visits to the thoracic surgery clinic. The tissues were
collected and stored at -80 °C until shipment on dry ice to the
University of Kentucky for analysis.
Preparation of Aldehyde-Derived DNA Adducts. Calf thymus
DNA (300 µg) was incubated with formaldehyde and acetaldehyde
(40 mM) for 3 h at 37 °C, followed by DNA precipitation with
sodium chloride and ethanol (18).
Reference DNA Adducts. Calf thymus DNAs adducted with
anti-diolepoxides of dibenz(a,h)anthracene (dibenz(a,h)anthracene-
1,2-dihydrodiol-3,4-epoxide; anti-DBADE), benz(a)anthracene (benz-
[a]anthracene-3,4-dihydrodiol-1,2-epoxide; anti-BADE), benzo(k)-
anti-BFDE), and benzo(a)pyrene (benzo(a)pyrene-7,8-dihydrodiol-
9,10-epoxide; anti-BPDE) were kindly provided by Drs. C. C.
Harris and A. Weston, National Cancer Institute, Bethesda, MD,
and DNAs adducted with N-hydroxy derivatives of N′-acetylben-
zidine (ABZ), 4-aminobiphenyl (ABP) and 2-aminofluorene (AF)
were obtained from Drs. F. F. Kadlubar and F. A. Beland, National
Center for Toxicological Research, AR. These reference DNA
adducts have been characterized in32P-postlabeled form previously
(19). The stock adducted DNAs (containing 1 adduct per 104-106
nucleotides) were diluted with untreated calf thymus DNA to a
desired level of approximately 1 adduct per 107nucleotides prior
to their use in this study. Two reference adduct mixtures were
prepared, one containing PAH-related adducts and the other
containing aromatic amines, with each adduct present at ap-
proximately 1 per 108nucleotides to 5 per 1010nucleotides. Adduct
levels were determined by32P-postlabeling as described (19).
DNA Isolation. DNA was isolated by a phenol extraction
procedure as described elsewhere (18). Briefly, lung tissues were
homogenized in 50 mM Tris-HCl (pH 8.0) containing 10 mM
EDTA and 5 mM butylated hydroxy toluene as a free radical
inhibitor. Isolated crude nuclei were digested with ribonucleases
A (150 µg/mL) and T1 (1 unit/µL) (Sigma Chemical Co., St. Louis,
MO), followed by digestion with proteinase K (150 µg/mL)
(Boehringer Mannheim Corp., Indianapolis, IN). The samples were
extracted sequentially with phenol, phenol/Sevag (chloroform/
isoamyl alcohol, 24:1), and Sevag, and DNA was precipitated with
1 vol of ethanol and 0.5 M sodium chloride. The DNA concentration
was estimated spectrophotometrically using 1 A260unit as equal to
50 µg of DNA.
DNA Adduct Analysis. DNA (10 µg) was digested with a
mixture of micrococcal nuclease (Sigma Chemical Co., St. Louis,
MO) and spleen phosphodiesterase (Boehringer Mannheim Corp.,
Indianapolis, IN) (enzyme/DNA ) 1:5, w/w, 5 h at 37 °C). For
human lung DNA and PAH- and aldehyde-adducted DNAs, adducts
were enriched by treatment with nuclease P1 (Calbiochem-
Novabiochem Corp., San Diego, CA) (enzyme/DNA ) 1:2.5, w/w,
45 min at 37 °C) (18). For selected human lung DNA and AA-
adducted DNA, adducts were also enriched by butanol extraction
(18). Adducts were labeled with molar excess of [γ-32P]ATP,
prepared enzymatically using32Pi (ICN Pharmaceutical, Inc., Costa
Mesa, CA) (18), and resolved by multidirectional polyethyleneimine
(PEI)-cellulose TLC in the following solvent systems. (i) System
I: D1 ) 1 M sodium phosphate, pH 5.7; D3 ) 4 M lithium formate/
8.5 M urea, pH 3.5; D4 ) 0.8 M lithium formate/8.5 M urea/0.5
M Tris-HCl, pH 8.0; and D5 ) 1 M sodium phosphate, pH 5.7.
(ii) System II: All solvents were the same as in system I, except
that D4 solvent was substituted with 2-propanol/4 M NH4OH (1.2:
1) 2 cm onto Whatman No. 1 paper wick. An aliquot of diluted
DNA digest (2 ng) was also labeled in parallel with adducts, and
normal nucleotides were resolved in 1.5 M ammonium formate,
pH 3.5. Adduct DRZs were visualized and quantified by Packard
InstantImager (Packard Instrument Co., Inc., Downers Grove, IL).
For quantification, the background radioactivity (cpm/mm2) from
the blank area of the chromatogram was subtracted from the
radioactivity of adduct DRZ. The relative adduct labeling (RAL)
was calculated as follows: RAL ) (cpm in adducts/cpm in total
nucleotides) × (1/dilution factor).
Co-Chromatography. Selected human lung DNA specimens
were analyzed alone or after mixing with reference PAH- and AA-
adducted DNAs, ranging from 5 adducts/1010to 1 adduct/108
nucleotides. DNA digestion, enrichment, and labeling conditions
were the same as described above. The labeled digests were
chromatographed in both solvent systems I and II.
Statistical Analysis. The transformed DNA adduct levels
(log[adduct level]) per 1010nucleotides were analyzed by means
of Generalized Regression models, and the computations were
carried out with SAS statistical software (SAS version 8). P < 0.05
was considered significant.
All samples of human lung DNA produced typical adduct
maps showing DRZs when analyzed by the nuclease P1-version
of32P-postlabeling and chromatographed in standard urea-based
solvent system I (Figure 1A,B). In general, more intense adduct
DRZs were observed in lung DNA from smokers (Figure 1A,B)
than in the ex- or never-smokers (not shown). However, D4
development in dilute ammonium hydroxide-based solvent
system II failed to yield any adduct spots even after a prolonged
autoradiographic exposure (Figure 1D,E). Use of butanol
extraction-mediated adduct enrichment resulted in diffused
296 Chem. Res. Toxicol., Vol. 19, No. 2, 2006 Arif et al.
adduct DRZs together with distinct adduct spots in urea-based
solvents (Figure 1G), but again, no adducts were detected when
D4 development was performed in dilute ammonium hydroxide-
based solvent system (Figure 1I).
Measurement of the adduct DRZ radioactivity revealed
significantly higher adduct levels in the current smokers (n )
24) compared to ex-smokers (n ) 22) (342 ( 38 versus 222 (
35 adducts/1010nucleotides; P < 0.026) (Figure 2). Adduct
levels in the never-smokers (n ) 3) (217 adducts/1010nucle-
otides) were not different than the ex-smokers. Comparison of
smokers and ex-smokers in each gender group showed that
female current smokers (n ) 11) had significantly higher levels
of DNA adduction than ex-smokers (n ) 9) (369 ( 76 versus
156 ( 49 adducts/1010nucleotides; P ) 0.02). However, no
significant differences were detected between male current
smokers (n ) 13) and ex-smokers (n ) 13) (323 ( 39 versus
267 ( 47 adducts/1010nucleotides, respectively; P ) 0.37).
Further statistical analysis revealed that male ex-smokers
possessed marginally (P ) 0.10) higher adduct levels than the
female counterparts (267 ( 47 versus 156 ( 49 adducts/1010
nucleotides). One female smoker lung tissue was highly
adducted (2930 adducts/1010nucleotides); this value was
excluded from the mean value presented in Figure 2. Further-
more, an 8-20-fold interindividual variability was observed in
the adduct levels, irrespective of the smoking status.
To identify the chemical nature of the smoke-associated
adducts, selected lung DNA samples were analyzed after mixing
with calf thymus DNA containing PAH-derived adducts at a
level of approximately 1 adduct/108nucleotides, using solvent
system I (urea-based solvents in both D3 and D4 directions)
and system II (urea-based solvent in D3 and ammonium
hydroxide-based in D4). In solvent system I, reference PAH
adducts migrated together with lung DNA adducts in the form
of DRZ (Figure 1C), but well-resolved, discrete reference adduct
spots were observed in the ammonium hydroxide-based solvent,
with no additional adducts associated with smoker lung DNA
(Figure 1F). DNA adducts derived from various PAHs were
characterized and numbered appropriately as BADE (no. 1),
BFDE (no. 2), BPDE (no. 3), and DBADE (no. 4) (Figure 1C,F).
AA-derived DNA adducts migrated in the chromatographic
region where smokers’ lung DNA adducts migrated in solvent
system I (Figure 1H), but unlike smoker lung DNA adducts
which were lost in solvent system II (Figure 1I), the AA-DNA
adducts were observed as well-resolved, distinct spots (Figure
1J). Under the experimental conditions used, reference DNA
adducts as low as 5 adducts/1010nucleotides were detectable
after prolonged autoradiographic exposure. These data suggest
an absence of PAH- and AA-derived DNA adducts in the
smoker lung DNA samples analyzed.
In attempts to explore the origin of smoke-associated adducts,
analysis of formaldehyde- and acetaldehyde-modified DNA by
32P-postlabeling showed typical adduct DRZs in the solvent
system I (Figure 3B,C), compared with vehicle treatment (Figure
3A). However, when adducts were resolved in ammonium
hydroxide-based solvent in D4 (system II), the DRZs disap-
peared from the chromatogram (Figure 3D-F). This chroma-
Figure 1. Representative32P-adduct maps from human smoker lung
DNA and reference adducts of selected polycyclic aromatic hydrocar-
bons (PAHs) and aromatic amines. Maps A-F were derived after
enrichment of the adducts by nuclease P1, while maps G-J were
derived following enrichment of adducts by butanol extraction. Panels
A and B represent two smoker lung DNA with varying degree of
adduction. DNA used in panel C was the same as used in panel A,
except that it was analyzed after mixing with calf thymus DNA adducted
with PAH diolepoxides listed below. Lung DNA in panel G was the
same as in panel B, except that the adducts were enriched by butanol
extraction. DNA in panel H represents calf thymus DNA adducted with
indicated arylamines. Following enzymatic digestion of DNA (10 µg),
adducts were enriched by either treatment with nuclease P1 or extraction
with butanol. Enriched adducts were labeled with molar excess of
[γ-32P]ATP in the presence of T4 polynucleotide kinase. The solvent
system I (urea-based solvents both in D3 and D4) and system II (urea-
based solvent in D3 and isopropyl alcohol/ammonium hydroxide in
D4) are described in detail in text. The figure was prepared by scanning
autoradiograms resulting from exposure of chromatograms to Cronex
4 X-ray films at -80 °C for 24 h. Reference adducts used were as
follows: spots 1-4 represent adducts resulting from anti-diolepoxides
of benz(a)anthracene, benzo(k)fluoranthene, benzo(a)pyrene, and dibenz-
(a,h)anthracene, respectively; spots 5, 6, and 7 represent the main
adducts resulting from N-hydroxy derivatives of 4-aminobiphenyl,
2-aminofluorene, and N′-acetylbenzidine, respectively.
Figure 2. Gender- and smoking status-associated differences in DNA
adduct levels in human lung tumors. Data were derived from nuclease
P1-mediated32P-postlabeling assay using high-salt, high urea-based
TLC, as described in Figure 1. Data are represented as the mean (
standard error, with each data point denoting adduct levels for individual
Figure 3. Representative32P-adduct autoradiograms from calf thymus
DNA alone (A and D) and DNA reacted with formaldehyde (B and E)
and acetaldehyde (C and F). Adducts were analyzed by nuclease P1-
mediated32P-postlabeling assay and chromatographed using the solvent
system I (A-C) and system II (D-F) described in the legend of Figure
1 and the text. Chromatograms were exposed to Cronex 4 X-ray films
at -80 °C for 24 h and were reproduced by scanning the autoradio-
DNA Adducts in Cigarette Smokers’ LungsChem. Res. Toxicol., Vol. 19, No. 2, 2006 297
tography behavior of the aldehyde-derived DNA adducts is
similar to that of the cigarette smoke-associated DNA adducts.
DNA damage as reflected by the formation of adducts is
believed to be an essential step in the initiation of the
carcinogenic process and has been positively correlated with
the mutation frequency and development of cancer (8). Several
investigators have reported the presence of BP- and 4-ABP-
derived DNA adducts sequestered in adduct DRZs in different
human tissues (9-12, 20). As yet, the complete and true
chemical nature of the DRZs is not known, although they are
found at higher levels in smokers’ tissues and have been
implicated in tobacco smoke carcinogenesis (21). Results from
the present study and from our earlier experimental studies
(reviewed in ref 22) prompted us to suggest that smoking-
associated DNA adducts may not be derived from typical PAHs
or AAs, normally present in tobacco smoke. These adducts are
also unrelated to polar oxidative adducts such as 8-oxo-
deoxyguanosine and cyclic DNA adducts resulting from lipid
peroxidation products, since such adducts are lost onto paper
wick during the chromatographic manipulations. On the basis
of the chromatographic resemblance, our data tend to suggest
that the smoke-associated DNA adducts may originate, at least
in part, from aldehydes such as formaldehyde and acetaldehyde,
which are present in significant quantities in cigarette smoke.
One of the limitations of the32P-postlabeling assay is its
inability to identify DNA adducts in the absence of reference
adducts of known chemical nature. The appearance of DNA
adduct spots on chromatograms depends on their mobilities and
can be variable depending on the choice of chromatography
solvents and the nature of the chromatographic matrix, for
example, cellulose and polyethyleneimine. A complex mixture
like tobacco smoke, which contains over 4000 compounds, can
potentially generate numerous unidentifiable DNA adducts in
the tissues (7). Most studies have, however, focused on common
environmental carcinogens, like PAHs and AAs, even though
aldehydes, catechols, free radicals, and others represent a
significant portion of inhaled tobacco smoke constituents (7).
BP- and 4-ABP-related DNA adducts reported in smokers were
identified mostly by32P-postlabeling in conjunction with high-
salt, high-urea TLC. Dilute ammonium hydroxide-based solvents
were introduced, which can resolve adduct DRZs into discrete
spots (17) and allow identification of adducts derived from
diverse classes of compounds, including PAHs and AAs.
To obtain a better understanding of the nature of smoking-
associated DNA adducts, select lung DNA samples from human
smokers were analyzed using ammonium hydroxide- and
conventional urea-based solvents, in the presence and absence
of PAH- and AA-derived reference adducts. As expected,
smoker lung DNA samples, with and without reference adducts,
produced prominent adduct DRZs in urea-based solvents.
However, when the same samples were analyzed in ammonium
hydroxide-based solvent as one of the eluting solvents, they did
not yield any adducts, but the same samples mixed with
reference adducts produced discrete spots of individual reference
adducts. These observations clearly suggested that the DRZs
produced by the same smoker lung DNA in urea-based solvents
do not contain typical PAH- and/or AA-derived adducts. Our
previous finding that BP-derived DNA adducts were undetect-
able in cigarette smoke-exposed rodent tissues is consistent with
the above observations (13-15).
This conclusion is supported by a recent study in which both
vapor phase and whole environmental tobacco smoke were
equally effective in producing lung tumors in the A/J mice (16).
This study also showed that chemopreventive agents that
inhibited model carcinogen-induced lung tumors failed to protect
against whole smoke-induced lung tumors. These data further
support our conclusion that agents other than PAHs may be
involved in smoke carcinogenesis.
The discrepancy over the presence of BPDE-DNA adducts
in smokers’ lung tissues in this report and other studies seems
to be method-oriented, particularly the chromatography condi-
tions. van Schooten et al. (23) performed co-chromatographic
experiments with reference BPDE-DNA adducts in only urea-
based solvents as opposed to our present and previous studies
(13-15) where we utilized multiple solvents, including am-
monium hydroxide-based solvents in co-chromatography experi-
An exhaustive analysis of published data on the presence of
BP-derived DNA adducts in human tissues by Boysen and Hecht
(24) demonstrates that 39% of 705 human tissues showed
detectable levels of BPDE-DNA adducts. These measurements
were based on tetrols from BPDE. The BPDE-DNA adducts
were detected in 45% smokers, 33% ex-smokers, 52% non-
smokers, 39% of occupationally exposed individuals, and 34%
of environmentally exposed people. The data further indicated
that BPDE-DNA adducts in smokers’ tissues are not consis-
tently different that in nonsmokers’ tissues and that due to the
environmental ubiquity of BP, it cannot be assumed that BPDE-
DNA adducts are present in human smokers’ tissues (24).
Several studies have identified PAH- and AA-derived protein
adducts in human smokers as well as smoke-exposed rat tissues
(25, 26). The undetectability of DNA adducts from these
carcinogens in human smokers in the present study and cigarette
smoke-exposed rat tissues in our previous studies (reviewed in
ref 22) suggests that electrophilic metabolites of PAHs and AAs
present in cigarette smoke may be consumed by the enormous
amounts of cellular proteins before they have a chance to cross
the nuclear membrane to react with the DNA.
A potential role of various short- and longer-lived free radicals
of cigarette smoke in forming adduct DRZs cannot be ruled
out, since cigarette smoke contains high levels of free radicals
(1014-1016radicals/puff) in both its gaseous and tar phases (27).
Findings from the laboratories of Randerath and Phillips reported
the detection of intrastrand DNA-DNA cross-links formed by
treatment of DNA with hydroxyl radicals (28, 29). Using urea-
based solvents, as employed in our studies, these investigators
were able to separate intrastrand cross-links on the chromato-
grams, suggesting that the smoke-related DNA adducts present
as adduct DRZs may chromatographically behave as certain
DNA-DNA cross-link products. DNA adducts produced by
reaction with formaldehyde and acetaldehyde in vitro showed
adduct DRZs as found in smokers’ lung DNA when chromato-
graphed in urea-based solvents but, when chromatographed in
ammonium hydroxide-based solvent did not yield any detectable
adducts. Additionally, cross-link adducts prepared by reaction
of rat lung nuclei with a known oxidant, potassium dichromate,
also behaved chromatographically similarly by producing adduct
DRZs in urea-based solvents and were abolished in ammonium
hydroxide-based solvents (Arif, J. M. and Gupta, R. C.,
unpublished data). Taken together, our data strongly suggest
that cigarette smoke-related adduct DRZs may represent DNA-
DNA and/or DNA-protein cross-links which can result from
either free radicals generated at the target site from catechols
and/or from smoke carcinogens such as aldehydes and butadiene,
all of which are present in levels that are orders of magnitude
298 Chem. Res. Toxicol., Vol. 19, No. 2, 2006Arif et al.
higher than the levels of lipophilic polyaromatic carcinogens Download full-text
In conclusion, our study clearly demonstrates higher levels
of adduct DRZs in lung tissues of current smokers than ex- or
never-smokers, consistent with the literature, but suggests that
the smoke-associated adducts are unrelated to typical polycyclic
aromatic hydrocarbons and aromatic amines. These DNA
adducts are implicated as products derived by cross-linking
agents such as aldehydes, butadiene, and catechols, which are
present in cigarette smoke in levels that are orders of magnitude
higher than the typical polyaromatic carcinogens. Our findings
clearly warrant a systematic study to determine the role of the
constituents in tobacco smoke other than typical PAHs, since
it would not only establish an association with tobacco smoke
lung carcinogenesis but also turn the focus of preclinical and
clinical intervention studies with appropriate chemopreventive
Acknowledgment. This work was supported by the USPHS
Grant CA-77114, NCI Master Agreement 97-N2-043, Kentucky
Lung Cancer Research Program, and in part from Agnes Brown
Duggan Endowment Funds (R.C.G.). Dr. Sibele Ina ´cio Meireles
is acknowledged for useful discussions.
(1) Jemal, A., Tiwari, R. C., Murray, T., Ghafoor, A., Samuels, A., Ward,
E., Feuer, E. J., and Thun, M. J. (2004) Cancer statistics, 2004. Ca
Cancer J. Clin. 54, 8-29.
(2) American Cancer Society (2005) Cancer Facts and Figuress2005,
pp 1-64, American Cancer Society, Atlanta, GA.
(3) Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer.
Carcinogenesis 91, 1194-1210.
(4) Alberg, A. J., Brock, M. V., and Samet, J. M. (2005) Epidemiology
of lung cancer: looking to the future. J. Clin. Oncol. 23, 3175-3185.
(5) Kruezer, M. P. B., Whitley, E., Ahrens, W., Gaborieau, V., Heinrich,
J., Jockel, K. H., et al. (2000) Gender differences in lung cancer risk
by smoking: a multicenter case-control study in Germany and Italy.
Br. J. Cancer 2, 227-233.
(6) Patel, J. D. (2005) Lung cancer in women. J. Clin. Oncol. 23, 3212-
(7) Hoffmann, D., Hoffmann, I., and El-Bayoumy, K. (2001) The less
harmful cigarette: a controversial issue. A tribute to Ernst L. Wynder.
Chem. Res. Toxicol. 14, 767-790.
(8) Hemminki, K., Koskinen, M., Rajaniemi, H., and Zhao, C. (2000)
DNA adducts, mutations, and cancer. Regul. Toxicol. Pharmacol. 32,
(9) Phillips, D. H., Hewer, A., Martin, C. N., Garner, R. C., and King,
M. M. (1988) Correlation of DNA adduct levels in human lung with
cigarette smoking. Nature 336, 790-792.
(10) Weston, A., and Bowman, E. D. (1991) Fluorescence detection of
benzo[a]pyrene-DNA adducts in human lung. Carcinogenesis 12,
(11) Santella, R. M., Grinberg-Funes, R. A., Young, T. L., et al. (1992)
Cigarette smoking-related polycyclic aromatic hydrocarbon-DNA
adducts in peripheral mononuclear cells. Carcinogenesis 13, 2041-
(12) van Schooten, F. J., Godschalk, R. W. L., Breedijk, A., et al. (1997)
32P-postlabeling of aromatic DNA adducts in white blood cells and
alveolar macrophages of smokers: saturation at high exposures. Mutat.
Res. 378, 65-75.
(13) Gupta, R. C., Sopori, M. L., and Gairola, C. G. (1989) Formation of
cigarette smoke-induced DNA adducts in the rat lung and nasal
mucosa. Cancer Res. 49, 1916-1920.
(14) Gairola, C. G., and Gupta, R. C. (1991) Cigarette smoke-induced DNA
adducts in the respiratory and non-respiratory tissues of rats. EnViron.
Mol. Mutagen. 17, 253-257.
(15) Arif, J. M., Gairola, C. G., Glauert, H. P., Kelloff, G. J., Lubet, R. A.,
and Gupta, R. C. (1997) Effects of dietary supplementation of
N-acetylcysteine on cigarette smoke-related DNA adducts in rat tissues.
Int. J. Oncol. 11, 1227-1233.
(16) Witschi, H. (2005) Carcinogenic activity of cigarette smoke gas phase
and its modulation by ?-carotene and N-acetylcysteine. Toxicol. Sci.
(17) Spencer-Beach, G. G., Beach, A. C., and Gupta, R. C. (1996) High-
resolution anion-exchange and partition thin-layer chromatography for
complex mixtures of32P-postlabeled DNA adducts. J. Chromatogr.,
B 677, 265-273.
(18) Gupta, R. C. (1996)32P-Postlabeling for detection of DNA adducts.
In Technologies for Detection of DNA Damage and Mutations (Pfeifer,
G. P., Ed.), pp 45-61, Plenum Press, New York.
(19) Gupta, R. C., and Earley, K. (1988)32P-adduct assay: comparative
recoveries of structurally diverse DNA adducts in the various
enhancement procedures. Carcinogenesis 9, 1687-1693.
(20) Culp, S. J., Roberts, D. W., Talaska, G., et al. (1997) Immunochemical,
32P-postlabeling, and GC-MS detection of 4-aminobiphenyl-DNA
adducts in human peripheral lung in relation to metabolic activation
pathways involving pulmonary N-oxidation, conjugation, and peroxi-
dation. Mutat. Res. 378, 97-112.
(21) Wiencke, J. K., Thurston, S. W., Kelsy, K. T., et al. (1999) Early age
at smoking initiation and tobacco carcinogen DNA damage in the lung.
J. Natl. Cancer Inst. 91, 614-619.
(22) Gupta, R. C., Arif, J. M., and Gairola, C. G. (1999) Enhancement of
pre-existing DNA adducts in rodents exposed to cigarette smoke.
Mutat. Res. 424, 195-205.
(23) . van Schooten, F. J., Hillebrand, M. J. X., van Leeuwen, F. F., et al.
(1992) Polycyclic aromatic hydrocarbon-DNA adducts in white blood
cells from lung cancer patients: no correlation with adduct levels in
lung. Carcinogenesis 13, 987-993.
(24) Boysen, G., and Hecht, S. S. (2003) Analysis of DNA and protein
adducts of benzo[a]pyrene in human tissues using structure-specific
methods. Mutat. Res. 543, 17-30.
(25) Myers, S. R., Spinnato, J. A., and Pinorini, M. T. (1996) Chromato-
graphic characterization of hemoglobin benzo[a]pyrene-7,8-diol-
9,10-epoxide adducts. Fundam. Appl. Toxicol. 29, 94-101.
(26) Myers, S. R., Spinnato, J. A., Pinorini-Godly, M. T., Cook, C., Boles,
B., and Rodgers, G. C. (1996) Characterization of 4-aminobiphenyl-
hemoglobin adducts in maternal and fetal blood-samples. J. Toxicol.
EnViron. Health 47, 553-566.
(27) Pryor, W. A. (1997) Cigarette smoke radicals and the role of free
radicals in chemical carcinogenicity. EnViron. Health Perspect. 105
(28) Randerath, K., Randerath, E., Smith, C. V., and Chang, J. (1996)
Structural origins of bulky oxidative DNA adducts (type II
I-compounds) as deduced by oxidation of oligonucleotides of known
sequence. Chem. Res. Toxicol. 9, 247-254.
(29) Lloyd, D. R., Phillips, D. H., and Carmichael, P. L. (1997) Generation
of putative intrastrand cross-links and strand breaks in DNA by
transition metal ion-mediated oxygen radical attack. Chem. Res.
Toxicol. 10, 393-400.
DNA Adducts in Cigarette Smokers’ LungsChem. Res. Toxicol., Vol. 19, No. 2, 2006 299