[CANCER RESEARCH 46, 5869-5877, November 1986]
Comparative 32P-Analysis of Cigarette Smoke-induced DNA Damage in Human
Tissues and Mouse Skin1
Erika Randerath, Tommie A. Avitts, M. Vijayaraj Reddy, Robert H. Miller, Richard B. Everson, and Kurt Randerath2
Departments of Pharmacology [E. K., T. A. A., M. V. R., K. R.] and Otorhinolaryngology ¡R.H. M.], Baylor College of Medicine, Houston, Texas 77030; and
Epidemiology Branch, Biometry and Risk Assessment Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
¡R.B. E.]
ABSTRACT
Previous studies using a highly sensitive "P-postlabeling assay for the
analysis of carcinogen/mutagen-induced DNA damage have shown the
presence of tobacco smoking-related DNA adducts in human placenta
(Everson, R. B., Randerath, E., Santella, R. M., Cefalo, R. C, Avitts, T.
A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The
occurrence of such adducts in smokers' bronchus and larynx is reported
here. Since the chemical nature of these adducts could not be character
ized by direct methods due to the extremely low levels of individual
adducts (<0.03 fmol per MUDNA), we have sought an experimental
animal model for studying the formation of tobacco-related DNA adducts.
Because cigarette smoke condensate is known to initiate tumors in mouse
skin, ICR mice were treated topically with cigarette tar equivalent to 1.5,
3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin
DNA was isolated 1 day after the last treatment. When DNA from
exposed mice was analyzed by the "P-postlabeling assay, 12 distinct ")'labeled DNA adduct spots, as well as a diagonal radioactive zone, which
presumably reflected the presence of incompletely resolved adducts, were
noted on polyethyleneimine-cellulose TLC fingerprints. One derivative
in particular (adduct 1) was seen to increase rapidly during the early
treatment phase and also to persist to 8 days after treatment. The
prominent adduct 1 was observed in the same location on the fingerprints
of DNA samples from smokers. Cochromatography experiments sug
gested identity of human and mouse DNA adduct 1. Similarly, several
other human and mouse adducts (adducts 3, 5, 6, and 9) appeared
identical, and the diagonal radioactive zone was also present on DNA
adduct maps from smokers. While absolute levels of individual human
adducts were too low to be accurately quantitated, semiquantitative
estimation of total tobacco-related aromatic DNA adducts in the human
specimens gave values of 1 adduct in (1.7-2.9) x Id nucleotides (0.100.18 fmol per pg DNA), with adduct 1 constituting 8.5-14% of the total.
On the basis of these results, it appears now feasible to determine the
chemical origin of smoking-induced DNA adducts in human tissues by
preparation of authentic 32P-labeled reference adducts from animals
treated with characterized subfractions of cigarette tar, "P-postlabeling,
and cochromatography of 32P-labeled human and animal adducts.
INTRODUCTION
Epidemiológica! studies have provided overwhelming evi
dence that tobacco smoking is the primary cause of lung cancer,
with about 85% of deaths from lung cancer being directly
attributable to smoking (1-3). In addition, in the United States,
tobacco smoking has been associated with an estimated 5070% of oral and laryngeal cancer deaths (1,3) and also with
cancer of the esophagus, bladder, pancreas, and kidney (1, 3).
Overall, tobacco smoking has been estimated to account for
30-40% of deaths from cancer (1-3) and 30-40% of deaths
from coronary heart disease (3, 4).
Received 4/21/86; revised 7/15/86; accepted 7/17/86.
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.
1This investigation was supported in part by a DuPont Occupational and
Environmental Health grant, USPHS grant CA 10893 (P6) awarded by the
National Cancer Institute, and a Laryngeal Research Fund of the Department of
Otorhinolaryngology.
2To whom requests for reprints should be addressed, at Department of
Pharmacology, Baylor College of Medicine, Texas Medical Center, Houston, TX
77030.
The molecular mechanism(s) of tobacco-associated carcinogenesis and other adverse health effects (3,4) in humans remain
unidentified, although the chemical composition of tobacco and
its combustion products have been extensively studied, and
more than 3800 compounds have been identified (1, 5-7). The
formation of covalent DNA addition products (adducts) has
been recognized as a key feature of the initiation of chemical
carcinogenesis for several years (8), and numerous studies have
shown the presence of mutagenic (9-11) and carcinogenic (2,
5, 12-15) compounds in tobacco smoke. The chemicals that
actually bind to human DNA as a consequence of exposure to
tobacco smoke have not been identified, however. No experi
ments have been reported to date that directly and unambigu
ously implicate known tobacco-associated carcinogens, e.g.,
PAHs,3 tobacco-specific nitrosamines, and aromatic amines, in
damage to human DNA in vivo and human carcinogenesis.
Knowledge of the composition of a complex mixture containing
genotoxic substances, such as tobacco smoke, is not sufficient
to pinpoint those chemicals that actually bind to DNA in vivo,
since mixtures may contain genotoxicants of varying potency
as well as modulators that effectively prevent or enhance the
formation of certain adducts in vivo. It is conceivable, for
example, that a quantitatively minor component of a mixture
may make a major contribution to total covalent DNA binding
and carcinogenesis. Therefore, DNA adduct formation needs
to be studied directly in the intact mammalian organism ex
posed to a mixture of genotoxicants in order to determine the
presence and nature of in vivo DNA-reactive chemicals, their
binding capacity relative to dose or intensity of exposure, and
the nature and properties of the adducts themselves. Until
recently, such investigations have not been conducted, in part
due to the lack of suitable methods for detecting and measuring
structurally unidentified DNA adducts in mammalian tissues.
A novel, highly sensitive "P-postlabeling assay for DNA
adduct analysis developed in our laboratory (16-22) has re
cently been used successfully to demonstrate the presence of
cigarette smoking-associated DNA adducts in human term
placentas (22, 23). The selectivity of the Chromatographie pro
cedures (18, 19) suggested that these adducts contained aro
matic carcinogen moieties. In the present investigation, ciga
rette smoke-induced DNA damage in humans and m mice was
further investigated by using this assay. A major goal of our
experiments was to find out whether genotoxic agents giving
rise to tobacco-related adducts in human DNA could be iden
tified via 32P-postlabeling assay. To this end, the assay was used
to search for DNA adducts in mouse tissue after treatment with
CSC. Since mouse skin is known to respond to initiating and
carcinogenic activities of tobacco smoke constituents (1, 123The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; CSC,
cigarette smoke condensate; BP, benzo(a)pyrene; BPDE I, a racemic mixture of
7/3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene
and its enantiomer; (+)anti-BPDE, (+)-enantiomer of BPDE I; PEI, polyethyleneimine; TLC,
thin-layer chromatography; RAL, relative adduct labeling; <RAL>, enhanced
relative adduct labeling under adduct intensification conditions; IF, adduct inten
sification factor; DRZ, diagonal radioactive zone.
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32P-ANALYSIS OF CIGARETTE
14), we compared skin DNA adducts from CSC-exposed mice
with adducts in tissues of smokers.
SMOKE-INDUCED
DNA DAMAGE
TLCSolvent1234567*Composition4.2
Table 1 Solvents for PEI-cellulose
urea.pH3.44.2
M lithium formate. 7.5 M
MATERIALS AND METHODS
Materials. Carrier-free [32P]phosphate (>300 mCi/ml) was obtained
from ICN Radiochemicals, Division of ICN Biomedicals, Inc., Irvine,
CA. [7-"P]ATP was prepared as described (18, 24). BP, benzo(gAi)perylene, 4-aminobiphenyl, 2-naph th>lamine, and trioctanoin
were from Sigma Chemical Co., St. Louis, MO, and benz(a)-anthracene, dibenz(oA)anthracene, chrysene, and fluoranthene were from
Aldrich Chemical Co., Milwaukee, WI. PEI-cellulose thin layers were
prepared in the laboratory (25) to ensure reproducibility of separations
and were freed from impurities by predevelopment in water. The sources
of materials for 32P-postlabeling assay, as well as safety precautions,
have been reported previously (16, 18, 19).
Preparation of Cigarette Smoke Condensate. CSC was prepared from
a common commercial brand of U.S. blended nonfilter cigarettes.
Smoke was collected from burning cigarettes in vacuo at -80°C, and
the condensate was taken up in acetone. After evaporation of acetone
in vacuo, the residue was dissolved in ether, and a small volume of
aqueous phase was discarded. After evaporation of ether, the material
was taken up in acetone to yield a concentration of about 30% (w/v),
corresponding to 0.75 cigarette/100 n\ condensate.
Animal Treatments. Female ICR mice (about 24 g) were maintained
on standard laboratory diet (Formulab) and water ad libitum. For
topical treatment with CSC, the backs of mice (3 per time point) were
shaved with clippers 3 days prior to treatment, and only mice in the
resting phase of the hair-growth cycle were used. CSC was applied to
the shaved area (5-6 cm2) with a capillary micropipet. Mice in group 1
received 2 doses, each equivalent to 0.75 cigarette/mouse, at 0 and 9 h
and were sacrificed at 28 h. Groups 2-4 received 2 doses, each equiva
lent to 0.75 cigarette, on days 1 and 2, then 1 dose, equivalent to 1.5
cigarettes, on subsequent days. Group 2 mice were treated for a total
of 3 days and sacrificed on day 4. Animals in groups 3 and 4 received
treatment over a total of 5 and 7 days, respectively, and were also
sacrificed 1 day after the last administration. Total exposure to CSC,
therefore, was equivalent to condensate from 1.5 (group 1), 3 (group
2). 6 (group 3), and 9 (group 4) cigarettes/mouse, respectively. While
the mice tolerated these doses, some acute toxic symptoms (probably
due to nicotine) were noted, and an increase in dosage by 30-40%
resulted in some deaths. Therefore, the doses indicated were high and
close to maximum tolerated amounts. Treatment for group 5 was the
same as for group 4, except that the animals were sacrificed 8 days
after the last treatment. Control mice received acetone alone and were
treated for the same length of time as were those in group 4. For the
preparation of skin DNAs adducted with reference carcinogens [BP,
benz(a (anthracene, dibenz(aA)anthracene,
chrysene, fluoranthene,
benzo(/?A/)perylene], mice (3 per group) were shaved, treated with 4
doses of 1.2 /¿molof test compound in 200 nI of acetone as described
(19, 21), and sacrificed 24 h or 2 weeks (BP) after the last treatment.
Tissues were collected and stored at —80°C
until DNA extraction. For
the preparation of liver DNAs adducted with 4-aminobiphenyl or 2naphthy lamine, mice were given ¡.p.injections of 25 nmol of chemical
in 0.1-0.2 ml trioctanoin/dimethyl sulfoxide (8/1, v/v) and sacrificed
24 h posttreatment. Livers were stored at —80°C.
urea.pH3.64.2
M lithium formate, 7.5 M
urea.pH3.80.8
M lithium formate, 7.5 M
TrisMCI,
M lithium chloride, 0.5 M
8.00.6 8.5 M urea, pH
TrisHCI,
M lithium chloride, 0.5 M
8.00.7 8.5 M urea, pH
urea.pH
M sodium phosphate, 7 M
6.42-Propanol/4
(28/22;by N ammonia
vol.)Direction*111222
' In two-dimensional PEI-cellulose TLC.
* Used for rechromatography.
laryngeal carcinoma was examined. All tissues were stored at -80°C
until DNA extraction.
DNA Isolation. DNA was isolated from 0.2-0.5 g of tissue by a
modification (26) of a standard solvent extraction procedure (27). DNA
concentrations were estimated spectrophotometrically using a value of
20 A26ounits of DNA per mg.
Enzymatic DNA Digestion and 32P-labeling of DNA Nucleotides.
DNA adduction was analyzed by a modification of the 32P-postlabeling
assay (16-22) under ATP-deficient conditions in order to achieve
enhanced adduct labeling (21, 28). DNA (4 ¿ig)
was digested to deoxyribonucleoside 3'-monophosphates in a total volume of 10 n\ as de
scribed (18, 28), except that the concentrations of micrococcal endonuclease and spleen exonuclease were 0.05 unii //i and 0.43 jig/nl,
respectively. For 32P-labeling, the reaction mixture (15 p\) contained
800 UMDNA-P, 3 iiM h-32P]ATP (4000 Ci/mmol), and 0.12 units of
polynucleotide kinase per ¡i\.Incubation at pH 9.5 and 38°Cwas for
30 min. For cochromatography of adducts from CSC-exposed mouse
skin DNA with adducts from smokers' placenta! DNAs or various
mouse skin PAH-DNAs [obtained by treatment with benz(a)-anthracene, dibenz(flA)anthracene, chrysene, fluoranthene, or benzo-(gAi)perylene], 4 ng each of the DNAs to be compared were combined before
digestion and labeling. Reaction volumes were doubled, but concentra
tions were not changed. In all other cochromatography experiments,
smaller amounts of reference DNAs (0.2-0.3 tig) were combined with
4 MgCSC-DNA, and digestion and labeling reactions were performed
as described above for 4 ng DNA.
Chromatography. The labeled digest was divided into a 2.5- and a
12.5-^1 aliquot for the analysis of total nucleotides and adducts, respec
tively. The 2.5-^1 portion was diluted with 1.0 ml of 20 mM Tris-HCl,
pH 9.5, and 5 nl of the diluted solution was applied to a PEI-cellulose
sheet in duplicate. Development was with 40 m\i ammonium sulfate,
which kept 32P-labeled normal nucleotides and adducts at the origin,
but removed a small amount of [32P]P,. Adducts in the 12.5-^1 aliquot
were freed from normal nucleotides and other impurities by chromatography on PEI-cellulose in l M sodium phosphate, pH 6.8. For 2directional chromatography, the adducts remaining at the origin were
contact-transferred to a fresh PEI-cellulose sheet by a magnet technique
(29, 30). Solvents have been listed in Table 1. Some remaining radio
active background material was removed by an additional development
in 1.7 M sodium phosphate, pH 6.0, similarly as described (31).
For rechromatography, 32P-labeled adduct spots were cut from PEIcellulose chromatograms and desalted by soaking in 30-50 ml water;
then the adducts were transferred to fresh PEI-cellulose acceptor sheets
by the magnet technique (29) and chromatographed in solvent 7 (Table
1).
Calculation of Relative Adduct Labeling. Relative adduct labeling
values, denoted RAL and (RAL), have been defined previously for 32P-
Human Tissues. Term placentas were obtained from healthy volun
teers (2 smokers and 1 nonsmoker). The smokers consumed an average
of 16 cigarettes/day during the third trimester of pregnancy. Placentas
that exhibited relatively high smoking-related adduci levels (23) were
selected to facilitate the cochromatography experiments. Bronchial
tissues were autopsy specimens. Smoker's bronchus was from a 66year-old white man who had smoked >1.5 packs of cigarettes per day
for over 20 years but had stopped smoking 3 weeks prior to his death
in preparation for surgery for an aortic aneurysm. Nonsmoker's bron
postlabeling assay under standard (18, 21) and adduct intensification
(28) conditions, respectively. Provided that adduct recovery was quan
chus was from a 70-year-old white woman. Aryepiglottic fold mucosa
was obtained from the larynx of a 56-year-old white woman who
titative, RAL represented the actual DNA adduct level, since both
smoked I pack of cigarettes per day. A small piece of tumor-free tissue
adducts and normal nucleotides were labeled quantitatively with excess
ATP, and was calculated according to Equation A or B (18):
that had been removed as part of a routine surgical procedure for
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"P-ANALYSIS
CPM
RAL =
addiicllsl
cleotides +
CPM
RAL =
CPM,0,a|
OF CIGARETTE
SMOKE-INDUCED
(A)
CPM.ddu.
adducili)
(B)
An adduct level of RAL = X x 10~7 is equivalent to X adducts in IO7
nucleotides or 1 adduct in (1/X) x IO7 nucleotides or 0.3 x X fmol
adduct per ^g DNA.
Since the levels of most DNA adducts analyzed here were too low to
be quantitated by the standard procedure (16, 18-21), their levels were
estimated by the more sensitive intensification version of the 32Ppostlabeling assay (21, 28). In this procedure, ATP-deficient conditions
result in preferential 32P-incorporation into adducts and hence en
hanced RAL (designated <RAL» values. (RAL) values were deter
mined according to Equation B, but the count rates used were from
ATP-deficient conditions. RAL was calculated from (RAL) by Equa
tion C (21, 28):
(C)
Intensification factors were determined in separate model experiments
by means of Equation C (28) or were estimated when this was not
possible (see "Results").
To derive time course data for adducts in CSC-exposed mouse skin
DNA, adduct spots as well as 0.7-cm2 areas of the maps located to the
right of adduct 1 (i.e., within the DRZ) and to the left of adduct 11,
respectively, were cut from 3 replicate chromatograms for each treat
ment time (see "Results"; Fig. 2, b-e) and counted by Cerenkov assay.
Count rates from these 2 areas were subtracted as blanks from count
rates of spot 1 and spots 10, 11, and 12, respectively. (RAL) values
were calculated from the count rates of adducts and total nucleotides
(18, 19) by Equation B. To estimate the levels of total (presumably
aromatic) tobacco-related DNA adducts, a (RAL) value was derived
from the DRZ plus all distinct adduct spots. For determining adduct
count rates, layer material corresponding to the DRZ, including the
distinct spots within this zone, was scraped from triplicate CSC-DNA
maps, while adduct spots located outside the DRZ were cut out.
Identical areas of control DNA maps (see "Results"; Fig. 2a) served as
blanks. (RAL) values for adduct 1 and total adducts of smoker's
bronchial and placenta! DNA's (see "Results"; Fig. 1) were determined
in a similar fashion.
To calculate the level of the reference BP-DNA adduct (see "Results";
Fig. 5), the adduct spots (panels c and d) and corresponding blank
areas (panels a and b) were cut from the chromatograms and counted
by Cerenkov assay. Count rates of the blanks were subtracted from the
sample count rates. (RAL) of the adduct was estimated as described
above, and RAL was derived from Equation C using a value of 11 for
IF, determined separately. The level of the BP-DNA adduct (see "Re
sults") represented the mean of 2 determinations.
RESULTS
DNA Damage in Smokers' Tissues. When DNA from bron
chus, aryepiglottic fold, and placenta of smokers was analyzed
by the intensification version of the 32P-postlabeling assay (21,
28), several radioactive spots were noted (Fig. 1, ¿>,
c, and e)
that were not present on maps of DNA from nonsmokers (Fig.
1, a and d). In addition to the distinct spots, the smokers'
samples exhibited a gray area extending from the origin region
to the upper right-hand margin of the autoradiograms. This
area, the DRZ, was particularly prominent on the autoradi
ogram from the bronchial DNA (Fig. 1¿>).
An overall similarity
of the adduct patterns was noted for the different tissues.
Specifically, the 3 smokers' DNA's appeared to have adduct
spots /, 3, and 9 in common.
Chromatographie
also shown by cochromatography experiments (see "Compari
son of Human and Mouse DNA Alterations"). But there were
also distinct differences. Thus, in the DNA of aryepiglottic fold
(panel c), adduct 2 was relatively strong, while adduct 5 was
not detected. The bronchial sample (panel b) had 2 additional
spots (spots 6 and 13). Spot 1 was a major adduct in smokers'
nuclcolio«.
RAL = (RAL)
IF
DNA DAMAGE
identity was
bronchus, aryepiglottic fold, and placenta and was also detected
in lung parenchyma and tonsils.4 This adduct had been previ
ously observed in 16 out of 17 term placentas of women who
smoked during pregnancy (23) but was absent from placentas
(23) and other tissues4 of nonsmokers. The DRZ appeared to
be related to smoking; it was never detected on maps from
nonsmokers (see, for example, panels a and d).
DNA Damage Induced in Mouse Skin during Topical Treat
ment with Cigarette Smoke Condensate. When DNA prepara
tions from female ICR mouse skin exposed to various amounts
of CSC were analyzed by the intensification version of the 32Ppostlabeling assay, the autoradiograms shown in Fig. 2, panels
b-e, were obtained. As evident from a comparison with the
acetone control (Fig. 2a), the number and amounts of individual
adducts as well as the intensity of the DRZ increased with
increasing length and extent of exposure. Several distinct ad
duct spots were clustered near the center of the DRZ. Individual
adducts formed at different rates and attained different extents
of 32P-labeling. After approximately 1 day of exposure (panel
b), only a single adduct (no. 1) was detectable. While exposure
to condensate from 3 cigarettes (panel c) resulted in a number
of additional minor adducts (adducts 2-8) DNA from skin
exposed to 6 (panel d) or 9 (panel e) cigarettes exhibited 4
additional adducts (adducts 9-12). These results showed that
the 32P-postlabeling technique detected specific and dose-de
pendent tissue DNA alterations elicited by exposure of mice to
a complex environmental mixture. Since the Chromatographie
procedure used was selective for aromatic DNA adducts (18,
19), the extra spots were presumably derived from aromatic
constituents of CSC. The presence of bulky unsaturated, nonaromatic substituents (but not small alkyl groups) in adducts
would also be consistent with their Chromatographie behavior.
The DRZ possibly reflected the presence of additional, incom
pletely resolved adducts.
Determination of the time course of relative adduct labeling
during CSC treatment (Fig. 3) indicated that 1 adduct in
particular (adduct 1) increased rapidly during the early phase
of treatment of mouse skin with CSC, while other adducts
formed more gradually. The intensification of the DRZ with
time resembled the time course of formation of adduct 1 (Fig.
3). Adduct 1 differed from the other distinct adducts not only
by its faster rate of formation, but also by its slower rate of
disappearance after withdrawal of CSC: 8 days after discontin
uation of treatment this adduct was still detectable at about
10% of its 8-day value, while other adducts could no longer be
detected (data not shown). Furthermore, adduct 1, but not the
other distinct adduct spots, was also noted in liver DNA from
mice exposed to CSC for 8 days (Fig. 4b). 32P-incorporation
into adduct 1 in liver DNA was 8-10 times lower than for the
corresponding skin DNA. Notably, a DRZ was also present on
maps from livers of CSC-exposed mice (Fig. 4b) but absent
from control samples, indicating that this zone reflected CSCinduced DNA alterations in a mouse tissue other than the skin.
Cochromatography Experiments with Adducted Reference
DNAs. To test whether distinct adducts noted on 32P-maps
(Fig. 2) were derived from aromatic carcinogens known to occur
* K. Randerath, T. A. Avitts, R. H. Miller, and E. Randerath; unpublished
experiments.
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"P-ANALYSIS
Fig. I. "P-Postlabeling
assay of DNA
damage induced in human (issues by cigarette
smoking. For sources of tissues, see text. DNA
(4 ng) was assayed by "P-postlabeling under
adduci intensification conditions. Chromatography was on PEI-cellulose in solvents 2 and
4 of Table 1, and screen-enhanced autoradiography was for 4.5 days at -80'C. Adduci spots
that appeared common to at least 2 of the
smokers' DNAs have been numbered. Note the
presence of several additional spots and a DRZ
in each of the fingerprints of smokers' DNAs.
OF CIGARETTE
SMOKE-INDUCED
Non-smoker
Bronchus
DNA DAMAGE
Smoker
Bronchus
Smoker
Aryepiglottic fold
Adduci numbers correspond to those in Fig. 2,
except those for adducts 2 and 13 (see also
legend to Fig. 6 and text). The presence of
adduci 6. which was not detected on the autoradiogram (panel e), in the smoker placental
DNA was observed when the sample was tested
in a different system (see Fig. 6, panels b and
e).
Non-smoker
Placenta
Fig. 2. Time dependence of specific covalent DNA damage in mouse skin induced dur
ing topical treatment with CSC, as determined
via J2P-postlabeling assay. Mice were treated
as described in the text. DNA (4 ^g) was ana
lyzed by "P-postlabeling assay under adduci
intensification conditions (see text). Labeled
adducts were separated on PEI-cellulose thin
layers in 2 directions in solvents 2 (from bot
tom to top) and 4 (from left to right) of Table
1 and located by screen-enhanced autoradiography for 4 days at -80°C.The control sample
exhibited several background spots that were
also present on the other autoradiograms. Adduct spots have been indicated by numbering.
The location of adduci 2 relative to that of
adduci 1 was somewhat variable. Note DRZ
on panels b-e extending from lower left to the
upper right.
Smoker
Placenta
a
Acetone
Control
b
CS Condensate
(1.5 cig., 28 h)
CS Condensate
(6cig., 6d)
CS Condensate
(3cig.,4d)
CS Condensate
(9cig-, 8d)
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"P-ANALYSIS
1.5
3
6
OF CIGARETTE SMOKE-INDUCED
9
a m
NUMBER OF CIGARETTES
Fig. 3. Time course of <RAL> for the major adduci fractions from Fig. 2, as
determined by Cerenkov counting. Spots were excised from the chromatograms,
and their radioactivity was evaluated by Cerenkov assay. HAI values, which
are proportional to adduci levels, were calculated from the count rates of adduci
spots, as described in the text. A 0.7-cm2 area immediately to the right of adduci
1 (located in the DRZ) was also evaluated. A, adduci 1; •,0.7-cm2 area to the
right of adduci I; O. adduci 10; D, adduci 11; V. adduci 12.
Acetone
Control
DNA DAMAGE
CS Condensate
(9cig.,8d)
Fig. 4. 32P-Postlabeling assay of DNA damage induced in mouse liver by
topical application to mouse skin of CSC. Fingerprints (panels a and />) were
derived from liver DNA (4 ^g) of acetone- (panel a) or CSC- (panel b) treated
mice. "P-Postlabeling assay was carried out under adduct intensification condi
tions. Chromatography on PEI-cellulose was in solvents 3 and 6 of Table 1, and
screen-enhanced autoradiography was for 4.5 days at —80"C.Spots located above
the DRZ (panel b) and in the corresponding location on panel a are due to
incorporation of label into unidentified background material.
in tobacco smoke, mixtures of adducted reference DNAs with
CSC-exposed mouse skin DNA were digested, labeled, and
fingerprinted on PEI-cellulose (19) in the solvent systems of
Table 1. The reference DNAs, prepared by treatment with BP,
benz(a)anthracene,
dibenz(a/i)anthracene,
chrysene, fluoran
thene, benzo(gA/)perylene, 4-aminobiphenyl, and 2-naphthyl
amine, respectively, as described in "Materials and Methods,"
contained 1 adduct in 2 x 105-108 nucleotides.
The location of the major BP-induced DNA adduct of mouse
skin, i.e., the reaction product of (-t-)anti-BPDE with N2 of
guanine (32, 33), in relation to adducts induced by CSC is
shown in Fig. 5. The BP adduct migrated in the lower portion
of the DRZ, but did not cochromatograph with any distinct
CSC-derived spot. The reference BP-DNA adduct seen in Fig.
5, panels c and d, corresponded to a level of 2.8 adducts in 10"
DNA nucleotides or 0.084 fmol adduct in 1 ¿tgDNA. If the
CSC-exposed mouse skin DNA (panelb) contained this adduct,
we estimate that its level was below 1 adduct in 5 x 108-109
nucleotides; otherwise the adduct would have been distinguish
able as a spot from the surrounding DRZ.
Among the reference adducts studied, only 1 of the 4-aminobiphenyl-DNA derivatives, 3',5'-bisphosphate
of N-(deoxyguanosin-8-yl)-4-aminobiphenyl,
and the major dibenz(aA)-
b
Acetone
Control
CS Condensate
(9cig., 8d)
a t BP
b t BP
Fig. 5. Comparison of [32P]adducts induced in mouse skin DNA by CSC and
BP, respectively, via cochromatography on PEI-cellulose TLC. Fingerprints were
derived from 4 ng of individual DNAs (panels a and A) or from mixtures of 4 ng
each of these DNAs with 0.16 ^g of BP-modified DNA (panels c and d). "PPostlabeling assay was carried out under adduci intensification conditions. l'Ino
matography was in solvents 1 and S of Table 1, and screen-enhanced autoradiography was for 4 days al -80'C. The major BP-induced DNA adduci, i.e., the
reaction product of (+)anti-BPDE with NJ of guanine, has been indicated by an
arrow, d, day.
anthracene-DNA adduct were found to overlap adduci 1 in the
solvents used for the maps of Fig. 2. However, the 4-aminobiphenyl adduct was separated from adduct 1 when concentra
tions of the first- and second-dimension solvents were reduced
to 85 and 65%, respectively, by the addition of water. Also, the
dibenz(a/i)anthracene
adduct and adduct 1 were resolved by
rechromatography on PEI-cellulose in solvent 7 (Table 1).
Thus, adduct 1 may not be related to any of the tobacco smoke
carcinogens listed above. On the other hand, most PAH- and
aromatic amine-induced DNA adducts, such as derivatives of
BP (Fig. 5), benz(a)anthracene, chrysene, fluoranthene, 4-aminobiphenyl, and 2-naphthylamine, chromatographed
in the
DRZ, but did not coincide with any distinct CSC-induced spot
(autoradiograms not shown).
Comparison of Human and Mouse DNA Alterations. When
DNA adduct maps from tissues of human smokers (Fig. 1) and
CSC-exposed mouse skin samples (Fig. 2) were compared under
identical Chromatographie conditions, similar locations of sev
eral adducts (adducts 1, 3, 5,6, and 9) and the DRZ were noted.
To investigate this observation further, human and mouse DNA
preparations were mixed, digested, 32P-labeled, chromato
graphed under different conditions, and autoradiographed. Se
lected results of such cochromatography experiments are shown
in Fig. 6. Here, vertical rows represent identical DNA samples,
while horizontal rows correspond to identical Chromatographie
conditions, as outlined in the figure legend. Placenta! DNA
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32P-ANALYSIS OF CIGARETTE SMOKE-INDUCED
DNA DAMAGE
•
2- 'I
-3
-5
-6
6
12-
12-
12-I
10-5
10
-14 I
-12
i
-14
Non-smoker
Placenta
ï
Smoker#l
Placenta
3 •
14
I
h|
n
10-
3
-12
-3
CS Condensate
(9cig.,8d)
Smoker#2
Placenta
6
Ir
m
-12
-3
-14
Smoker #1
Placenta
t CSC
L
Smoker #2
Placenta
t CSC
Fig. 6. Comparison of smoking-induced DNA lesions in human placentas with DNA damage in mouse skin treated with CSC: cochromatography of labeled DNA
digests on PEI-cellulose in 3 solvent combinations. Individual DNAs or mixtures of the two DNAs to be compared (4 *igeach) were subjected to 32P-postlabeling as
described in the text. The solvent combinations used were: (I) Solvents 1 and 5 of Table 1 (panels a-f)\ (II) solvents 3 and S (panels g-m); and (III) solvents I and 6
(panels n-s). Screen-enhanced autoradiography was for 3-4 days at —
80"C. Three relatively intense and several weaker background spots may be noted on
autoradiograms from nonsmoker placenta! DNA (panels a, g, and n); these spots were present on the other autoradiograms, where they were readily distinguished
from tobacco-related adducts and the DRZ. The background spots, which were also obtained from labeled digest of acetone-treated mouse skin DNA under identical
Chromatographie conditions (Figs. Sa and da), may have been derived from assay-related enzymes containing bound "P. Smoker 1 placenta! DNA was the same as
that represented in Fig. I, panel e.
from nonsmokers (see examples in the first vertical row of Fig.
6) displayed 3 radioactive background spots, which were noted
on all other maps also and served as reference points for the
location of adducts. Placenta! DNA samples from smoking
women (second and third vertical rows) exhibited a prominent
adduci 1 spot and a DRZ in each Chromatographie system
examined. Comparison of the fingerprints obtained with the 3
solvent combinations (Fig. 6) showed that minor variations of
the solvent compositions and pH greatly influenced the location
of adducts and background spots. In each of the 3 solvent
combinations, adduct I as well as the DRZ from human and
mouse tissues comigrated exactly (Fig. 6). In addition, in sys
tems I and II, other distinct adduct spots (spots 3, 5, and 6),
also chromatographed identically. With the exception of adduct
3, this was not readily apparent in system III because of overall
poorer resolution. Spot 9 from human and mouse sources also
showed coincidence in solvents 2 and 4 (Table 1; see Figs. 1
and 2). This adduct was not sufficiently separated from the
DRZ in systems I, II, and III (Fig. 6). An additional adduct
(adduct 14) was detected in smoker 1 placenta! DNA in these
systems. Analogous experiments with mixed DNA from human
bronchus and placenta showed Chromatographie identity of
adducts 1, 2, 3, 5, and 9 (autoradiograms not shown). Taken
together, these results strongly suggest identity of tobaccorelated covalent DNA alterations in human tissue and mouse
skin. To substantiate this conclusion, the major 32P-labeled
adduct (adduct 1) from individual and mixed human and mouse
DNA preparations containing tobacco-related DNA alterations
were rechromatographed in solvent 7 (Table 1). This solvent,
which provided high resolution by partition chromatography of
many aromatic DNA adducts, did not resolve human and mouse
adduct 1. Such experiments were also attempted for the other
adducts shown in Fig. 1, but no conclusive results were obtained
because of contaminating label contributed by the DRZ.
Each of the 3 two-dimensional TLC systems gave distinct
patterns of background and adduct spots (Fig. 6), which was
also true for the combination of solvents 2 and 4 (Figs. 1 and
2). While systems I and II led to comparable adduct separations,
the DRZ was compressed along the diagonal of the maps in
system III. This resulted in an improved separation specifically
of adduct 1 from the DRZ (Fig. 6). Combined use of the solvents
listed in Table 1 thus contributed to the Chromatographie
characterization of tobacco-related DNA adducts in human and
mouse tissues and thus may eventually aid in the identification
of the chemicals responsible for the formation of the adducts.
Levels of Cigarette Smoke-induced DNA Adducts. Approxi
mate levels of (presumably aromatic) human and mouse DNA
adducts, which were too low to be analyzed by the standard 32Ppostlabeling assay, were estimated by 12P-postlabeling assay
under ATP-deficient conditions (28). Preferential labeling of
adducts over normal nucleotides (28) was evident because the
extent of labeling of adducts, but not normal nucleotides, de
pended on the DNA concentration when [ATP] was kept con
stant and was <sc [DNA-P] (data not shown). According to
experience gained in our laboratory with about 30 bulky and
aromatic adducts derived from carcinogens of diverse structure
[(21, 28, 34) and unpublished work5], adduct IPs usually range
5 K. Randerath, J. A. Weaver, L.-J. W. Lu, M. E. Schurdak, and E. Randerath;
unpublished experiments.
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"P-ANALYSIS
OF CIGARETTE SMOKE-INDUCED
from 10 to 30. Assuming an IF of 20, we calculated the level of
adduct 1 in mouse skin DNA exposed to 9 cigarettes from the
data of Fig. 3. The (RAL) value of 1.56 (±0.23)x IO"7 for this
adduct would correspond to 1 adduct in 1.3 (±0.20) x 10*
nucleotides according to Equation C. With the same assump
tion, the approximate total tobacco-related DNA damage in
this tissue, detectable by the technique described in "Materials
and Methods," was calculated to correspond to 1 adduct in 6.53
(±0.58)x IO6 nucleotides (RAL = 1.53 x 10~7). The approxi
mate level of smoking-induced DNA adduct 1 in human bron
chus and placenta samples (Fig. 1) was estimated similarly to
be 1 adduct in about 2 x IO8 nucleotides. Since the 2 placenta!
specimens included in this study had levels of adduct 1 that
were among the highest values observed earlier (23), the levels
of placenta! adducts presented here were probably higher than
would be typically expected. The other adducts marked on the
maps shown in Fig. 1 were not estimated individually, since
their location within the DRZ made measurement difficult.
Likewise, the amount of adduct 1 in aryepiglottic fold (Fig. le)
was insufficient for estimation. However, when total tobaccorelated radioactivity present on these maps was evaluated,
(RAL) values of (7-12) x 10~7 were obtained, which corre
sponds to approximately 1 adduct in (1.7-2.9) x IO7 DNA
nucleotides. Thus, adduct 1 constituted about 5 and 8.5-14%
of the total covalent DNA lesions estimated for mouse skin and
human tissues, respectively.
DISCUSSION
The finding of distinct cigarette-smoke associated DNA ad
ducts in target tissue of smoking-related carcinogenesis (Fig. 1)
is consistent with the generally accepted notion that covalent
DNA lesions play a key role in the initiation of chemical
carcinogenesis (8, 35-38). The persistence of major smokinginduced adducts in human bronchial DNA 3 weeks after ces
sation of smoking (Fig. 1B) is also in accord with correlations
between long-time persistence of adducts in target cells and the
maintenance of the initiated state in a number of animal models
(28, 38-43). In addition, ex-smokers are at a prolonged risk of
developing smoking-related cancer (3). Since smoking-induced
adducts may form part of the molecular link between specific
components of tobacco smoke and human genetic damage and
cancer, their identification appears important for the elucida
tion of the mechanism(s) of smoking-induced carcinogenesis.
As a first step toward this end, experimental approaches need
to be designed that would permit the identification of those
genotoxicants in tobacco smoke that actually bind to the tissue
DNA in intact mammalian organisms exposed to this complex
mixture. As pointed out earlier, a study of the genotoxic activity
of individual compounds, while providing valuable information,
may not be adequate to pinpoint the most active genotoxic
components of the mixture.
In theory, the most direct way to identify the structures of
the adducts formed would be to isolate them and then determine
their structures by established physicochemical methods. How
ever, this does not appear practical because the isolation of 1
Mgof an adduct present at a level of 10~s mol/mol DNA-P [as
estimated for the most prominent adduct (adduct 1) investigated
here] would require about 100 g of DNA or 100 kg of tissue.
Therefore, alternative indirect methods are required to elucidate
the chemical origin of tobacco-associated adducts in human
DNA. One possibility, suggested by the results reported here,
would entail the Chromatographie characterization, e.g., via
TLC, of "P-postlabeled adducts from tissue of experimental
DNA DAMAGE
animals exposed to mixed tobacco genotoxicants in vivo, fol
lowed by the application of the same technique to purified
subfractions of the mixture so as to identify the components
responsible for specific adducts elicited by the mixture. Adducts
formed with authentic compounds in experimental animals
would then be subjected to a detailed Chromatographie com
parison with human DNA adducts.
Since mouse skin has been the tissue of choice for the study
of initiating activity of tobacco smoke constituents (1, 12-14),
and since our previous work had shown i2P-postlabeling assay
to be suitable for the analysis of DNA adducts in mouse skin
(19-22, 28, 34, 39, 44), we applied CSC to mouse skin and
then searched for DNA adducts in the treated tissue by means
of this assay. The detection of a number of distinct adduct spots
on the CSC-DNA fingerprints (Fig. 2) raised the question of
whether some of these adducts were derived from known car
cinogens in tobacco smoke (2, 5, 12-15). In particular, we
wondered whether CSC-DNA contained detectable amounts of
the major BP adduct, i.e., the reaction product of (+)anti-BPDE
(BPDE I) with N2of guanine (32, 33), because of the possibility
that smoking-induced cancer may be linked to the presence of
BP in tobacco smoke (45). While this adduct was not detected
as a well-defined spot (Fig. 5b), the DRZ may conceivably have
masked its presence. Our estimation of a maximal level of 1
BPDE I-adduct in 5 x 10*-10" DNA nucleotides (see "Results")
implies that the contribution of this compound (if present) to
the estimated total tobacco-related aromatic DNA alterations
detected by the 32P-postlabeling assay (RAL » 1.5 x 10~7)
amounted to 0.7-1.3%. The low level or absence of this adduct
may have been due to the modulation of the formation of BPDNA adducts by other components of tobacco smoke, such as
inhibitors of BP metabolism (46) or inducers of arylhydrocarbon hydroxylases that may interfere with BPDE I-DNA adduct
formation (47). Therefore, DNA lesions other than those in
duced by BP may make a more significant contribution to the
initiation of tobacco carcinogenesis in mouse skin.
Our interpretation that the DRZ presumably consisted of a
number of incompletely resolved adducts is based on several
facts: (a) the Chromatographie location of the various reference
adducts within the DRZ; (b) the presence of several dozen
aromatic carcinogens in cigarette smoke (1), each of which may
give rise to adducts on the thin-layer maps; and (c) the strong
dose and time dependence of formation of the DRZ (Figs. 2
and 3). The DRZ was indicative of exposure of DNA to tobacco
products and was not detected in any other adducted DNA
studied to date (18-21, 28, 30, 31, 34, 39, 44, 48-50). Its
formation was dependent on the action of T4 polynucleotide
kinase in the labeling reaction. In view of the absolute specificity
of this enzyme for labeling of 5'-hydroxyl groups of nucleotides
(51, 52), labeling of nonnucleotide contaminants can be ex
cluded as a source of 32P-label in the DRZ. Further characteri
zation of this material will probably require both subfractionation of CSC and refinement of Chromatographie conditions for
the "P-labeled compounds. The electrolyte/urea systems de
veloped by us for the resolution of aromatic carcinogen-DNA
adducts on PEI-cellulose TLC afford high resolution of struc
turally related nucleotide derivatives of individual or closely
related carcinogens (18-21, 31, 34). Chromatographie condi
tions may be refined through a combination of PEI-cellulose
TLC with regular or reversed-phase partition chromatography
(see Ref. 50).
The results reported here provide evidence for a remarkable
similarity of tobacco smoke-related covalent DNA alterations
in different human and mouse tissues. Many additional exper-
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"P-ANALYSIS
OF CIGARETTE
SMOKE-INDUCED
iments that have not been presented in "Results" all showed
Chromatographie identity of adducts 1, 3, 5, 6, and 9 as well as
the DRZ from the different sources in the solvents of Table 1.
These results presumably indicate that certain metabolic path
ways leading to cigarette smoke-associated DNA adduction
were common to mouse skin and both target and nontarget
human tissues. The mouse skin bioassay may thus become a
valuable tool in the preparation of reference compounds for the
characterization of human adducts, provided that the same
adducts can be unambiguously shown to be present in mouse
DNA.
When total aromatic adduci levels in mouse skin (Fig. le)
and human tissues of smokers were estimated by 32P-postlabeling assay, similar values [1 adduci in (0.7-2.9) x IO7 DNA
nucleotides] were obtained. This represents our best estimate,
since intensification factors are different for each individual
aromatic adduct, but an average value of 20 appears reasonable
on the basis of our experience (see "Results"). In view of the
overall Chromatographie similarities, the mouse/human com
parison appears valid. (It may be noted that no other method
is currently available to measure traces of unidentified DNA
adducts formed in mammalian tissue by exposure to a complex
genotoxic mixture in vivo.) Since the value for the bronchial
DNA was from a former smoker, the adduct levels were prob
ably higher during the active phase of smoking and may well
have exceeded those present in the mouse skin DNA. These
results demonstrate substantial cigarette smoking-related DNA
damage in human target tissue. The contribution of these
genetic alterations, which are presumably present for many
years of an active smoker's life, to smoking-induced cancer
appears firmly established on the basis of current knowledge of
chemical carcinogenesis (8, 35-37, 40, 53, 54).
This paper provides further evidence for the measurement of
human carcinogen exposure (55) by 32P-postlabeling assay. Key
features of this approach are: (a) Application to complex mix
tures the components of which may not have been adequately
characterized as to chemistry and/or genotoxic activities in the
intact mammalian organism; (/»)quantitative or semiquantitative estimation of DNA binding; (c) sensitivity of detection for
aromatic derivatives of 1 adduct in 109-10'°nucleotides (0.3-3
attorno! adduct/V g DNA); and (d) exposure-specific Chromat
ographie fingerprints that may aid in the identification of the
DNA-reactive chemicals (or their metabolites) in the genotoxic
mixture. In view of these properties, it is our belief that postlabeling technology is likely to yield valuable insights into levels
of carcinogen exposure in humans.
ACKNOWLEDGMENTS
The authors are grateful to Dr. James G. Lewis, Pathology Depart
ment, Duke University Medical Center, for providing access to autopsy
specimens of human bronchus.
REFERENCES
1. United States Public Health Service. The Health Consequences of Smoking:
Cancer. A Report of the Surgeon General. Rockville, MD: United States
Department of Health and Human Services, Office on Smoking and Health,
1982.
2. Loeb, L. A., Ernster, V. L., Warner, K. E.. Abbotts, J., and Laszlo, J.
Smoking and lung cancer: An overview. Cancer Res., 44: 5940-5958, 1984.
3. Fielding, J. E. Smoking: Health effects and control (First of two parts). N.
Engl. J. Med., 313:491-498, 1985.
4. United States Public Health Service. The Health Consequences of Smoking:
Cardiovascular Disease. A Report of the Surgeon General. Rockville, MD:
United Stales Department of Health and Human Services, Office on Smoking
and Health. 1983.
5. Hoffmann, D., and Hecht, S. S. Nicotine-derived ¿V-nitrosaminesand to-
DNA DAMAGE
bacco-related cancer: current status and future directions. Cancer Res 45935-944, 1985.
Dube, M. F., and Green, C. R. Methods of collection of smoke for analytical
purposes. Recent Adv. Tobacco Sci., 8:42-102, 1982.
Hoffmann, D., Schmeltz, I., Hecht, S. S., and Wynder, E. L. Tobacco
carcinogenesis. ¡n:H. V. Gelboin and P. O. Tso (eds.), Polycyclic Hydrocar
bons and Cancer, Vol. l, pp. 85-117. New York: Academic Press, 1978.
Singer, B., and Grunberger, D. Molecular Biology of Mutagens and Carcin
ogens. New York: Plenum Press, 1983.
Kier, L. D., Yamasaki, E., and Ames, B. N. Detection of mutagenic activity
in cigarette smoke condensâtes.Proc. Nati. Acad. Sci. USA, 71:4159-4163
1974.
10. DeMarini, D. M. Genotoxicity of tobacco smoke and tobacco smoke conden
sate. Mutât.Res., 114: 59-89, 1983.
11. Obe, G., Heller, W. D., and Vogt, H. J. Mutagenic activity of cigarette
smoke. In: G. Obe (ed.), Mutations in Man. New York: Springer-Verlag
1984.
12. Hoffmann, D., and Wynder, E. L. A study of tobacco carcinogenesis. XI.
Tumor initiators, tumor accelerators and tumor-promoting activity of con
densate fractions. Cancer (Phila.), 27: 848-864, 1971.
13. Hoffmann, D., Schmeltz, I., Hecht, S. S., and Wynder, E. L. Chemical
studies on tobacco smoke. XXXIX. On the identification of carcinogens,
tumor promoters and cocarcinogens in tobacco smoke. In: DHEW Publica
tion (NIH)-76-1221, Smoking and Health. Vol. I: Modifying the Risk for
the Smoker, pp. 125-146. Rockville, MD: United States Department of
Health, Education, and Welfare, 1976.
14. Hoffmann, D., Wynder, E. L., Rivenson. A., LaVoie, E. J., and Hecht, S. S.
Skin bioassays in tobacco carcinogenesis. Prog. Exp. Tumor Res., 26- 4367, 1983.
15. Hoffmann, D., Hecht, S. S., Haley, N. J., Brunnemann, K. D., Adams, J.
D., and Wynder, E. L. Tobacco carcinogenesis: metabolic studies in humans.
In: C. C. Harris and H. N. Autrup (eds.). Human Carcinogenesis, pp. 809832. New York: Academic Press, 1983.
16. Randerath, K., Reddy, M. V., and Gupta, R. C. "P-Labeling test for DNA
damage. Proc. Nati. Acad. Sci. USA, 78:6126-6129, 1981.
17. Reddy, M. V., Gupta, R. C., and Randerath, K. "P-Base analysis of DNA.
Anal. Biochem., 117: 271-279, 1981.
18. Gupta, R. C., Reddy, M. V.. and Randerath, K. 32P-Post-labeling analysis of
non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis (Lond.),
3: 1081-1092. 1982.
19. Reddy, M. V., Gupta, R. C., Randerath, E., and Randerath, K. 32P-Postlabeling test for covalent DNA binding of chemicals in vivo: application to a
variety of aromatic carcinogens and methylating agents. Carcinogenesis
(Lond.), 5:231-243, 1984.
20. Randerath, K., Randerath, E, Agrawal, H. P.. and Reddy, M. V. Biochemical
(postlabeling) methods for analysis of carcinogen-DNA adducts. In: A. Berlin,
M. Draper, K. Hemminki, and H. Vainio (eds.). Monitoring Human Expo
sure to Carcinogenic and Mutagenic Agents, IARC Scientific Publications
59, pp. 217-231. Lyon, France: International Agency for Research on Cancer,
1984.
21. Randerath, K., Randerath, E., Agrawal. H. P., Gupta, R. C., Schurdak, M.
E., and Reddy. M. V. Postlabeling methods for carcinogen-DNA adduct
analysis. Environ. Health Perspect., 62: 57-65, 1985.
22. Randerath, K., Reddy, M. V., Avitts, T. A., Miller, R. H., Everson, R. B.,
and Randerath, E. 32P-Postlabeling test for smoking-related DNA adducts in
animal and human tissues. Banbury Rep. In: Mechanisms in Tobacco Car
cinogenesis. D. Hoffmann and C. C. Harris (eds.). Cold Spring Harbor
Laboratory, pp. 85-95. 1986.
23. Everson, R. B., Randerath, E., Santella, R. M., Cefalo, R. C., Avitts, T. A.,
and Randerath, K. Detection of smoking-related covalent DNA adducts in
human placenta. Science (Wash. DC), 231: 54-57, 1986.
Johnson,
R. A., and Walseth, T. F. The enzymatic preparation of [a-32P]
24.
ATP, [a-32P]GTP, [32P]cAMP, and |32P)cGMP. and their use in assay of
adenylate and guanylate cyclases, and cyclic nucleotide phosphodiesterases.
Adv. Cyclic Nucleotide Res., 10: 135-167, 1979.
Randerath,
K., and Randerath, E. Ion-exchange thin-layer chromatography.
25.
XV. Preparations, properties, and applications of paper-like PEI-cellulose
sheets. J. Chromatogr., 22: 110-117, 1966.
26. Gupta, R. C. Non-random binding of the carcinogen Ar-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Nati. Acad.
Sci. USA, SI: 6943-6947, 1984.
27. Marmur, J. A procedure for the isolation of deoxyribonucleic acid from
micro-organisms. J. Mol. Biol., 3: 208-218, 1961.
28. Randerath, E., Agrawal, H. P., Weaver, J. A., Bordelon, C. B., and Rander
ath, K. 32P-Postlabeling analysis of DNA adducts persisting for up to 42
weeks in the skin, epidermis, and dermis of mice treated topically with 7,12dimethylbenz(a)anthracene. Carcinogenesis (Lond.), 6: 1117-1126, 1985.
Randerath,
K., Gupta, R. C., and Randerath, E. 3H- and 32P-derivative
29.
methods for base composition and sequence analysis of RNA. Methods
Enzymol., 65:638-680, 1980.
30. Lu, L.-J. W., Disher, R. M., Reddy, M. V., and Randerath, K. 32P-Postlabeling assay of transplacental DNA damage induced by the environmental
carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene. Cancer Res., 46:
3046-3054, 1986.
31. Randerath, K., Haglund, R. E., Phillips, D. H., and Reddy, M. V. 32PPostlabeling analysis of DNA adducts formed in the livers of animals treated
with safrole, estragóle and other naturally-occurring alkenylbenzenes. I.
Adult female CD-I mice. Carcinogenesis (Lond.), 5: 1613-1622, 1984.
5876
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.
"P-ANALYSIS
OF CIGARETTE
SMOKE-INDUCED
32. Weinstein, I. B., Jeffrey, A. M., Jennelte. K. W., Blobstein, S. H., Harvey,
R. G., Harris, C. C, Autrup, H., Kasai, H., and Nakanishi, K. Benzo(o)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and
in vivo. Science (Wash. DC). 193: 592-595, 1976.
33. Jeffrey, A. M., Weinstein, I. B., Jennette, K. W., Grzeskowiak. K.. Nakanishi,
K., Harvey, R. G., Autrup, H., and Harris, C. C. Structures of benzo(a)pyrene-nucleic acid adducts formed in human and bovine bronchial explants. Nature (Lond.), 269: 348-350, 1977.
34. Schurdak, M. E., and Randerath, K. Tissue-specific DNA adduci formation
in mice treated with the environmental carcinogen, 7H-dibenzo(c/?)carbazole.
Carcinogenesis (Lond.), 6: 1271-1274, 1985.
35. Miller, E. C., and Miller. J. A. Searches for ultimate chemical carcinogens
and their reactions with cellular macromolecules. Cancer (Phila.), 47: 23272345, 1981.
36. Berenblum, I. Sequential aspects of chemical carcinogenesis: Skin. In: F. F.
Becker (ed.). Cancer I. A Comprehensive Treatise. Etiology: Chemical and
Physical Carcinogenesis, Ed. 2, pp. 451-483. New York: Plenum Press,
1982.
37. Slaga, T. J. Mechanisms involved in two-stage carcinogenesis in mouse skin.
In: T. J. Slaga (ed.). Mechanisms of Tumor Promotion. Vol. 2. Tumor
Promotion and Skin Carcinogenesis. pp. 1-16. Boca Raton, FL: CRC Press,
1984.
38. Wogan, G. N.. and Gorelick, N. J. Chemical and biochemical dosimetry of
exposure to genotoxic chemicals. Environ. Health Perspect., 62: 5-18, 1985.
39. Randerath. E., Agrawal, H. P., Reddy, M. V., and Randerath, K. Highly
persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin:
detection by "P-postlabeling analysis. Cancer Lett., 20: 109-114, 1983.
40. Swenberg, J. A.. Richardson. F. C., Boucheron, J. A., and Dyroff, M. C.
Relationship between DNA adduci formation and carcinogenesis. Environ.
Health Perspect.. 62: 177-183, 1985.
41. Singer. B. Alkylalion of the <>'' of guanine is only one of many chemical
events that may initiate carcinogenesis. Cancer Invest., 2: 233-238, 1984.
42. Pegg. A. E. Methylation of the <)" position of guanine in DNA is the most
likely event in carcinogenesis by methylating agents. Cancer Invest., 2: 223231. 1984.
DNA DAMAGE
43. Kleihues, P., and Bucheler, J. Long-term persistence of O'-methylguanine in
rat brain DNA. Nature (Lond.), 269: 625-626, 1977.
44. Randerath, K., Agrawal. H. P.. and Randerath, E. 12-O-Tetradecanoylphorbol-13-acetate-induced
rapid loss of persistent 7,l2-dimethylbenz(a)anthracene-DNA adducts in mouse epidermis and dermis. Cancer Lett., 27:
35-43, 1985.
45. Perera, F. P., and Weinstein, I. B. Molecular epidemiology and carcinogenDNA adduci detection: New approaches to studies of human cancer causa
tion. J. Chronic Dis., 35: 581-600, 1982.
46. Bialer, M., Sloneker, S. D.. and Kostenbauder, H. B. Isolation of a cigarette
smoke fraction responsible for the inhibition of benzo(a)pyrene metabolism
in the isolated perfused rabbit lung. Chem.-Biol. Interact., SI: 309-320,
1984.
47. Wilson, A. G. E., Rung, H.-C. Boroujerd, M., and Anderson, M. W.
Inhibition in vivo of the formation of adducts between metabolites of
benzo(a)pyrene and DNA by aryl hydrocarbon hydroxylase inducers. Cancer
Res., 41: 3453-3469, 1981.
48. Reddy, M. V., Irvin, T. R., and Randerath, K. Formation and persistence of
sterigmatocystin-DNA adducts in rat liver determined via 32P-postlabeling
analysis. Mutât.Res., 752:85-96, 1985.
49. Liehr, J. G., Randerath, K.. and Randerath, E. Target organ-specific covalent
DNA damage preceding diethylstilbestrol-induced carcinogenesis. Carcino
genesis (Lond.), 6: 1067-1069, 1985.
50. Liehr, J. G., Avitts. T. A., Randerath, E., and Randerath, K. Estrogeninduced endogenous DNA adduction: Possible mechanism of hormonal
cancer. Proc. Nati. Acad. Sci. USA, 83: 5301-5305, 1986.
51. Richardson, C. C. Polynucleotide kinase from Escherichia coli infected with
bacteriophage T4. Prog. Nucleic Acid Res., 2: 815-828, 1971.
52. Kleppe, K., and Lillehaug, J. R. Polynucleotide kinase. Adv. Enzymol. Relat.
Areas Mol. Biol., 48: 245-275, 1979.
53. Farber, E. Cellular biochemistry of the stepwise development of cancer with
chemicals: G. H. A. Clowes Memorial Lecture. Cancer Res., 44:5463-5474,
1984.
54. Weinberg, R. A. The action of oncogenes in the cytoplasm and nucleus.
Science (Wash. DC), 230: 770-776, 1985.
55. Garner, R. C. Assessment of carcinogen exposure in man. Carcinogenesis
(Lond.), 6: 1071-1078, 1985.
5877
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Comparative 32P-Analysis of Cigarette Smoke-induced DNA
Damage in Human Tissues and Mouse Skin
Erika Randerath, Tommie A. Avitts, M. Vijayaraj Reddy, et al.
Cancer Res 1986;46:5869-5877.
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