Identification of carcinogen DNA adducts in human saliva by linear quadrupole ion trap/multistage tandem mass spectrometry

Article abstract of DOI:10.1021/tx100098f

DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified by liquid chromatography-electrospray ionization/multistage tandem mass spectrometry (LC-ESI/MS/MSⁿ) in saliva samples from 37 human volunteers on unrestricted diets. The N-(deoxyguanosin-8-yl) (dG-C8) adducts of the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3,8- dimethylmidazo[4,5-f]quinoxaline (MeIQx), and the aromatic amine, 4-aminobiphenyl (4-ABP), were characterized and quantified by LC-ESI/MS/MS ⁿ, employing consecutive reaction monitoring at the MS³ scan stage mode with a linear quadrupole ion trap (LIT) mass spectrometer (MS). DNA adducts of PhIP were found most frequently: dG-C8-PhIP was detected in saliva samples from 13 of 29 ever-smokers and in saliva samples from 2 of 8 never-smokers. dG-C8-AαC and dG-C8-MeIQx were identified solely in saliva samples of three current smokers, and dG-C8-4-ABP was detected in saliva from two current smokers. The levels of these different adducts ranged from 1 to 9 adducts per 10⁸ DNA bases. These findings demonstrate that PhIP is a significant DNA-damaging agent in humans. Saliva appears to be a promising biological fluid in which to assay DNA adducts of tobacco and dietary carcinogens by selective LIT MS techniques.

Full text of DOI:10.1021/tx100098f

1234
Chem. Res. Toxicol. 2010, 23, 1234–1244
Identification of Carcinogen DNA Adducts in Human Saliva by
Linear Quadrupole Ion Trap/Multistage Tandem Mass
Spectrometry
Erin E. Bessette,† Simon D. Spivack,‡ Angela K. Goodenough,†,§ Tao Wang,|
Shailesh Pinto,⊥ Fred F. Kadlubar,# and Robert J. Turesky*,†
DiVision of EnVironmental Health Sciences, Wadsworth Center, New York State Department of Health, Albany,
New York 12201, DiVision of Pulmonary Medicine, Albert Einstein College of Medicine/Montefiore Medical
Center, Bronx, New York 10461, DiVision of Biostatistics, Department of Epidemiology and Population Health,
Albert Einstein College of Medicine, 1300 Morris Park AVenue, Bronx, New York 10461, Pulmonary
Medicine-Centennial 4, Montefiore Medical Center, 3332 Rochambeau AVenue, Bronx, New York 10467, and
DiVision of Medical Genetics, UniVersity of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
ReceiVed March 14, 2010
DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified by liquid
chromatography-electrospray ionization/multistage tandem mass spectrometry (LC-ESI/MS/MSn) in saliva
samples from 37 human volunteers on unrestricted diets. The N-(deoxyguanosin-8-yl) (dG-C8) adducts
of the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-
9H-pyrido[2,3-b]indole (ARC), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and the aromatic
amine, 4-aminobiphenyl (4-ABP), were characterized and quantified by LC-ESI/MS/MSn, employing
consecutive reaction monitoring at the MS3 scan stage mode with a linear quadrupole ion trap (LIT)
mass spectrometer (MS). DNA adducts of PhIP were found most frequently: dG-C8-PhIP was detected
in saliva samples from 13 of 29 ever-smokers and in saliva samples from 2 of 8 never-smokers. dG-
C8-ARC and dG-C8-MeIQx were identified solely in saliva samples of three current smokers, and dG-
C8-4-ABP was detected in saliva from two current smokers. The levels of these different adducts ranged
from 1 to 9 adducts per 108 DNA bases. These findings demonstrate that PhIP is a significant DNA-
damaging agent in humans. Saliva appears to be a promising biological fluid in which to assay DNA
adducts of tobacco and dietary carcinogens by selective LIT MS techniques.
The oral cavity is the portal of entry for carcinogens that are
ingested in the diet or inhaled through smoking. Epithelial buccal
cells and leukocytes are the principal mammalian cells present
in saliva (10, 11). Cells of the oral cavity have been employed
to measure biomarkers of genetic damage and molecular and
cellular changes following exposure to tobacco smoke or arsenic
as well as to assess the efficacy of chemopreventive agents
(11–17). The expression of mRNA of several cytochrome P450
(P450) enzymes, including P450s 1A1 and 1A2, which bioac-
tivate carcinogens, has been detected in buccal cells (18, 19).
When placed in primary culture, buccal cells were shown to
bioactivate aflatoxin B1 (18), benzo[a]pyrene (B[a]P)1 (20), and
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (21) to me-
tabolites that bind to DNA or protein. More recently, the P450
1A2 protein was detected in human salivary glands by immu-
Introduction
The covalent modification of DNA by chemical mutagens is
recognized as the initiating step in chemical carcinogenesis (1).
DNA adduct measurement is an important end point, both for
cross-species extrapolation of the biologically effective dose and
for the human risk assessment of exposure to chemical
carcinogens (2, 3). Identification and quantification of chemical-
specific adducts in the target tissue are the most relevant findings
for risk assessment (3). However, the opportunity to measure
carcinogen DNA adducts in human tissues is often precluded
by the unavailability of biopsy samples. Thus, accessible
biological fluids, such as blood (4), urine (5), exfoliated bladder
epithelial cells in urine (6), or exfoliated mammary epithelial
cells in the milk of lactating women (7, 8), have served as
surrogate matrices in which to assess exposure to chemicals or
their metabolites or the formation of DNA adducts. The
identification of DNA adducts clearly demonstrates exposure
to the biologically active metabolite; however, the adduct must
correlate with cancer risk, if it is considered valid as a biomarker
of health risk (9).
1 Abbreviations: 4-ABP, 4-aminobiphenyl; MeIQx, 2-amino-3,8-dim-
ethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine; ARC, 2-amino-9H-pyrido[2,3-b]indole; B[a]P, benzo[a]pyrene;
(()-anti-B[a]PDE, (()-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-
benzo[a]pyrene; CT-DNA, calf thymus DNA; dG, deoxyguanosine; ESI,
electrospray ionization; HAA, heterocyclic aromatic amine; LC-ESI/MS/
MSn, liquid chromatography-electrospray ionization/multistage tandem
mass spectrometry; LIT MS, linear quadrupole ion trap mass spectrometer;
LOQ, limit of quantification; MS, mass spectrometer; ppb, parts-per-billion;
SPE, solid-phase extraction; SRM, selected reaction monitoring; TSQ MS,
triple-stage quadrupole mass spectrometer; TSQ/MS/MS, triple-stage quad-
rupole/tandem mass spectrometry; dG-C8-4-ABP, N-(deoxyguanosin-8-yl)-
4-ABP;dG-C8-MeIQx,N-(deoxyguanosin-8-yl)-MeIQx;dG-C8-ARC,N-(deox-
yguanosin-8-yl)-ARC; dG-C8-PhIP, N-(deoxyguanosin-8-yl)-PhIP; dG-N2-
* To whom correspondence should be addressed. Tel: 518-474-4151.
Fax: 518-473-2095. E-mail: Rturesky@wadsworth.org.
† New York State Department of Health.
‡ Albert Einstein College of Medicine/Montefiore Medical Center.
§ Current address: Bristol-Myers Squibb, P.O. Box 4000, Princeton, New
Jersey 08543.
| Albert Einstein College of Medicine.
⊥ Montefiore Medical Center.
B[a]P,
tetrahydrobenzo[a]pyrene.
10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-
# University of Arkansas for Medical Sciences.
10.1021/tx100098f  2010 American Chemical Society
Published on Web 05/05/2010

DNA Adducts in SaliVa
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1235
(dG), DNase I (Type IV, bovine pancreas), alkaline phosphatase
(from Escherichia coli), and nuclease P1 (from Penicillium citri-
num) were purchased from Sigma (St. Louis, MO). [13C10]-dG was
purchased from Cambridge Isotopes (Andover, MA). Phosphodi-
esterase I (from Crotalus adamanteus venom) was from GE
Healthcare (Piscataway, NJ). All solvents used were high-purity B
& J Brand from Honeywell Burdick and Jackson (Muskegon, MI).
ACS reagent-grade formic acid (88%) was purchased from J. T.
Baker (Phillipsburg, NJ). HyperSep filter SpinTips C18 (20 mg)
were from Thermo Scientific (Palm Beach, FL). Oragene-DNA
saliva kits were from Genotek Inc. (Ontario, Canada).
Preparation of the DNA Adduct Standards. N-(Deoxygua-
nosin-8-yl)-PhIP (dG-C8-PhIP) (36, 37), N-(deoxyguanosin-8-yl)-
MeIQx (dG-C8-MeIQx) (38, 39), and N-(deoxyguanosin-8-yl)-ARC
(dG-C8-ARC) (40) were prepared by reaction of their N-acetoxy-
HAA derivatives with dG or [13C10]-dG (5 mg) in 100 mM
potassium phosphate buffer (pH 8.0). N-(Deoxyguanosin-8-yl)-4-
aminobiphenyl (dG-C8-4-ABP) was prepared by reaction of N-hy-
droxy-4-ABP with pyruvonitrile, followed by reaction with dG or
[13C10]-dG (41). For the case of dG-C8-MeIQx, the internal standard
was prepared by reaction of the N-acetoxy derivative of 3-[2H3C]-
MeIQx with dG. The (()-anti-B[a]PDE-derived dG adducts, 10-
(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroben-
zo[a]pyrene (dG-N2-B[a]P), were prepared by reaction of (()-anti-
B[a]PDE with dG as described (42). The adducts were purified by
solid-phase extraction (SPE), followed by HPLC purification
(38, 39), and were isolated as a mixture of unresolved isomers.
The isotopic purity of the [13C10]-dG-labeled internal standards
exceeded 99%, whereas the isotopic purity of the dG-C8-[2H3C]-
MeIQx adduct was 96.5%.
nohistochemical methods (22). P450 1A2 is a principal enzyme
involved in the bioactivation of heterocyclic aromatic amines
(HAAs) and the aromatic amine 4-aminobiphenyl (4-ABP)
(23, 24).
Both 32P-postlabeling (25–27) and immunohistochemical
techniques (28, 29) have been employed to screen for DNA
adducts in cells of the oral cavity. Several of the studies reported
differences in total DNA adduct levels between smokers and
nonsmokers; however, the complexity of the adduct profiles and
the inability to identify specific DNA adducts precluded any
interpretation on the principal DNA damaging agents and their
significance in the risk of the development of oral cancer or
cancers of other organs (25–27, 30). It is noteworthy that high
levels of DNA adducts were detected in the oral cavity of
nonsmokers: Many of these lesions could be attributed to
genotoxicants in the diet.
Electrospray ionization (ESI) is a soft ionization technique
employed to detect nonvolatile and thermally labile compounds
(31). The online coupling of liquid chromatography with
ESI-tandem mass spectrometry (MS/MS) can be used to
provide structural information on DNA adducts, and the
incorporation of stable, isotopically labeled internal standards
allows precise and accurate quantification of DNA adducts
(32, 33). While the levels of sensitivity of recent vintage MS
instruments are sufficient to detect carcinogen DNA adducts at
trace levels in human tissues, there is still a need to establish
DNA adduct biomarkers in surrogate tissues or fluids that can
be obtained noninvasively, to permit assessment of DNA
damage posed by chemical carcinogens.
We recently employed a linear quadrupole ion trap mass
spectrometer (LIT MS) to screen for DNA adducts of environ-
mental, dietary, and endogenous genotoxicants by data-depend-
ent constant neutral loss scanning, followed by triple-stage mass
spectrometry from oral cells of smokers (34). Cyclic DNA
adducts of acrolein, a highly reactive R,ꢀ-unsaturated aldehyde
that is formed endogenously through lipid peroxidation and that
also arises in cigarette smoke (35), were identified. However,
DNA adducts of other tobacco-related or meat-derived carcino-
gens, which include B[a]P, 4-ABP, 2-ARC (2-amino-9H-
pyrido[2,3-b]indole), 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine (PhIP), and 2-amino-3,8-dimethylimidazo[4,5-
f]quinoxaline (MeIQx) (34), were not detected. In this present
study, we have screened for DNA adducts of the above tobacco
and meat-associated carcinogens in saliva samples of subjects,
employing liquid chromatography-electrospray ionization/
multistage MS/MS (LC-ESI/MS/MSn) at the MS3 scan stage
with the LIT MS. DNA adducts of all of these carcinogens,
except for B[a]P, were identified in a number of human saliva
samples. Human saliva appears to be a highly promising
biological fluid with which we can monitor exposure to various
carcinogens through detection of their DNA adducts.
Tritium-Labeled PhIP-Modified, Tritium-Labeled 4-ABP-Modi-
fied, and Tritium-Labeled B[a]P-Modified CT-DNA. [3H]-PhIP-
modified CT-DNA was prepared as described (36). The extent of
[3H]-PhIP modification was estimated at 1 adduct per 106 unmodi-
fied DNA bases. [3H]-4-ABP-modified CT-DNA (62 adducts per
108 unmodified DNA bases) (43) and [3H]-B[a]P-modified CT-DNA
(111 adducts per 108 unmodified DNA bases) (44) were provided
by Dr. F. Beland (National Center for Toxicological Research).
Isolation of DNA from Saliva. Saliva (∼4 mL) was collected
in Oragene-DNA kits and stored at -20 or -80 °C prior to isolation
of DNA. The DNA was isolated following the manufacturer’s
instructions. However, further purification of the DNA was required
for the efficient enzymatic hydrolysis of DNA to its mononucleo-
sides (unpublished observations). The purified salivary DNA was
further processed by proteinase K treatment and then treated with
RNase A and RNase T1, followed by extraction with phenol and
chloroform (45). The DNA was precipitated in 9:1 C2H5OH:3 M
sodium acetate buffer (pH 6.0), followed by washing of the DNA
filament with C2H5OH:H2O (7:3). The amount of DNA recovered
from saliva samples ranged from 9 to 51 µg.
Enzyme Digestion and SPE of DNA Adducts. Isotopically
labeled internal standards were added to the DNA solution prior to
enzyme digestion, at a level of 10 adducts per 108 bases. The
enzymatic digestion conditions used for the hydrolysis of DNA
(9-51 µg) in 5 mM Bis-Tris-HCl buffer (pH 7.1, 50 µL) employed
DNase I for 1.5 h, followed by incubation with nuclease P1 for
3 h, and then treatment with alkaline phosphatase and phosphodi-
esterase for 18 h (37). The DNA digest solution was diluted with
high-purity water (200 µL; Burdick and Jackson), and the adducts
were purified by SPE, using HyperSep filter SpinTips. The DNA
digest extracts were applied to a SpinTip, which was placed on a
vacuum manifold and prewashed with CH3OH containing 0.1%
HCO2H (0.5 mL), followed by 10% CH3OH in 0.1% HCO2H (0.5
mL). The SpinTip was then washed with 10% CH3OH in 0.1%
HCO2H (2 × 0.25 mL) to remove nonmodified 2′-deoxynucleosides.
Then, the desired adducts were eluted with CH3OH containing 0.1%
HCO2H (0.3 mL) into silylated glass insert capillary LC vials
(Microliter Analytical Supplies, Suwanee, GA). Samples were
evaporated to dryness by vacuum centrifugation and then recon-
stituted in 1:1 DMSO/H2O (20 µL).
Materials and Methods
Caution: ARC, 4-ABP, B[a]P, MeIQx, PhIP, and their deriVa-
tiVes are carcinogens, and they should only be handled in a well-
Ventilated fume hood with the appropriate protectiVe clothing.
Chemicals. MeIQx, 3-[2H3C]-MeIQx, and PhIP were purchased
from Toronto Research Chemicals (Toronto, ON, Canada). 2-Nitro-
9H-pyrido[4,5-b]indole was a kind gift from Dr. D. Miller (National
Center for Toxicological Research, Jefferson, AR). (()-r-7,t-8-
Dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(()-
anti-B[a]PDE] was purchased from the NCI Chemical Carcinogen
Reference Standards Repository, Midwest Research Institute (Kan-
sas City, MO). 4-Nitrobiphenyl was purchased from Aldrich
(Milwaukee, WI). Calf thymus DNA (CT-DNA), deoxyguanosine

1236 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Bessette et al.
Figure 1. Chemical structures of tobacco and meat carcinogens and their DNA adducts monitored in human saliva.
LC/MS Parameters. Chromatography was performed with an
Agilent 1100 Series capillary LC system (Agilent Technologies,
Palo Alto, CA) equipped with an Aquasil C18 column (0.32 mm
× 250 mm) from Thermo Fisher (Bellafonte, PA). Samples (6 µL)
were injected, and analytes were separated with a gradient. The
solvent conditions were held at 100% A (solvent composition:
0.01% HCO2H and 10% CH3CN) for 2 min, followed by a linear
gradient to 100% B (solvent composition: 95% CH3CN containing
0.01% HCO2H) over 30 min at a flow rate of 6 µL/min. The MS
instrumentation was an LTQ MS (ThermoElectron, San Jose, CA),
and Xcalibur version 2.07 software was used for data manipulations.
Analyses were conducted in the positive ionization mode and
employed an Advance nanospray source from Michrom Bioresource
Inc. (Auburn, CA). Representative optimized instrument tuning
parameters were as follows: capillary temperature, 220 °C; source
spray voltage, 1.5 kV; source current, 2.8 µA; no sheath gas, sweep
gas, or auxiliary gas was employed; capillary voltage, 32 V; tube
lens voltage, 110 V; and in-source fragmentation, 10 V.
We employed the LIT MS in the tandem MS/MS scan mode to
monitor the loss of deoxyribose from the protonated molecules of
the adducts ([M + H - 116]+), followed by the consecutive reaction
monitoring scan mode at the MS3 scan stage to characterize the
product ions of the aglycone adducts [BH2]+. The top two or three
most abundant ions produced at the MS3 scan stage were used for
quantitative measurements. The ions monitored in MS > MS2 >
MS3 scan modes were as follows: dG-C8-PhIP (m/z 490.1 > 374.1
> 250.2, 329.2, 357.2); [13C10]-dG-C8-PhIP (m/z 500.1 > 379.1 >
251.2, 333.3, 362.2); dG-C8-MeIQx (m/z 479.1 > 363.1 > 239.2,
318.3, 346.3); dG-C8-[2H3C]-C8-MeIQx (m/z 482.1 > 366.1 >
242.2, 321.2, 349.3); dG-ARC (m/z 449.1 > 333.1 > 288.2, 316.3);
[13C10]-dG-ARC (m/z 459.1 > 338.1 > 292.3, 321.2); dG-C8-4-ABP
(m/z 435.1 > 319.1 > 208.2, 274.2, 302.2); [13C10]-dG-C8-4-ABP
(m/z 445.1 > 324.1 > 210.2, 278.2, 307.2), dG-N2-B[a]P (m/z 570.1
> 454.1 > 257.2, 285.2, 303.1); and [13C10]-dG-N2-B[a]P (m/z 580.1
> 459.1 > 257.2, 285.2, 303.1).
For DNA adducts of HAAs and 4-ABP, the normalized collision
energies were set at 32 and 40, and the isolation widths were set at
3.0 and 1.0 Da, respectively, for the MS2 and MS3 scan modes.
The activation Q was set at 0.35, and the activation time was 5 ms
for both scan modes. For dG-N2-B[a]P, the normalized collision
energies were set at 35 and 40, respectively, for the MS2 and MS3
scan modes. The isolation width was 6 Da and the activation Q
was 0.35 for the MS2 scan mode, and the isolation width was 5.0
Da and the activation Q was 0.20 for the MS3 scan mode. The
activation times were 20 ms for MS2 and 5 ms for MS3. Helium
was used as the collision damping gas in the ion trap and was set
at a pressure of 1 mTorr. One µscan was used for data acquisition.
The automatic gain control settings were full MS target 30000 and
MSn target 10000, and the maximum injection time was 50 ms.
With these MS parameters, about 12 scans were acquired for each
adduct and its corresponding internal standard.
approved by the Institutional Review Boards at the Wadsworth
Center and the Albert Einstein College of Medicine.
Accuracy and Performance of the Analytical Method. Cali-
bration curves were constructed with unlabeled DNA adducts added
to 15 µg of DNA digest (45 nmol of deoxynucleoside), which was
assayed by LC-ESI/MS/MS3, following SPE. The range in the level
of spiking, reported as adducts per 108 DNA bases (and average
amount of adduct in pg) per 15 µg of DNA was as follows: 0 (0
pg), 0.3 (0.06 pg), 0.6 (0.12 pg), 1.0 (0.21 pg), 3.0 (0.62 pg), 6.0
(1.2 pg), and 10 (2.1 pg). The internal standards were added at a
level equivalent to 10 adducts per 108 bases for dG-C8-MeIQx and
dG-C8-PhIP (2.2 pg), 7 adducts per 108 bases for dG-C8-ABP (1.4
pg), and 13 adducts per 108 bases (2.7 pg) for dG-C8-ARC. The
calibration curves were done in triplicate for each calibrant level,
and the data were fitted to a straight line (area of response of the
adduct/internal standard versus the amount of adduct/internal
standard) using ordinary least-squares with equal weightings. The
coefficient of determination (r2) values of the slopes exceeded 0.98
(Supporting Information, Figure S-1). The dissociation efficiency
of the dG-N2-B[a]P adduct was poor, and the recovery of total ion
counts, when going from MSn to Mn+1 scan stage mode with the
LIT MS, was very low in comparison to the other adducts
investigated (unpublished observations). The poor sensitivity
prevented the acquisition of a complete calibration curve for the
dG-N2-B[a]P adduct. Therefore, the estimates of B[a]P adducts in
DNA were based upon peak area and assumed that the response of
the adduct was equal to the response of the internal standard, with
injection of the equivalent of 10 adducts per 108 bases (2.6 pg of
[13C10]-dG-N2-B[a]P).
The accuracy of the method and the limit of quantification (LOQ)
values of the DNA adducts were determined with DNA isolated
from saliva from a nonsmoker, which contained nondetectable levels
of DNA adducts (<3 adducts per 109 DNA bases). CT-DNA
modified with PhIP, 4-ABP, and B[a]P with defined levels of dG-
C8-PhIP (36), dG-C8-ABP (43), and dG-N2-B[a]P (44) was diluted
with salivary DNA from the volunteer to achieve a level of
carcinogen DNA modification of 0, 1, 3, or 10 adducts per 108
DNA bases in 50 µg of DNA.
Results
Method Development and Validation. PhIP, 4-ABP, ARC,
MeIQx, and B[a]P were selected for screening, because these
carcinogens arise in tobacco smoke (35, 46–48) and/or are
formed in cooked meats (49). These compounds undergo
metabolic activation by cytochrome P450 enzymes to produce
electrophiles that react with DNA (32, 44). The structures of
these carcinogens and their DNA adducts are shown in Figure
1. We employed the LIT MS in tandem MS/MS to monitor the
loss of deoxyribose from the protonated molecules of the adducts
([M + H - 116]+), followed by the consecutive reaction
monitoring scan mode at the MS3 stage, to characterize and
measure product ions of the aglycone adducts [BH2]+.
Subjects. Both men and women were participants in this study.
Some subjects were residents of New York City or the immediate
vicinity; other subjects resided in Albany, New York. The subjects
were either current smokers, former smokers, or never smokers.
All of the subjects were on unrestricted diets. This study was
The estimates and LOQ values of the DNA adducts were
determined with CT-DNA modified with [3H]-PhIP, [3H]-ABP,

DNA Adducts in SaliVa
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1237
Table 1. Estimates of DNA Adducts in Human Salivary
DNA Mixed with Carcinogen-Modified CT-DNA
ranged from 32 to 84 years. The subjects were categorized as
current smokers, former smokers, or never smokers, on the basis
of self-report. Some demographics of the volunteers and the
estimates of DNA adducts measured are reported in Table 2.
Representative reconstructed ion chromatograms of the LC-ESI/
MS/MS3 traces of carcinogen DNA adducts found in saliva from
two current smokers are depicted in Figure 3A,B. The dG-C8
adducts of PhIP, MeIQx, ARC, and 4-ABP were identified in
both saliva samples.
target adduct
level per 108
independent
DNA bases dG-N2-B[a]P dG-C8-PhIP dG-C8-4-ABP measurements
1.0
3.0
10.0
NAa
NA
15.7(1.0
1.0 ( 0.1
2.9 ( 1.1
11.7 ( 1.2
0.8 ( 0.3
1.9 ( 0.4
6.0 ( 0.8
5
6
3
a Not assayed (NA), the LOQ value for dG-N2-B[a]P was 5 adducts
per 108 DNA bases.
The MS3 scan stage mode was employed both for quantifica-
tion of the adducts and for characterization of the product ion
spectra of the aglycone adducts [BH2]+. The product ion spectra
of the salivary DNA adducts and the respective internal
standards are shown in Figure 4. The MS3 product ion spectra
provide rich structural information about these adducts and
corroborate their identities. The proposed pathways of mass
fragmentation of the DNA adducts have been previously
reported (32, 34, 37, 52): The product ion spectra of the salivary
DNA adducts were in excellent agreement with the spectra of
the synthetic adducts.
The dG-C8-PhIP adduct was detected most frequently; it was
found in 15 subjects at levels that ranged from 1 to 9 adducts
per 108 DNA bases. dG-C8-ARC and dG-C8-MeIQx were
detected in saliva samples of three current smokers, followed
by dG-C8-4-ABP, which was identified in saliva samples from
two current smokers.
Pilot Exposure Analyses. Subjects were sorted into two
groups based on the concentration of PhIP adducts (not detected
versus detected). Logistic regression models were used for
testing association between PhIP adducts and other variables.
Because of the limited sample size, covariates including age,
race, gender, and lung cancer status were first tested. After
univariate logistic analyses, covariates, including gender and
lung cancer status, were not included because they are not likely
to be confounder (p > 0.25). The multivariate logistic model
examined parameters, including age, race, tobacco smoking
status [never, former (quit × 1 year), and current] and
cumulative dose (pack years), quit years (recent, <10 years, or
greater), as well as dietary factors such as meat, grilled meat,
and fruit and vegetable intake and amount, along with alcohol
intake and quantity. No parameters were clearly significant
predictors of the most commonly detected DNA adduct (PhIP),
although the multivariate model suggested some borderline
relationships between PhIP adduct detectability and smoking
(current, p ) 0.062; former, p ) 0.060) and current alcohol
intake (p ) 0.078).
and [3H]-B[a]P, with known levels of dG-C8-PhIP (36), dG-
C8-4-ABP (43), and dG-N2-B[a]P (44), and diluted with salivary
DNA from a volunteer who harbored nondetectable levels of
adducts (<3 adducts per 109 bases). The level of carcinogen
DNA modification was established at 0, 1, 3, or 10 adducts per
108 DNA bases in 50 µg of DNA. The spiking of salivary DNA
with carcinogen-modified CT-DNA enabled us to determine the
efficiency of enzyme hydrolysis, the accuracy of the method,
and the recovery of DNA adducts. The estimates of DNA
adducts are presented in Table 1. The estimates of dG-C8-PhIP
were within 10% of the target value for all levels of spiking.
The level of dG-C8-4-ABP was underestimated by about 30%
at all levels of spiking, whereas the estimate of the dG-N2-B[a]P
was ∼60% greater than the target value. The target value of
dG-C8-PhIP in [3H]-PhIP-modified CT-DNA was determined
by liquid scintillation counting of dG-C8-[3H]-PhIP, isolated
by HPLC after enzymatic digestion of the DNA (36); the target
values of dG-C8-4-ABP and dG-N2-B[a]P were based upon the
levels of adducts determined by triple stage quadrupole tandem
mass spectrometry (TSQ/MS/MS) in an independent laboratory
(43, 44). We previously estimated, by TSQ/MS/MS (50), the
amount of dG-N2-B[a]P adduct in this B[a]P-modified CT-DNA
at a level of 15 ( 2.1 adducts per 108 DNA bases (mean ( SD,
n ) 3): the target value was 10 adducts per 108 bases. The
response of the dG-N2-B[a]P adduct obtained by LIT MS at
the MS3 scan stage was about 10-fold weaker than the responses
observed for the other DNA adducts investigated. The inter-
laboratory estimates of the levels of these DNA adducts are
quite comparable, if we consider that the assays employed
different sets of internal standards, different enzymes and DNA
digestion conditions, and different MS instruments for analyses.
Our data reveal that potential constituents copurified with the
human salivary DNA samples do not impair the hydrolysis of
the DNA or the subsequent analysis of DNA adducts.
The reconstructed ion chromatograms of the LC-ESI/MS/MS3
traces of salivary DNA adducts from the volunteer, with and
without addition of CT-DNA modified with PhIP and 4-ABP,
diluted to a level of 1 adduct per 108 bases, are shown in Figure
2. Neither adduct is detected in the unspiked DNA sample
(Figure 2A); however, both adducts are readily measured in the
salivary DNA sample spiked with the CT-DNA modified with
PhIP and 4-ABP (Figure 2B). The LC-ESI/MS/MS3 traces of
dG-C8-MeIQx and dG-C8-ARC adducts spiked at a level of 1
adduct per 108 bases, in the same salivary DNA sample prior
to enzyme digestion and SPE, were also readily measured
(unpublished observations). On the basis of the level of the
signal of the DNA adducts to the background signal (51), the
LOQ values of all of the adducts, except for dG-N2-B[a]P, are
∼5-10 adducts per 109 DNA bases, and the LOQ value of dG-
N2-B[a]P was ∼50 adducts per 109 bases when 15 µg of DNA
was injected on the column.
Discussion
Saliva is a rich source of DNA (53), is easily obtained, and
is routinely used for genetic analyses (45) and evaluation of
DNA damage (17). The oral cavity is directly exposed to
carcinogens present in tobacco smoke and in the diet. Over the
past 20 years, cells of the oral cavity have been screened by
32P-postlabeling or immunohistochemical methods (25–29) for
DNA adducts. A plethora of lesions have been detected;
however, the identities of these presumed DNA adducts have
never been confirmed by MS techniques. The objective of our
study was to determine whether DNA adducts derived from
several prototypical carcinogens present in tobacco smoke or
cooked meat could be detected in the saliva of subjects on
unrestricted diets by application of selective LIT MS scanning
techniques.
Analysis of Carcinogen DNA Adducts in Human
Saliva. Salivary DNA samples were obtained from 37 volunteers
(Table 2): 19 subjects were male, and 18 were female. The ages
Our findings demonstrate that LC-ESI/MS/MS3 with the LIT
MS can be used to screen for carcinogen DNA adducts in human

1238 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Bessette et al.
Figure 2. Reconstructed ion chromatograms of the (A) LC-ESI/MS/MS3 traces of DNA adducts present in the saliva of a never-smoker with
nondetectable DNA adducts and (B) LC-ESI/MS/MS3 traces of DNA adducts in the saliva of the same subject, after spiking with PhIP- and
4-ABP-carcinogen-modified CT-DNA, at a level of 1 adduct per 108 bases. The dG-C8 adducts of MeIQx and ARC and their internal standards
were also monitored. The peak observed for dG-C8-MeIQx (tR ) 20.5 min) is attributed to the isotopic impurity of the dG-C8-[2H3C]-MeIQx
internal standard (96.5% purity), which is contaminated with the unlabeled adduct at a level of 3.5%. The retention time (tR) and area (A)
are reported.

DNA Adducts in SaliVa
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1239
Table 2. Levels of Carcinogen DNA Adducts in Human Saliva
self-reported smoking
adducts per 108 DNA bases
subject gender racea
age
historyb
cig/day
DNA assayed (µg)c dG-C8-PhIP dG-C8-MeIQx dG-C8-ARC dG-C8-4-ABP
27
11
23
19
37
26
17
33
36
28
3
F
H
H
51
57
never
current
0
30
10
20-40
0
20
10
0
0
0
5
20-40
3
14
0
20
20-40
0
20
20
3
40
0
30
10
40
51
47
44
42
36
35
34
33
33
29
29
27
27
27
26
26
26
26
26
24
24
21
21
20
20
20
19
18
18
17
16
14
13
13
12
12
9
2.7
ND
8.5
4.5
ND
7.8
2.0
ND
1.1
ND
ND
1.2
ND
ND
ND
7.7
6.5
ND
2.4
ND
9.7
ND
ND
ND
2.8
ND
2.0
ND
ND
1.8
ND
ND
ND
ND
ND
1.8
ND
NDd
ND
ND
1.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
5.8
M
M
F
M
M
F
W
W
W
W
B
W
W
W
H
W
H
B
W
W
W
H
W
H
unknown current
63
53
34
48
43
49
53
75
75
84
68
55
current
never
current
current
never
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
5.1
F
F
never
never
M
F
M
M
M
F
M
F
F
M
F
F
F
F
20 years
current
20 years
5 years
never
21
1
9
34
25
20
30
24
14
15
12
29
13
35
8
16
32
31
22
10
5
unknown current
63
44
current
never
ND
ND
ND
3.5
ND
ND
ND
5.4
ND
ND
ND
5.5
unknown current
69
48
63
74
48
43
78
62
47
87
30 years
current
current
H
W
W
B
W
H
W
H
W
B
B
B
H
H
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
never
F
F
7 years
10 years
20 years
19 years
<20 years
<20 years
M
M
M
F
M
M
F
M
M
M
F
40-60
1 time only
1
10
10
10-20
3
unknown 17 years
65
58
80
45
70
50
50
current
current
current
current
<3 months 10
3 years
2
7
6
18
4
20
B
B
H
20-40
20
M
<1 years
a W, white; B, black; and H, Hispanic. b Reported time since quitting. c Samples with the highest DNA contents were assayed first, in batches of five
d
or six samples. ND, not detected; <5 adducts per 109 DNA bases.
saliva. An important advantage of the LIT MS over triple stage
quadrupole mass spectrometer (TSQ MS) instruments, which
have been more commonly used for the quantification of DNA
adducts (32, 33), is the ability of the LIT MS to acquire MS3
product ion spectra. We have used this multistage tandem
scanning technique for structural characterization and identifica-
tion of aglycone adducts [BH2]+ of several different carcinogen
DNA adducts in tissues of experimental animals and humans
(34, 37, 54). We also employed LIT MS at the MS3 scan stage
for quantitative measurements of dG-C8-PhIP (37). The intraday
and interday precision values (coefficient of variation %) were
<10% at the LOQ: This level of performance is close to the
precision achieved by TSQ MS instruments (37). However, the
slow duty cycle of the sequence of scanning events of the LTQ
MS, which is termed the µscan and includes the time periods
for the initial prescan event, the ion injection, isolation,
activation, and mass analysis, restricts the number of scans
acquired. The paucity of scans can lead to an insufficient number
of data points acquired for each peak and result in imprecise
measurements when multiple DNA adducts are measured in a
single scan segment. The injection time of the LIT MS, the
most time-consuming event of the µscan, can be reduced, so as
to shorten the duty cycle and augment the number of scans
acquired across the peaks. However, the sensitivity of response
can decrease concomitantly, if the duration of the injection time
is too brief. A too-brief injection time can adversely affect the
quality and reproducibility of the product ion spectra, and the
quantitative estimates become less precise. Indeed, we have
observed a reduction in the precision of adduct measurements
(Table 1) when the number of DNA adducts and internal
standards assayed in the same scan segment is increased from
1 to 4 or 5 adducts, even though ∼12 scans were acquired for
each adduct and its internal standard. In contrast to the extensive
spectral data acquired by LIT MS at the MS3 scan stage for
analyte characterization, the criteria used for DNA adduct
identification using TSQ MS instruments are generally limited
to a monitoring of the loss of deoxyribose ([M + H]+ f [M +
H - 116]+) in the selected reaction monitoring (SRM) mode
combined with the characteristic tR of the adduct (32, 33).
Product ion spectra can be obtained with the TSQ MS in the
tandem MS/MS mode (39, 55); however, the slow duty cycle
of the TSQ MS in the full-scan mode, typically of the order of
0.1% (depending upon the monitored m/z range), results in a
drastic reduction in sensitivity (56). Hence, the product ion
spectra of DNA adducts are not routinely acquired in biomoni-
toring studies with TSQ MS instruments. However, the duty
cycle of the TSQ MS is extremely rapid in the SRM mode, and
the high level of sensitivity of the SRM mode allows accurate
and precise quantitation when multiple analytes are measured
simultaneously (56). Thus, both LIT MS and TSQ MS instru-
ments have important applications in the biomonitoring of DNA
adducts.
In the present study, we have identified dG-C8-PhIP adduct
in the saliva of about 45% of the ever-smokers. DNA adducts
of ARC, MeIQx, and 4-ABP were also detected but at lower
frequency. Firm conclusions about the source(s) of exposure to

1240 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Bessette et al.
Figure 3. Reconstructed ion chromatograms of the LC-ESI/MS/MS3 traces of DNA adducts present in saliva samples from two current smokers.
All DNA adducts were present at levels above the LOQ.
PhIP and other dietary carcinogens cannot be made as the
concentrations of HAAs in cooked meats can vary over a 100-
fold range (49, 57): We did not detect a relationship between
PhIP adduct formation and self-reported frequent grilled meat
or total meat intake. The approximation of tobacco usage
through self-reported smoking history is also likely to lead to
uncertainty in the estimate of carcinogen exposure, but there
may be a relationship among PhIP adduct formation, tobacco

DNA Adducts in SaliVa
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1241
Figure 4. MS3 product ion spectra of DNA adducts identified in saliva DNA samples of current smokers and MS3 product ion spectra of the
corresponding internal standards (after background subtraction), from upper to lowest panel: dG-C8-4-ABP, [13C10]-dG-C8-4-ABP, dG-C8-ARC,
[13C10]-dG-C8-ARC, dG-C8-MeIQx, dG-C8-[2H3C]-MeIQx, dG-C8-PhIP, and [13C10]-dG-C8-PhIP.
exposure, and alcohol intake, albeit of borderline statistical
significance in this pilot study. Moreover, both well-done meat
consumption (58) and tobacco usage (59) induce the levels of
expression of P4501A2, which bioactivate HAAs and 4-ABP
(60) and can lead to elevated levels of DNA adducts.
mechanisms of PhIP formation during combustion remain to
be determined.
The high frequency of detection of dG-C8-PhIP in salivary
DNA is noteworthy. PhIP has also been detected with high
frequency in hair samples of omnivores (66–68), and a high
percentage of mammary gland (69) and prostate gland biopsy
(70) samples have tested positive for putative PhIP-DNA adducts
by immunohistochemical techniques. The presumed dG-C8-PhIP
adduct was also detected, by 32P-postlabeling, in about 50% of
the exfoliated mammary epithelial cells sampled from the milk
of lactating mothers (8). These data demonstrate that PhIP can
damage DNA in multiple tissues of humans.
The bioactivation of PhIP, other HAAs, and 4-ABP is
catalyzed by several enzymes present in different organs of the
body. The liver is the most metabolically active organ in the
biotransformation of HAAs and 4-ABP to genotoxicants. The
carcinogenic N-hydroxy metabolites form principally by action
of P450 1A2 (23, 60) in the liver and can reach the oral cavity
through systemic circulation (71), followed by phase II activa-
tion in cells of the oral cavity. However, P450s 1A1, 1A2, or
1B1, which are expressed in the buccal cells or salivary glands
of the oral cavity (18, 22), can also directly bioactivate PhIP
and the other procarcinogens noted above (72, 73). Moreover,
The diet is considered to be the major source of exposure to
PhIP. The amount of PhIP formed in cooked meats can range
from less than 1 part-per-billion (ppb) up to 500 ppb (49),
depending upon the type of meat, the cooking temperature, and
the method of cooking (57, 61). ARC and MeIQx also form in
cooked meats and generally occur at <10 ppb levels. To our
knowledge, 4-ABP has not been reported in cooked meats. PhIP,
ARC, and 4-ABP also arise in mainstream tobacco smoke. The
levels of PhIP have been reported to range from 11 to 23 ng/
cig (47), whereas ARC can arise at levels as high as 258 ng/cig
(48). The levels of 4-ABP occur at 0.1-4.3 ng/cig (46, 62, 63).
There are no reports of MeIQx formation in tobacco smoke (47).
On the basis of studies with model systems, creatinine, a
constituent of muscle, is thought to be an essential precursor
for the formation of PhIP (64). However, PhIP has been detected
in incineration ash and in airborne and diesel exhaust particles
(65), in addition to tobacco smoke (47). These findings suggest
that PhIP is an ubiquitous environmental contaminant. The

1242 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Bessette et al.
adducts, and urinary mutagenicity. Cancer Epidemiol. Biomarkers
PreV. 1, 61–66.
saliva contains peroxidases (74), which can catalyze the
bioactivation of all of these compounds (75).
(7) Thompson, P. A., DeMarini, D. M., Kadlubar, F. F., McClure, G. Y.,
Brooks, L. R., Green, B. L., Fares, M. Y., Stone, A., Josephy, P. D.,
and Ambrosone, C. B. (2002) Evidence for the presence of
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The vast majority of DNA isolated from saliva with the
Oragene kit is of human origin, with a median bacterial
content of 11.8% (http://www.dnagenotek.com/pdf_files/
PDWP002_BacterialContent.pdf). Epithelial buccal cells and
leukocytes are the two principal mammalian cell types found
in saliva (10, 11). Like other epithelial cell types, buccal
cells constantly and rapidly generate. For healthy oral
epithelia, the time frame from new cell production to
exfoliation of the buccal cell from the mucosal surface is
estimated to be between 5 and 12 days (76). The leukocytes,
which originate mainly from the gingival crevice (77) and
then migrate into the oral cavity, are predominantly short-
lived neutrophils and other granulocytes. Given the short
lifespans of both buccal and leukocyte cell types, we believe
that the DNA adducts present in saliva are likely to occur
from recent exposures to carcinogens. Studies will be required
to determine whether adducts are formed in both cell types
or whether they preferentially form in one type.
In summary, human saliva appears to be a promising fluid in
which we can monitor DNA adducts of tobacco and meat-
associated carcinogens through the use of selective LIT MS
methods. The high percentage of samples that are positive for
dG-C8-PhIP is striking. Future studies that examine the kinetics
of PhIP-DNA adduct formation in cells of the oral cavity of
humans exposed to defined amounts of PhIP, combined with
studies that can unravel the myriad of plausible enzymes that
contribute to PhIP adduct formation in oral cells, will be required
before this biomarker can be exploited in human population
studies.
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Acknowledgment. This work was funded by R01 CA122320
(R.J.T.) from the National Cancer Institute, by R21 ES014438
(E.E.B., S.D.S, A.K.G., and R.J.T.) from the National Institute
of Environmental Health Sciences, and by Grant 2007/58 funded
by the World Cancer Research Fund International (E.E.B.,
R.J.T., and F.F.K.).
Note Added after ASAP Publication. This paper was
published on the Web on May 5, 2010, with an error in Figure 1.
The corrected version was reposted on June 4, 2010.
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