32P-Postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: a comparative study of labeling efficiencies.

Article abstract of DOI:10.1021/tx990038t

32P-Postlabeling is an extremely powerful technique for the detection of DNA adducts. Typically, the quantitation of DNA adducts by (32)P-postlabeling is achieved by relative adduct labeling, via comparison of the radioactivity incorporated into the adducts to that associated with the normal nucleotides. This approach is based on a number of assumptions, the foremost being that normal and adducted nucleotide 3'-phosphates are converted to 3', 5'-bisphosphates with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted a comparative study of the kinetics of phosphorylation by T(4) polynucleotide kinase using 2'-deoxyguanosine 3'-phosphate (dG3'p) and a series of N-(deoxyguanosin-8-yl)arylamine 3'-phosphate adduct standards (dG3'p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methylaniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and the dG3'p-C8-Ar adducts followed Michaelis-Menten kinetics. The apparent turnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts compared to that when labeling dG3'p. The apparent specificity constant calculated for dG3'p-C8-4-aminobiphenyl (1.4 microM(-)(1) min(-)(1)) was approximately 4-fold lower than that (5. 4 microM(-)(1) min(-)(1)) found for dG3'p. Apparent specificity constants for the monoarylamine adducts were even lower (0.043-0.23 microM(-)(1) min(-)(1)) and decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline > 2, 4-dimethylaniline. Similar experiments conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results indicate that (32)P-postlabeling of dG3'p-C8-Ar adducts is less efficient than that of dG3'p and suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency upon substitution ortho to the amino group (e.g., 2, 4-dimethylaniline). In addition, the ATP concentrations required for optimal labeling were found to be substantially higher than those used in typical (32)P-postlabeling assays. Since the high specific activity of carrier-free [gamma-(32)P]ATP precludes increasing the ATP concentration to a significant extent, these data emphasize the need for using highly efficient adduct enrichment procedures when conducting (32)P-postlabeling analyses of DNA adducts.

Full text of DOI:10.1021/tx990038t

Chem. Res. Toxicol. 1999, 12, 661-669
661
32P -P ostla belin g of N-(Deoxygu a n osin -8-yl)a r yla m in e
Ad d u cts: A Com p a r a tive Stu d y of La belin g Efficien cies
Lu´ısa L. G. Mourato,† Frederick A. Beland,*,‡ and M. Matilde Marques*,†
Centro de Qu´ımica Estrutural, Complexo I, Instituto Superior Te´cnico, Av. Rovisco Pais,
1049-001 Lisboa, Portugal, and Division of Biochemical Toxicology, National Center for
Toxicological Research, J efferson, Arkansas 72079
Received March 2, 1999
32P-Postlabeling is an extremely powerful technique for the detection of DNA adducts.
Typically, the quantitation of DNA adducts by 32P-postlabeling is achieved by relative adduct
labeling, via comparison of the radioactivity incorporated into the adducts to that associated
with the normal nucleotides. This approach is based on a number of assumptions, the foremost
being that normal and adducted nucleotide 3′-phosphates are converted to 3′,5′-bisphosphates
with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted
a comparative study of the kinetics of phosphorylation by T4 polynucleotide kinase using 2′-
deoxyguanosine 3′-phosphate (dG3′p) and a series of N-(deoxyguanosin-8-yl)arylamine 3′-
phosphate adduct standards (dG3′p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methylaniline,
and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3′p and the dG3′p-C8-Ar adducts
followed Michaelis-Menten kinetics. The apparent turnover numbers were 40-240-fold lower
when labeling dG3′p-C8-Ar adducts compared to that when labeling dG3′p. The apparent
specificity constant calculated for dG3′p-C8-4-aminobiphenyl (1.4 µM-1 min-1) was ap-
proximately 4-fold lower than that (5.4 µM-1 min-1) found for dG3′p. Apparent specificity
constants for the monoarylamine adducts were even lower (0.043-0.23 µM-1 min-1) and
decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline >
2,4-dimethylaniline. Similar experiments conducted with dG3′p-C8-Ar standards for 2-methyl-
aniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results
indicate that 32P-postlabeling of dG3′p-C8-Ar adducts is less efficient than that of dG3′p and
suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under
kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency
upon substitution ortho to the amino group (e.g., 2,4-dimethylaniline). In addition, the ATP
concentrations required for optimal labeling were found to be substantially higher than those
used in typical 32P-postlabeling assays. Since the high specific activity of carrier-free [γ-32P]-
ATP precludes increasing the ATP concentration to a significant extent, these data emphasize
the need for using highly efficient adduct enrichment procedures when conducting 32P-
postlabeling analyses of DNA adducts.
In addition, since the 32P-postlabeling step does not rely
on carcinogen-specific properties, the technique is re-
markably versatile, being suitable for the detection of
adducts from a wide variety of structurally distinct
chemical carcinogens (3). This combination of high sen-
sitivity and versatility has been instrumental in the
emergence of 32P-postlabeling as an important tool for
monitoring human exposure to chemical carcinogens.
Since chronic exposure to specific carcinogens appears
to yield dose-related steady-state levels of DNA adducts
in target tissues for tumorigenicity (4), the identification
and accurate quantitation of human DNA adducts may
be of great utility in estimating human cancer risk.
In tr od u ction
In the past two decades, 32P-postlabeling (1, 2) has
become a widely used methodology for detecting carcino-
gen-DNA adducts. Although several improvements in
the assay have appeared since its introduction, the basic
procedure involves enzymatic hydrolysis of carcinogen-
modified DNA to 3′-nucleotides, most often followed by
an adduct enrichment step, subsequent 5′-32P-phospho-
rylation with [γ-32P]ATP and T4 polynucleotide kinase
(PNK)1, and chromatographic separation of carcinogen-
nucleotide adducts from residual normal nucleotides prior
to adduct detection and quantitation (3). By virtue of the
high specific activity of [γ-32P]ATP, the assay is extremely
sensitive, and adduct levels as low as one in 1010 normal
nucleotides have been reported using 5-10 µg of DNA (3).
A major weakness of the 32P-postlabeling methodology
is that limited structural information is obtained. Selec-
1
* To whom correspondence should be addressed. F.A.B.: HFT-110,
NCTR, J efferson, AR 72079; telephone, (870) 543-7205; fax, (870) 543-
7136; e-mail, fbeland@nctr.fda.gov. M.M.M.: CQE, Complexo I, IST,
Av. Rovisco Pais, 1049-001 Lisboa, Portugal; telephone, 351-1-841-
9200; fax, 351-1-846-4455; e-mail, qmdmar@beta.ist.utl.pt.
† Instituto Superior Te´cnico.
Abbreviations: ABP, 4-aminobiphenyl; Ar, arylamine; dG3′p, 2′-
deoxyguanosine 3′-phosphate; dG3′p-C8-Ar, N-(2′-deoxyguanosin-8-yl)-
arylamine 3′-phosphate; dG3′,5′p, 2′-deoxyguanosine 3′,5′-bisphosphate;
dG3′,5′p-C8-Ar, N-(2′-deoxyguanosin-8-yl)monoarylamine 3′,5′-bisphos-
phate; diMeA, dimethylaniline; dT3′p, thymidine 3′-phosphate; dTT,
dithiothreitol; MeA, methylaniline; PEI, polyethyleneimine; PNK, T4
polynucleotide kinase; RAL, relative adduct labeling.
‡ National Center for Toxicological Research.
10.1021/tx990038t CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/25/1999

662 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Mourato et al.
tive chemical or enzymatic tests [e.g., the susceptibility
of arylamine 3′-nucleotide adducts to nuclease P1 (5, 6)],
as well as the chromatographic behavior in selected
solvents, may suggest a particular class of chemical
carcinogens from which an unknown adduct is derived.
However, although such tests form the basis of group-
specific detection procedures (3), the assignment of
adduct structures cannot be accomplished in the absence
of standards.
One additional concern about 32P-postlabeling is the
accuracy of reported measurements of DNA adduct levels.
Typically, the quantitation of DNA adducts is achieved
by relative adduct labeling (RAL), in which the ratio of
the adduct-associated radioactivity to the total nucleotide
radioactivity is corrected using an appropriate dilution
factor (7). Absolute levels of binding are often reported
using this procedure, by assuming an average concentra-
tion of 3 × 106 fmol of nucleotides/µg of DNA (8).
However, this approach is based on a number of poten-
tially incorrect assumptions, the foremost being that
adducted and normal nucleotide 3′-phosphates are con-
verted to 3′,5′-bisphosphates with equal efficiency. Al-
though reports about the quantitative aspects of the
PNK-catalyzed phosphorylation step are relatively scarce,
there is sufficient evidence that the labeling efficiency is
adduct-dependent. For instance, some aromatic (9) and
bulky (10) adducts appear to be better substrates for PNK
than normal nucleotides, a property underlying the use
of ATP-deficient postlabeling strategies. By contrast,
other bulky and nonbulky adducts seem to be labeled
with low efficiencies (11-16).
Aromatic amines and amides are chemical carcinogens
to which humans are exposed from a variety of sources,
including tobacco smoke (17-21). These carcinogens have
been implicated in human bladder carcinogenesis (22)
and are thought to play a role in the onset of bladder
cancer in smokers (22-24). It has been suggested that
aromatic amines may be also involved in the induction
of breast cancer in postmenopausal women who smoke
(25), and recently, single-ring aromatic amines have been
reported to be present in human breast milk (26).
Analyses of exfoliated bladder cells and autopsy and
biopsy samples of human bladder (27-30) have indicated
the presence of several putative arylamine-DNA ad-
ducts, among which only N-(deoxyguanosin-8-yl)-4-ami-
nobiphenyl has been identified (28-30). N-(Deoxygua-
nosin-8-yl)arylamine adducts are the major persistent
adducts formed upon metabolic activation of aromatic
amine carcinogens (31-33), and in recent work, we have
demonstrated that these are also the major adducts from
the in vitro reaction of DNA with electrophilic derivatives
of alkylanilines present in tobacco smoke (34, 35).
Although the presence of alkylaniline-derived DNA ad-
ducts in human samples has yet to be established,
hemoglobin-alkylaniline adducts have been detected,
with the adduct levels from some of the alkylanilines
being higher in smokers (36, 37).
study of DNA samples modified in vitro and in vivo with
4-[3H]aminobiphenyl, using different DNA adduct detec-
tion methodologies (39). The results obtained by 32P-
postlabeling consistently indicated DNA adduct levels
that were 3-5% of the values determined by measuring
3
the extent of H incorporation. Although other factors,
such as incomplete recovery of adducts during the hy-
drolysis and enrichment steps, may contribute to the
underestimation of arylamine-DNA adduct levels by
RAL, the intrinsic affinities of the adducts for PNK could
play a major role in the process.
With the aim of evaluating relative labeling efficiencies
of arylamine-DNA adducts, this study describes a
comparative kinetic analysis of the PNK-catalyzed phos-
phorylation, using 2′-deoxyguanosine 3′-phosphate (dG3′p)
and a number of N-(deoxyguanosin-8-yl)arylamine 3′-
phosphate adduct standards (dG-3′p-C8-Ar, Ar being
4-aminobiphenyl, 3- and 4-methylaniline, and 2,4- and
3,4-dimethylaniline) derived from arylamines present in
tobacco smoke.
Ma ter ia ls a n d Meth od s
Ca u tion : N-Arylamines, N-hydroxyarylamines, and N-acet-
oxyarylamines are potentially carcinogenic. They should be
handled with protective clothing, in a well-ventilated fumehood.
Exposure to 32P should be kept as low as possible, by working in
a confined laboratory area, with protective clothing, plexiglass
shielding, Geiger counters, and body dosimeters. Wastes must
be discarded according to appropriate safety procedures.
Ch em ica ls. 2′-Deoxyguanosine 3′-phosphate (dG3′p), thymi-
dine 3′-phosphate (dT3′p), and apyrase (11.8 units/mg) were
purchased from Sigma Chemical Co. (St. Louis, MO). ATP was
acquired from Boehringer Manheim Gmbh (Indianapolis, IN).
[γ-32P]ATP was synthesized from ADP (Sigma) and carrier-free,
HCl-free [32P]orthophosphate (ICN Pharmaceuticals, Costa
Mesa, CA) (2, 8). The [γ-32P]ATP was diluted 500-1000-fold
before being used in the labeling reactions. PNK (90 090 units/
mg, 35 000 mg/mmol of monomer) was purchased from Amer-
sham U.S. Biochemical (Cleveland, OH) at a concentration of
30 units/µL. Dilutions of the stock solution were performed as
needed.
N-(2′-Deoxyguanosin-8-yl)-4-aminobiphenyl
3′-phosphate
(dG3′p-C8-4ABP) and the N-(2′-deoxyguanosin-8-yl)monoaryl-
amine 3′-phosphate adducts (dG3′p-C8-Ar, Ar being 3MeA,
4MeA, 2,4diMeA, and 3,4diMeA) were synthesized by reaction
of the corresponding N-acetoxyarylamines with dG3′p and
purified by reversed-phase HPLC as described previously (34).
Adduct concentrations were determined by UV spectroscopy,
using the extinction coefficients of the corresponding nucleoside
adducts (34, 35, 40). All other chemicals and biochemicals were
of analytical grade and were used without further purification.
In str u m en ta tion . UV spectra were recorded with a Beck-
man DU-40 UV/vis spectrophotometer. Reaction mixtures were
incubated at 37 °C in 150 µL polypropylene Eppendorf tubes,
using a multiblock heater (Lab-Line Instruments, Inc., Melrose
Park, IL). Liquid scintillation counting was performed on a
Packard Tri-Carb model 1600TR instrument (Packard Instru-
ment Co., Meriden, CT), using Ultima Gold (Packard) as the
scintillation fluid. TLC plates were exposed to intensifying
screens at room temperature for variable periods of time (1-12
h) in Kodak X cassettes. Visualization and quantitation of the
chromatograms were then conducted with a phosphorimager
(Storm 860 Imager, Molecular Dynamics, Sunnyvale, CA).
The quantitation of 32P-postlabeled arylamine-modified
DNA adducts using the RAL method seems to underes-
timate adduct levels in a consistent manner. For in-
stance, when DNA adducts were assessed in mice treated
with radiolabeled 2-acetylaminofluorene as part of an
interlaboratory trial of 32P-postlabeling procedures, the
values obtained by RAL were 4-8-fold lower than those
Ch r om a togr a p h y. PEI-cellulose TLC plates, manufactured
by Macherey-Nagel or Merck, were purchased from Alltech
Associates, Inc. (Deerfield, IL). The Macherey-Nagel plates were
washed with deionized water and dried prior to use. Specific
chromatographic conditions were as follows.
3
determined on the basis of H incorporation (38). More
recently, we reported a comparative adduct quantitation

32P-Postlabeling Efficiency of DNA Adducts
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 663
Sch em e 1. Syn th esis of d G3′p -C8-Ar Ad d u ctsa
(1) d G3′,5′p . The samples were applied to Merck plates
(length of 20 cm), and the elution was conducted overnight onto
a Whatman #1 paper wick using 150 mM sodium phosphate
(pH 6.0).
(2) d G3′,5′p -C8-4ABP . The samples were applied to Mach-
erey-Nagel plates (length of 10 cm). The elution was conducted
on a Whatman #1 paper wick (direction D1) with 900 mM
sodium phosphate (pH 6.8), and then in the opposite direction
(D2) with 3.6 or 5.3 M lithium formate and 8.5 M urea (pH 3.5).
(3) d G3′,5′p -C8-Ar (Ar bein g 3MeA, 4MeA, 2,4d iMeA, a n d
3,4d iMeA). The samples were applied to Macherey-Nagel plates
(length of 20 cm). The elution was conducted onto a Whatman
#1 paper wick (direction D1) with 2.5 M sodium phosphate (pH
6.0), and then in the opposite direction (D2) with 1.8 M lithium
formate and 4.2 M urea (pH 3.5).
Kin etic Stu d ies. (1) P h osp h or yla tion a s a F u n ction of
Tim e. The time dependence of the conversion of dG3′p to the
corresponding bisphosphate (dG3′,5′p) was determined by an
adaptation of a literature procedure (41). A stock labeling
solution (55 µL) containing PNK (60 milliunits/µL, 275 fmol)
and ATP (16.1 µM, 886 pmol) in 80 mM bicine-NaOH, 40 mM
MgCl2, 40 mM dithiothreitol (dTT), 4 mM spermidine (pH 9.4)
buffer was prepared. A 35 µL aliquot of this labeling solution
was added to 105 µL of a 6.67 µM aqueous solution of dG3′p
(700 pmol, 5 µM final concentration). The mixture was vortexed,
centrifuged for 5-10 s, and incubated at 37 °C. Following
incubation periods of 2, 5, 10, 20, 30, 60, and 120 min, 20 µL
aliquots of the reaction mixture were removed, the reaction was
quenched by mixing with 2 µL of 673 µM dT3′p (1346 pmol, 57.3
µM final concentration) and 30 milliunits of apyrase (1.5 µL),
and the mixtures were further incubated for 30 min at 37 °C.
Aliquots of each mixture (15 µL) were then spotted at 2 cm
intervals on a 20 cm × 20 cm PEI-cellulose TLC plate and eluted
as described above. Control incubations were conducted for 120
min in the absence of dG3′p, either with or without 100 pmol of
dT3′p.
a
R is 3-methyl, 4-methyl, 2,4-dimethyl, 3,4-dimethyl, or 4-phenyl.
of dG3′p and dG3′-C8-4ABP to the corresponding bisphosphates
using the assay conditions described for the substrate concen-
tration dependence study. Control incubations were performed
in the absence of PNK.
(5) Ca lcu la tion s. The specific activity of the [γ-32P]ATP in
each labeling mixture was determined by liquid scintillation
counting of diluted aliquots, whose concentration had been
established by UV spectroscopy. Following elution, and prior
to exposure of the TLC plate to the intensifying screen, an
aliquot of the labeling mixture was spotted onto the plate to
serve as a quantitation standard. Whenever necessary, correc-
tions for 32P decay were introduced in the calculations.
The kinetic parameters (Vmax and Km) were calculated using
the weighted least-squares fitting (relative weights) program
Leonora (42). Similar results were obtained with version 1.0 of
the kinetics program developed by Brooks (43) by excluding
outliers from the initial data sets and recalculating the kinetic
parameters as needed.
Resu lts
The goal of this work was to compare the kinetics of
phosphorylation of dG3′p and a number of dG3′p-C8-Ar
(Ar being 4ABP, 3MeA, 4MeA, 2,4diMeA, and 3,4diMeA)
adducts from arylamines present in tobacco smoke under
experimental conditions similar to those used in typical
32P-postlabeling protocols. Therefore, the PNK-catalyzed
phosphorylation reactions were conducted at 37 °C and
pH 9.4, in a bicine-NaOH buffer containing MgCl2, dTT,
and spermidine (3). All the adducts were synthesized by
reaction of the corresponding N-acetoxyarylamines with
dG3′p (Scheme 1), as described in detail in our previous
work (34, 35).
The characterization of an enzyme-catalyzed reaction
usually includes the determination of the maximum
initial reaction velocity (Vmax) and Michaelis constant (Km)
for each substrate. To obtain satisfactory estimates of
these parameters, some preliminary experiments were
necessary to ensure that the basic assumptions underly-
ing the Michaelis-Menten relationship were observed.
Specifically, an incubation time had to be selected within
a range where the measured initial velocity was constant
with time and varied linearly with the enzyme concen-
tration (44). In addition, suitable chromatographic condi-
tions had to be established for each substrate.
Ch r om a togr a p h y. The elution conditions for the
separation of dG3′,5′p and dG3′,5′p-C8-4ABP from the
residual [γ-32P]ATP and inorganic phosphate were adapted
from literature procedures (3, and references therein).
Due to the low lipophilicity of dG3′,5′p, a single elution
in a phosphate buffer with a low ionic strength was
sufficient to achieve substantial migration of the normal
nucleotide (not shown). By contrast, a second elution with
a lithium formate buffer containing 8.5 M urea was
necessary to promote migration of the lipophilic dG3′p,5′p-
C8-4ABP (Figure 1a). To prevent overmigration of the
less lipophilic adducts (i.e., those derived from methyl-
ated anilines), a phosphate buffer with a high ionic
The time dependence of the dG3′p-C8-4ABP phosphorylation
was studied in a similar manner. With this substrate, three
independent experiments were performed, using 27.5, 275, and
2750 fmol of PNK (6, 60, and 600 milliunits/µL, respectively) in
the stock labeling solution.
(2) P h osp h or yla tion a s a F u n ction of ATP Con cen tr a -
tion . The effect of ATP concentration on the initial velocity of
the phosphorylation reaction was studied for the conversion of
dG3′p and dG3′p-C8-4ABP to the corresponding bisphosphates.
Several labeling mixtures (10 µL) were prepared, each contain-
ing 500 fmol of PNK (600 milliunits/µL) and different ATP
concentrations in 80 mM bicine-NaOH, 40 mM MgCl2, 40 mM
dTT, 4 mM spermidine (pH 9.4) buffer. The total ATP concen-
trations were 4-2000 µM (0.04-20 nmol) for experiments
conducted with dG3′p and 200-6800 µM (2-68 nmol) for
experiments conducted with dG3′p-C8-4ABP. Following a 10
min equilibration at 37 °C, each labeling mixture was added to
a preincubated 30 µL aqueous solution containing 200 pmol of
the nucleotide substrate. The mixtures were then incubated for
5 min and treated subsequently as described for the time
dependence study.
(3) P h osp h or yla tion a s a F u n ction of Su bstr a te Con -
cen tr a tion . The effect of substrate concentration on the phos-
phorylation reaction was studied for dG3′p, dG3′p-C8-4ABP, and
a variety of dG3′p-C8-Ar adducts (Ar being 3MeA, 4MeA,
2,4diMeA, and 3,4diMeA) using the methodology described
above. The labeling solution contained 600 milliunits/µL (50
fmol) PNK and 406 µM (dG3′p) or 955 µM (arylamine adducts)
ATP in 80 mM bicine-NaOH, 40 mM MgCl2, 40 mM dTT, 4 mM
spermidine (pH 9.4) buffer. Aliquots (5 µL) of this solution were
added to 15 µL of various substrate solutions (typically 2-3600
pmol, 0.13-240 µM), and the mixtures were incubated and
subsequently processed.
(4) P h osp h or yla tion a s a F u n ction of P NK Con cen tr a -
tion . The effect of PNK concentration on the initial velocity of
the phosphorylation reaction was analyzed for the conversion

664 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Mourato et al.
F igu r e 1. Representative TLC chromatograms for the phos-
phorylation of dG3′p-C8-4ABP (a) and dG3′p-C8-4MeA (b). The
elution conditions are outlined in Materials and Methods. The
substrate concentrations (micromolar) are denoted. QS indicates
the quantitation standard.
F igu r e 3. Variation of the initial velocity of phosphorylation
as a function of the ATP concentration using (a) 5 µM dG3′p
and (b) 5 µM dG3′p-C8-4ABP as the substrate. The incubation
conditions are outlined in Materials and Methods.
proximately the same interval (0-10 min) for both dG3′p
and dG3′p-C8-4ABP, an incubation time of 5 min was
selected to conduct the subsequent experiments with
these substrates and the remaining dG3′p-C8-Ar adducts.
Selection of th e P NK Con cen tr a tion . The initial
velocity of the dG3′p phosphorylation reaction was found
to vary linearly with the PNK concentration from 25 to
800 milliunits/µL (2.0-64 nM). Similar results were
obtained for the phosphorylation of dG3′p-C8-4ABP.
Since the accumulation of bisphosphate products had to
be avoided to maintain stationary-state conditions, the
PNK concentration was selected to be approximately 2
orders of magnitude lower than the range of substrate
concentrations to be used in the kinetic measurements.
Therefore, all the subsequent experiments were con-
ducted using 150 milliunits/µL (12 nM) PNK in the
incubation mixture. This enzyme concentration lies
within the range of PNK concentrations (100-1000
milliunits/µL) typically used in 32P-postlabeling protocols
(3, 47).
Selection of th e ATP Con cen tr a tion . The effect of
the ATP concentration on the initial velocity of the
phosphorylation reaction was studied using dG3′p and
dG3′p-C8-4ABP at a concentration of 5 µM. The experi-
mental conditions were adapted from those reported by
Lillehaug and Kleppe (41) and from typical 32P-postla-
beling protocols (3, and references therein), taking into
account the preliminary time course and enzyme linearity
tests described above. With both substrates, more data
points were obtained in the low-ATP concentration region
to achieve better accuracy in the definition of the
experimental curves (42). The results are displayed in
Figure 3 (panels a and b), and the calculated kinetic
parameters are shown in Table 1. The analysis of both
curves indicates that Michaelis-Menten kinetics oc-
curred; however, it is clear that a higher ATP concentra-
F igu r e 2. Expanded region (0-20 min) of the time course curve
for the phosphorylation of dG3′p-C8-4ABP. The incubation
conditions are outlined in Materials and Methods.
strength was used for D1, whereas the urea and lithium
formate concentrations were decreased in the D2 buffer
(Figure 1b), compared to those used for elution of
dG3′,5′p-C8-4ABP (Figure 1a). This type of strategy is
normally successful for the analysis of adducts containing
less than two aromatic rings (45, 46).
Selection of th e In cu ba tion Tim e. The time course
of the PNK-catalyzed phosphorylation reaction was ana-
lyzed for the period from 0 to 120 min using either 5 µM
dG3′p or dG3′p-C8-4ABP as the substrate and 15 milli-
units/µL (1.2 nM) PNK. Both species underwent fast
phosphorylation during the first 20-30 min. This was
followed by a decline in the rate of substrate conversion
as time progressed, with the product concentration
tending toward a plateau. In both instances, no decrease
in the concentration of the bisphosphate product was
detected during the time of observation. This contrasts
with a report by Hemminki et al. (13), who detected a
decline in the concentration of dG3′,5′p-C8-4ABP after
approximately 45 min, presumably due to a time-depend-
ent dephosphorylation of the adduct. Regardless of the
substrate, the measured velocity of phosphorylation was
constant with time up to approximately 10 min, as
illustrated for dG3′p-C8-4ABP (Figure 2). Similar results
were obtained for this substrate at two additional PNK
concentrations (0.12 and 12 nM, not shown). Since the
linear region of the time course curves spanned ap-

32P-Postlabeling Efficiency of DNA Adducts
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 665
Ta ble 1. Kin etic P a r a m eter s Obta in ed for ATP Usin g Differ en t Su bstr a tes
Km
Vmax
kcat
kcat/Km
b
c
d
substratea
dG3′p
dG3′p-C8-4ABP
(µM)b
(µM min-1
)
(min-1
)
(µM-1 min-1
)
77.3 ( 30.6
179 ( 58.7
0.076 ( 0.020
0.14 ( 0.02
6.39
11.5
0.083
0.065
a
b
The substrate concentration was 5 µM. Calculated for the Michaelis-Menten equation using the weighted least-squares (relative
weights) fitting program Leonora (42). Both Km and Vmax are apparent values and are presented ( the standard error. c Apparent turnover
d
number, defined as Vmax/[E0]. The PNK concentration in the incubation mixture was 150 milliunits/µL (12 nM). Apparent specificity
constant.
tion was required to reach the apparent Vmax plateau
when using dG3′p-C8-4ABP as a substrate. This is
reflected in the higher apparent Km value (179 µM)
calculated for ATP in the presence of dG3′p-C8-4ABP,
as compared to that (77.3 µM) obtained in the presence
of dG3′p. Interestingly, the apparent Vmax was higher
when dG3′p-C8-4ABP was used as a substrate, which
resulted in a higher apparent turnover number (kcat
;
Table 1). Nonetheless, within experimental error, the
apparent specificity constant (kcat/Km; Table 1) for ATP
appeared to be unaffected by the type of nucleotide
substrate (i.e., normal vs adducted dG3′p). In view of the
results obtained for the apparent Km constants, subse-
quent experiments to establish the kinetic parameters
for the phosphorylation of dG3′p were conducted using
101 µM ATP, whereas all the incubations conducted with
dG3′p-C8-Ar adducts were performed using 240 µM ATP.
Although not strictly corresponding to ATP-saturating
conditions, the selected ATP concentrations were higher
than the apparent Km constants listed in Table 1 and
provided satisfactory kinetic data without an excessive
increase in the amount of 32P handled in each incubation.
Deter m in ation of th e Kin etic P ar am eter s of P h os-
p h or yla tion for d G3′p a n d th e d G3′p -C8-Ar Ad -
d u cts. Representative curves displaying the variation of
the initial velocity of phosphorylation (incubation time
of 5 min) as a function of substrate concentration are
shown in Figure 4 for dG3′p and two of the dG3′p-C8-Ar
adducts (Ar being 4ABP and 3,4diMeA). Substrate con-
centrations in the incubation mixtures ranged from 0.1
µM to as high as 180 µM, with the higher concentration
dictated in each case by the need to reach the Vmax
plateau. Similar to the case in the ATP dependence study,
more data points were obtained in each experiment in
the region of low substrate concentration (Figure 4). The
kinetic parameters calculated for all the substrates are
shown in Table 2. In every instance, the phosphorylation
reaction appeared to follow Michaelis-Menten kinetics.
In addition to the substrates listed in Table 2, similar
experiments were conducted using dG3′p-C8-Ar adduct
standards derived from 2-methylaniline and 2,3-dimeth-
ylaniline. Very poor and irreproducible labeling was
obtained with these two substrates, which precluded the
calculation of reliable kinetic parameters.
F igu r e 4. Variation of the initial velocity of phosphorylation
as a function of the substrate concentration using (a) dG3′p as
the substrate, in the presence of 101 µM ATP, (b) dG3′p-C8-
4ABP as the substrate, in the presence of 240 µM ATP, and (c)
dG3′p-C8-3,4-diMeA as the substrate, in the presence of 240 µM
ATP. The incubation conditions are outlined in Materials and
Methods.
when labeling the dG3′p-C8-Ar adducts than when
labeling dG3′p (Table 2). Moreover, the apparent enzyme
specificity constant (kcat/Km) was approximately 4-fold
lower for dG3′p-C8-4ABP and 20-130-fold lower for the
monoarylamine adducts compared to that for dG3′p.
These findings strongly suggest that 32P-postlabeling of
dG3′p-C8-Ar adducts is less efficient than that of dG3′p,
and therefore, kinetically controlled conditions are likely
to lead to preferential labeling of normal nucleotides,
whenever these are present in the incubation mixture.
In addition, the lowest labeling efficiency was found for
adducts containing a substituent ortho to the arylamine
nitrogen. This is inferred from the small apparent PNK
specificity constant (0.043 µM-1 min-1; Table 2) deter-
The data in Table 2 indicate apparent Km values
ranging from 0.86 µM for dG3′p-C8-4ABP to 20.4 µM for
dG3′p-C8-2,4diMeA, with the normal nucleotide dG3′p
exhibiting an intermediate apparent Km (10.5 µM). This
suggests that a higher substrate concentration is required
to reach Vmax when labeling dG3′p, compared to most of
the adducts investigated in this study. However, the
apparent Vmax was found to be much greater with dG3′p
than with any of the adducted substrates, leading to a
greater extent of substrate conversion during the incuba-
tion period. This is clearly reflected in the apparent
turnover numbers (kcat), which were 40-240-fold lower

666 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Ta ble 2. Kin etic P a r a m eter s Obta in ed for d G3′p a n d d G3′p -C8′-Ar Ad d u cts
Mourato et al.
Km
Vmax
kcat
kcat/Km
b
c
d
substratea
dG3′p
(µM)b
(µM min-1
)
(min-1
)
(µM-1 min-1
)
10.5 ( 2.8
0.86 ( 0.23
5.3 ( 1.0
5.6 ( 1.8
20.4 ( 6.1
1.5 ( 0.3
0.67 ( 0.13
56.6
1.22
0.51
1.30
0.87
0.24
5.38
1.41
0.095
0.232
0.043
0.156
dG3′p-C8-4ABP
0.014 ( 0.002
0.0060 ( 0.0007
0.015 ( 0.003
0.010 ( 0.002
0.0028 ( 0.0003
dG3′p-C8-3MeA
dG3′p-C8-4MeA
dG3′p-C8-2,4diMeA
dG3′p-C8-3,4diMeA
a
Substrate concentrations ranged from 0.1 to 180 µM in the incubation mixtures. The ATP concentration was 101 (dG3′p) or 240 µM
b
(arylamine adducts). Calculated for the Michaelis-Menten equation using the weighted least-squares (relative weights) fitting program
Leonora (42). Both Km and Vmax are apparent values and are presented ( the standard error. c Apparent turnover number, defined as
d
V
max/[E0]. The PNK concentration in the incubation mixture was 150 milliunits/µL (12 nM). Apparent specificity constant.
mined with dG3′p-C8-2,4diMeA, and also from the irre-
producible labeling obtained when the 2-methylaniline
and 2,3-dimethylaniline standards were used as sub-
strates.
phorylation step is fast, with the product concentration
tending toward a plateau after 20-30 min. This is in good
agreement with the results of Hemminki and co-workers
for dG3′p-C8-4ABP (13), although we were unable to
detect a subsequent decline in the concentration of the
bisphosphate product reported by those authors. This
may be due to differences in labeling conditions because
Hemminki et al. (13) conducted their incubations at pH
7.9, which would favor phosphatase activity compared
to the pH 9.4 used in our study. In any case, since the
PNK-catalyzed phosphorylation is fast and because dG-
C8-Ar adducts are known to be unstable under alkaline
conditions (55), it is apparent that long incubation times
can and should be avoided when quantifying this type of
adduct by 32P-postlabeling.
Discu ssion
In this study, we compared the kinetics of the PNK-
catalyzed phosphorylation of nucleotide 3′-monophos-
phates, using dG3′p and a variety of dG3′p-C8-Ar adducts
(Ar being 4ABP, 3MeA, 4MeA, 2,4diMeA, and 3,4diMeA)
derived from arylamines present in tobacco smoke.
dG3′p-C8-4ABP was selected for analysis because 4-ami-
nobiphenyl is considered to be a human bladder carcino-
gen (22) and dG3′p-C8-4ABP has been identified in
human DNA (28-30). Presently, it is not known if
adducts derived from alkylanilines are part of the puta-
tive arylamine-DNA adducts detected in human samples
(27-30). Although, as a class, single-ring aromatic
amines are considered weak carcinogens (48), some (e.g.,
2-methylaniline and 2,6-dimethylaniline) are rodent (49-
51) and possibly human (51, 52) carcinogens. Since
alkylated anilines are major contributors to the aromatic
amine fraction of cigarette smoke (18-20) and because
their electrophilic derivatives can bind to DNA (34, 35),
it is important to determine if typical 32P-postlabeling
conditions are adequate for the detection and quantita-
tion of alkylaniline-DNA adducts.
Using dG3′p and dG3′p-C8-4ABP as substrates, we
found the initial velocity of phosphorylation to be linearly
dependent on PNK over a broad range of enzyme
concentrations. The value selected to conduct the kinetic
studies (150 milliunits/µL) was sufficiently low to prevent
accumulation of bisphosphate products and was well
within the range used in typical 32P-postlabeling protocols
(3, 47). By contrast, whereas typical 32P-postlabeling
protocols use ca. 2-3 µM [γ-32P]ATP (3, 47), the apparent
Km values obtained for ATP with dG3′p and dG3′p-C8-
4ABP as substrates (77.3 and 179 µM, respectively; Table
1) suggest that optimal labeling requires ATP concentra-
tions much higher than those normally used. A similar
observation has been reported by Reddy and co-workers
(56), who demonstrated that increasing the concentration
of ATP to 60 µM resulted in maximal labeling of thymine
glycol adducts. If we assume that our results with dG3′p-
C8-4ABP can be extrapolated to other dG3′p-C8-Ar
adducts, the requirement for higher ATP concentrations
may be even more critical for the adducts than for normal
nucleotides.
Kinetic parameters for the phosphorylation of normal
nucleotide 3′-monophosphates have not been reported,
with the exception of a study by Lillehaug and Kleppe
(41), who obtained an apparent Km of 22.2 µM for dT3′p
in the presence of 66 µM ATP. Preliminary work on the
phosphorylation of dG3′p-C8-4ABP was conducted by
Hemminki and co-workers (13, 15), but Km or Vmax values
were not obtained.
We performed initial experiments using dG3′p and
dG3′p-C8-4ABP as substrates, to establish incubation
times, as well as PNK and ATP concentrations suitable
for estimating the kinetic parameters. In the labeling
step, we used a bicine-NaOH buffer containing MgCl2,
dTT, and spermidine, which allows the maintenance of
a high pH (ca. 9-9.5). Although optimum labeling of
normal and adducted nucleotides has been reported to
occur at pH 7.4-8.4 (53), most 32P-postlabeling assays
are conducted under alkaline conditions with the aim of
minimizing the 3′-phosphatase activity of PNK (54). Since
our goal was to compare the kinetics of phosphorylation
of dG3′p and dG3′p-C8-Ar adducts under conditions
typical of current 32P-postlabeling protocols, an alkaline
pH was maintained in all incubations. Regardless of the
substrate type (i.e., normal vs adducted dG3′p), our initial
time dependence measurements indicated that the phos-
On the basis of these results, 240 µM ATP was used
in the incubations to establish the kinetic parameters of
the arylamine adducts. This compares to the 101 µM ATP
used in the incubations conducted with dG3′p. These ATP
concentrations were lower than those (44) required for
saturation, and as a result, all the kinetic parameters
listed in Table 2 must be regarded as apparent values.
Nonetheless, of the parameters presented in Table 2, the
apparent specificity constants (kcat/Km) are the most
useful for comparative purposes. Since specificity con-
stants can be used in reactions with two substrates under
nonsaturating concentrations (57), we chose not to in-
crease the ATP concentrations to minimize exposure to
32P.
In all instances (see Figure 4 for representative ex-
amples), the phosphorylation reactions followed Michae-

32P-Postlabeling Efficiency of DNA Adducts
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 667
lis-Menten kinetics. Taking into account the fact that
the active form of PNK is a tetramer (58), we also used
Hill’s equation to treat the experimental data obtained
for the various substrates but found no evidence of
allosteric interactions (not shown). This is consistent with
the results reported by Lillehaug and Kleppe (41) for
dT3′p and the ribonucleotide 3′-monophosphates.
These findings suggest that normal nucleotides will be
labeled preferentially to dG3′p-C8-Ar adducts under
kinetically controlled conditions, which is consistent with
the underestimation of arylamine adduct levels by RAL,
when compared to measurements of the extent of 3H
incorporation (38, 39). If the high specific activity of
carrier-free [γ-32P]ATP is taken into account, increasing
the ATP concentration in current protocols to achieve
optimal labeling does not seem feasible. Therefore, our
data emphasize the need for using highly efficient adduct
enrichment procedures prior to 32P-postlabeling reactions.
The apparent specificity constant (kcat/Km) obtained for
dG3′p was found to be approximately 4-fold higher than
that obtained for dG3′p-C8-4ABP and 20-125-fold higher
than those determined for the monoarylamine adducts.
This strongly suggests that dG3′p-C8-Ar adducts are
intrinsically poorer substrates for PNK than dG3′p.
Although the lability of the adducts under alkaline
conditions cannot be dismissed, it should be noted that
the incubations were conducted for a short time (5 min)
and that we did not detect a significant decrease in the
concentration of dG3′,5′p-C8-4ABP over a period of 2 h.
When it is considered that all the adducts investigated
were structurally similar, and that 4-aminobiphenyl is
bulkier than single-ring arylamines, the reasons for the
better labeling efficiency of dG3′p-C8-4ABP compared to
those obtained for monoarylamine adducts are unclear.
It is noteworthy that the apparent specificity constants
obtained with the alkylaniline-derived adducts exhibited
substantial variation with the position of the alkyl
substituents, decreasing in the following order: 4MeA
> 3,4diMeA > 3MeA > 2,4diMeA. In addition, we
obtained very poor and irreproducible labeling with
dG3′p-C8-2MeA and dG3′p-C8-2,3diMeA, for which no
kinetic parameters could be obtained. Taken together,
these observations suggest that arylamine adducts con-
taining an alkyl substitution ortho to the arylamine
nitrogen are poorer substrates for PNK than adducts not
containing ortho substituents, and that substitution para
to the arylamine nitrogen (e.g., 4-aminobiphenyl and
4-methylaniline) is less likely to hamper the labeling
process. In previous work (34, 35), we found that mono-
arylamine adducts containing ortho alkyl substituents
have a higher propensity to adopt low-energy syn con-
formations about the glycosyl bond. Thus, the decrease
in the labeling efficiencies observed with the ortho-
substituted adducts may be related, in part, to steric
factors. In this context, it may be significant that the
phenyl rings in 4-aminobiphenyl are not coplanar (40,
59), which may result in a less hindered environment
near the active site than would be expected for a bulky
aromatic segment.
Ack n ow led gm en t. We thank Letha Couch for the
synthesis of [γ-32P]ATP and Cindy Hartwick for helping
to prepare the manuscript. This work was supported in
part by NATO (Collaborative Research Grant 960165).
In addition, L.L.G.M. received financial assistance from
FCT (Portugal) and the Luso-American Foundation for
Development (FLAD).
Refer en ces
(1) Randerath, K., Reddy, M. V., and Gupta, R. C. (1981) 32P-Labeling
test for DNA damage. Proc. Natl. Acad. Sci. U.S.A. 78, 6126-
6129.
(2) Gupta, R. C., Reddy, M. V., and Randerath, K. (1982) 32P-
postlabeling analysis of non-radioactive aromatic carcinogen-DNA
adducts. Carcinogenesis 3, 1081-1092.
(3) Beach, A. C., and Gupta, R. C. (1992) Human biomonitoring and
the 32P-postlabeling assay. Carcinogenesis 13, 1053-1074.
(4) Poirier, M. C., and Beland, F. A. (1992) DNA adduct measure-
ments and tumor incidence during chronic carcinogen exposure
in animal models: implications for DNA adduct-based human
cancer risk assessment. Chem. Res. Toxicol. 5, 749-755.
(5) Gupta, R. C., and Earley, K. (1988) 32P-adduct assay: comparative
recoveries of structurally diverse DNA adducts in the various
enhancement procedures. Carcinogenesis 9, 1687-1693.
(6) Gallagher, J . E., J ackson, M. A., George, M. H., Lewtas, J ., and
Robertson, I. G. C. (1989) Differences in detection of DNA adducts
in the 32P-postlabelling assay after either 1-butanol extraction
or nuclease P1 treatment. Cancer Lett. 45, 7-12.
(7) Reddy, M. V., Gupta, R. C., Randerath, E., and Randerath, K.
(1984) 32P-Postlabeling test for covalent DNA binding of chemi-
cals in vivo: application to a variety of aromatic carcinogens and
methylating agents. Carcinogenesis 5, 231-243.
(8) Gupta, R. C. (1985) Enhanced sensitivity of 32P-postabeling
analysis of aromatic carcinogen:DNA adducts. Cancer Res. 45,
5656-5662.
(9) Randerath, E., Agrawal, H. P., Weaver, J . A., Bordelon, C. B.,
and Randerath, K. (1985) 32P-Postlabeling analysis of DNA
adducts persisting for up to 42 weeks in the skin, epidermis and
dermis of mice treated topically with 7,12-dimethylbenz[a]an-
thracene. Carcinogenesis 6, 1117-1126.
(10) Reddy, M. V., and Randerath, K. (1987) 32P-Analysis of DNA
adducts in somatic and reproductive tissues of rats treated with
the anticancer antibiotic, mitomycin C. Mutat. Res. 179, 75-88.
(11) Hemminki, K., Peltonen, K., and Mustonen, R. (1990) 32P-
Postlabelling of 7-methyl-dGMP ring-opened 7-methyl-dGMP and
platinated dGpdG. Chem.-Biol. Interact. 74, 45-54.
(12) Koivisto, P., and Hemminki, K. (1990) 32P-Postlabelling of 2-hy-
droxyethylated, ethylated and methylated adducts of 2′-deoxy-
guanosine 3′-monophosphate. Carcinogenesis 11, 1389-1392.
(13) Hemminki, K., Szyfter, K., and Kadlubar, F. F. (1991) Quanti-
tation of the 32P-postlabeling reaction using cyclic N1,N2 and C8
modified deoxyguanosine 3′-monophosphates as substrates. Chem.-
Biol. Interact. 77, 51-61.
(14) Szyfter, K., Hemminki, K., Crane, A. E., and Watson, W. P. (1991)
Quantitative and kinetic examination of 32P-postlabeling of
etheno-substituted nucleotides. Chem.-Biol. Interact. 80, 99-107.
(15) Hemminki, K., Fo¨rsti, A., Lo¨fgren, M., Segerba¨ck, D., Vaca, C.,
and Vodicka, P. (1993) Testing of quantitative parameters in the
32P-postlabelling method. In Postlabelling Methods for Detection
of DNA Adducts (Phillips, D. H., Castegnaro, M., and Bartsch,
H., Eds.) pp 51-63, IARC Scientific Publication 124, International
Agency for Research on Cancer, Lyon, France.
Con clu sion s
We have conducted a comparative kinetic study of the
PNK-catalyzed 32P-postlabeling of dG3′p and a series of
dG3′p-C8-Ar adducts (Ar being 4ABP, 3MeA, 4MeA,
2,4MeA, and 3,4diMeA) from aromatic amines present
in tobacco smoke. The results indicate that optimal
labeling requires [γ-32P]ATP concentrations substantially
higher than those used in typical 32P-postlabeling pro-
tocols and confirm that the adducts are labeled with
lower efficiencies than dG3′p, with the apparent specific-
ity constants decreasing in the following order: 4ABP >
4MeA > 3,4diMeA > 3MeA > 2,4diMeA. Substitution
ortho to the arylamine nitrogen further decreased the
labeling efficiency, which may be related, in part, to the
higher propensity of ortho-substituted adducts to adopt
low-energy syn conformations about the glycosyl bond.

668 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Mourato et al.
(16) Shields, P. G., Harris, C. C., Petruzzelli, S., Bowman, E. D., and
Weston, A. (1993) Standardization of the 32P-postlabeling assay
for polycyclic aromatic hydrocarbon-DNA adducts. Mutagenesis
8, 121-126.
(17) Searle, C. E., and Teale, O. J . (1990) Occupational carcinogens.
In Handbook of Experimental Pharmacology. Chemical Carcino-
genesis and Mutagenesis I (Cooper, C. S., and Grover, P. L., Eds.)
Vol. 94/I, pp 103-151, Springer-Verlag, Heidelberg, Germany.
(18) Patrianakos, C., and Hoffmann, D. (1979) Chemical studies on
tobacco smoke LXIV. On the analysis of aromatic amines in
cigarette smoke. J . Anal. Toxicol. 3, 150-154.
(19) Luceri, F., Pieraccini, G., Moneti, G., and Dolara, P. (1993)
Primary aromatic amines from side-stream cigarette smoke are
common contaminants of indoor air. Toxicol. Ind. Health 9, 405-
413.
(20) Grimmer, G., Schneider, D., Naujack, K.-W., Dettbarn, G., and
J acob, J . (1995) Intercept-reactant method for the determination
of aromatic amines in mainstream tobacco smoke. Beitr. Tabak-
forsch. Int. 16, 141-156.
(35) Marques, M. M., Mourato, L. L. G., Amorim, M. T., Santos, M.
A., Melchior, W. B., J r., and Beland, F. A. (1997) Effect of
substitution site upon the oxidation potentials of alkylanilines,
the mutagenicities of N-hydroxyalkylanilines, and the conforma-
tions of alkylaniline-DNA adducts. Chem. Res. Toxicol. 10, 1266-
1274.
(36) Stillwell, W. G., Bryant, M. S., and Wishnok, J . S. (1987) GC/MS
analysis of biologically important aromatic amines. Application
to human dosimetry. Biomed. Environ. Mass Spectrom. 14, 221-
227.
(37) Bryant, M. S., Vineis, P., Skipper, P. L., and Tannenbaum, S. R.
(1988) Hemoglobin adducts of aromatic amines: associations with
smoking status and type of tobacco. Proc. Natl. Acad. Sci. U.S.A.
85, 9788-9791.
(38) Phillips, D. H., and Castegnaro, M. (1993) Results of an inter-
laboratory trial of 32P-postlabelling. In Postlabelling Methods for
Detection of DNA Adducts (Phillips, D. H., Castegnaro, M., and
Bartsch, H., Eds.) pp 35-49, IARC Scientific Publication 124,
International Agency for Research on Cancer, Lyon, France.
(39) Beland, F. A., Doerge, D. R., Churchwell, M. I., Poirier, M. C.,
Schoket, B., and Marques, M. M. (1999) Synthesis, characteriza-
tion, and quantitation of a 4-aminobiphenyl-DNA adduct stan-
dard. Chem. Res. Toxicol. 12, 68-77.
(21) Felton, J . S., Knize, M. G., Roper, M., Fultz, E., Shen, N. H., and
Turteltaub, K. W. (1992) Chemical analysis, prevention, and low-
level dosimetry of heterocyclic amines from cooked food. Cancer
Res. 52 (Suppl.), 2103s-2107s.
(40) Shapiro, R., Underwood, G. R., Zawadzka, H., Broyde, S., and
Hingerty, B. E. (1986) Conformation of d(CpG) modified by the
carcinogen 4-aminobiphenyl: a combined experimental and theo-
retical analysis. Biochemistry 25, 2198-2205.
(41) Lillehaug, J . R., and Kleppe, K. (1975) Kinetics and specificity of
T4 polynucleotide kinase. Biochemistry 14, 1221-1225.
(42) Cornish-Bowden, A. (1995) Analysis of Enzyme Kinetic Data, pp
1-198, Oxford University Press, New York.
(43) Brooks, S. P. J . (1992) A simple computer program with statistical
tests for the analysis of enzyme kinetics. BioTechniques 13, 906-
911.
(44) Segel, I. H. (1993) Enzyme Kinetics. Behavior and Analysis of
Rapid Equilibrium and Steady-State Enzyme Systems, pp 1-957,
Wiley Classics Library Edition, New York.
(22) Hoffmann, D., and Hecht, S. S. (1990) Advances in tobacco
carcinogenesis. In Handbook of Experimental Pharmacology.
Chemical Carcinogenesis and Mutagenesis I (Cooper, C. S., and
Grover, P. L., Eds.) Vol. 94/I, pp 63-102, Springer-Verlag,
Heidelberg, Germany.
(23) Mommsen, S., and Aagaard, J . (1983) Tobacco as a risk factor in
bladder cancer. Carcinogenesis 4, 335-338.
(24) Vineis, P. (1992) Epidemiological models of carcinogenesis: the
example of bladder cancer. Cancer Epidemiol., Biomarkers Prev.
1, 149-153.
(25) Ambrosone, C. B., Freudenheim, J . L., Graham, S., Marshall, J .
R., Vena, J . E., Brasure, J . R., Michalek, A. M., Laughlin, R.,
Nemoto, T., Gillenwater, K. A., Harrington, A. M., and Shields,
P. G. (1996) Cigarette smoking, N-acetyltransferase 2 genetic
polymorphisms, and breast cancer risk. J . Am. Med. Assoc. 276,
1494-1501.
(26) DeBruin, L. S., Pawliszyn, J . B., and J osephy, P. D. (1999)
Detection of monocyclic aromatic amines, possible mammary
carcinogens, in human milk. Chem. Res. Toxicol. 12, 78-82.
(27) Cuzick, J ., Routledge, M. N., J enkins, D., and Garner, R. C. (1990)
DNA adducts in different tissues of smokers and non-smokers.
Int. J . Cancer 45, 673-678.
(28) Talaska, G., Al-J uburi, A. Z. S. S., and Kadlubar, F. F. (1991)
Smoking related carcinogen-DNA adducts in biopsy samples of
human urinary bladder: identification of N-(deoxyguanosin-8-
yl)-4-aminobiphenyl as a major adduct. Proc. Natl. Acad. Sci.
U.S.A. 88, 5350-5354.
(29) Talaska, G., Schamer, M., Skipper, P., Tannenbaum, S., Caporaso,
N., Unruh, L., Kadlubar, F. F., Bartsch, H., Malaveille, C., and
Vineis, P. (1991) Detection of carcinogen-DNA adducts in exfo-
liated urothelial cells of cigarette smokers: association with
smoking, hemoglobin adducts, and urinary mutagenicity. Cancer
Epidemiol., Biomarkers Prev. 1, 61-66.
(30) Lin, D., Lay, J . O., J r., Bryant, M. S., Malaveille, C., Friesen, M.,
Bartsch, H., Lang, N. P., and Kadlubar, F. F. (1994) Analysis of
4-aminobiphenyl-DNA adducts in human urinary bladder and
lung by alkaline hydrolysis and negative ion gas chromatography-
mass spectrometry. Environ. Health Perspect. 102 (Suppl. 6), 11-
16.
(31) Beland, F. A., and Kadlubar, F. F. (1990) Metabolic activation
and DNA adducts of aromatic amines and nitroaromatic hydro-
carbons. In Handbook of Experimental Pharmacology. Chemical
Carcinogenesis and Mutagenesis I (Cooper, C. S., and Grover, P.
L., Eds.) Vol. 94/I, pp 267-325, Springer-Verlag, Heidelberg,
Germany.
(32) Kadlubar, F. F. (1994) DNA adducts of carcinogenic aromatic
amines. In DNA Adducts: Identification and Biological Signifi-
cance (Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F.
F., Segerba¨ck, D., and Bartsch, H., Eds.) pp 199-216, IARC
Scientific Publication 125, International Agency for Research on
Cancer, Lyon, France.
(33) Delclos, K. B., and Kadlubar, F. F. (1997) Carcinogenic aromatic
amines and amides. In Comprehensive Toxicology, Volume 12:
Chemical Carcinogens and Anticarcinogens (Bowden, G. T., and
Fischer, S. M., Eds.) pp 141-170, Elsevier Science, Ltd., Oxford,
U.K.
(34) Marques, M. M., Mourato, L. L. G., Santos, M. A., and Beland,
F. A. (1996) Synthesis, characterization, and conformational
analysis of DNA adducts from methylated anilines present in
tobacco smoke. Chem. Res. Toxicol. 9, 99-108.
(45) Randerath, K., Haglund, R. E., Phillips, D. H., and Reddy, M. V.
(1984) 32P-Post-labelling analysis of DNA adducts formed in the
livers of animals treated with safrole, estragole and other
naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice.
Carcinogenesis 5, 1613-1622.
(46) Phillips, D. H., Reddy, M. V., and Randerath, K. (1984) 32P-
Post-labelling analysis of DNA adducts formed in the livers of
animals treated with safrole, estragole and other naturally-
occurring alkenylbenzenes. II. Newborn male B6C3F1 mice.
Carcinogenesis 5, 1623-1628.
(47) Phillips, D. H., and Castegnaro M. (1999) Standardization and
validation of DNA adduct postlabelling methods: report of
interlaboratory trials and production of recommended protocols.
Mutagenesis 14, 301-315.
(48) Garner, R. C., Martin, C. N., and Clayson, D. B. (1984) Carcino-
genic aromatic amines and related compounds. In Chemical
Carcinogens (Searle, C. E., Ed.) pp 175-276, ACS Monograph 182,
American Chemical Society, Washington, DC.
(49) Weisburger, E. K., Russfield, A. B., Homburger, F., Weisburger,
J . H., Boger, E., Van Dongen, C. G., and Chu, K. C. (1978) Testing
of twenty-one environmental aromatic amines or derivatives for
long-term toxicity or carcinogenicity. J . Environ. Pathol. Toxicol.
2, 325-356.
(50) Keenaghan, J . B., and Boyes, R. N. (1972) The tissue distribution,
metabolism and excretion of lidocaine in rats, guinea pigs, dogs
and man. J . Pharmacol. Exp. Ther. 180, 454-463.
(51) International Agency for Research on Cancer (1993) 2,6-Di-
methylaniline (2,6-xylidine). IARC Monographs on the Evaluation
of Carcinogenic Risks to Humans. Occupational Exposures of
Hairdressers and Barbers and Personal Use of Hair Colourants;
Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and
Aromatic Amines, Vol. 57, pp 323-335, International Agency for
Research on Cancer, Lyon, France.
(52) Ward, E., Carpenter, A., Markowitz, S., Roberts, D., and Halperin,
W. (1991) Excess number of bladder cancers in workers exposed
to ortho-toluidine and aniline. J . Natl. Cancer Inst. 83, 501-506.
(53) Lee, C. K., Brown, B. G., Bombick, B. R., and Doolittle, D. J . (1995)
Effect of pH of the T4 polynucleotide kinase reaction on the 32P-
postlabeling assay of DNA adducts. Anal. Biochem. 227, 156-
161.
(54) Cameron, V., and Uhlenbeck, O. C. (1977) 3′-Phosphatase activity
in T4 polynucleotide kinase. Biochemistry 16, 5120-5126.
(55) Shibutani, S., Gentles, R. G., Iden, C. R., and J ohnson, F. (1990)
Facile aerial oxidation of the DNA-base adduct N-(2′-deoxygua-
nosin-8-yl)-2-aminofluorene [dG(C8)AF]. J . Am. Chem. Soc. 112,
5667-5668.

32P-Postlabeling Efficiency of DNA Adducts
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 669
(56) Reddy, M. V., Bleicher, W. T., and Blackburn, G. R. (1991) 32P-
Postlabeling detection of thymine glycols: evaluation of adduct
recoveries after enhancement with affinity chromatography,
nuclease P1, nuclease S1, and polynucleotide kinase. Cancer
Commun. 3, 109-117.
(57) Cornish-Bowden, A. (1984) Enzyme specificity: its meaning in
the general case. J . Theor. Biol. 108, 451-457.
(58) Panet, A., van de Sande, J . H., Loewen, P. C., Khorana, H. G.,
Raae, A. J ., Lillehaug, J . R., and Kleppe, K. (1973) Physical
characterization and simultaneous purification of bacteriophage
T4 induced polynucleotide kinase, polynucleotide ligase, and
deoxyribonucleic acid polymerase. Biochemistry 12, 5045-5050.
(59) Cho, B. P., Beland, F. A., and Marques, M. M. (1992) NMR
structural studies of a 15-mer DNA sequence from a ras proto-
oncogene, modified at the first base of codon 61 with the
carcinogen 4-aminobiphenyl. Biochemistry 31, 9587-9602.
TX990038T