Detection of DNA and globin adducts of polynuclear aromatic hydrocarbon diol epoxides by gas chromatography - Mass spectrometry and [3H]CH3I postlabeling of released tetraols

Article abstract of DOI:10.1021/tx950165z

Gas chromatography - negative ion chemical ionization mass spectrometry - selected ion monitoring (GC-NICI-MS-SIM) was employed to detect tetramethyl ether derivatives of tetraols formed upon hydrolysis of DNA and globin adducts derived from diol epoxides of benzo[α]-pyrene (BP) and other polynuclear aromatic hydrocarbons (PAH). The tetramethyl ether derivatives could also be detected by [³H]CH₃I postlabeling. The methodology involves the following steps: (1) isolation of DNA or globin; (2) mild acid hydrolysis under vacuum; (3) isolation of the resulting tetraols and derivatization to the corresponding tetramethyl ethers using methyl sulfinyl carbanion and unlabeled or ³H-labeled CH₃I; (4) analysis by GC-NICI-MS-SIM or HPLC with radioflow detection. The optimum conditions for hydrolysis of adducts and derivatization of the resulting tetraols as well as the feasibility of this approach for detecting PAH adducts in mice and humans were explored. Using the set of four BP-tetraols that can be formed upon hydrolysis of adducts formed from r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[α]pyrene (anti-BPDE) or r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo-[α]pyrene (syn-BPDE) as models, the stability of the tetraols under the hydrolysis conditions was investigated. Adducts derived from anti-BPDE yield predominantly the stable r-7,t-8,9-c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[α]pyrene (trans-anti-BP-tetraol), while adducts derived from syn-BPDE released cis-syn-BP-tetraol as a major hydrolysis product. Hydrolysis under vacuum significantly increased the recovery of tetraols. Conditions for derivatization of the BP-tetraols as well as tetraols derived from several other PAH anti-diol epoxides were investigated. Tetramethyl ethers proved to be superior derivatives that were stable, easy to prepare in high yields, and detectable with high sensitivity by GC-NICI-MS-SIM (1-50 fmol per injection). Alternatively, these derivatives could be detected by HPLC with radioflow detection if [³H]CH₃I were employed for derivatization. The methodology was tested by comparing levels of DNA and globin adducts in mice treated with either unlabeled or ³H-labeled BP. Good agreement was obtained among the GC-NICI-MS-SIM, [³H]CH₃I postlabeling, and conventional radiometric methods. Moreover, analysis of human hemoglobin by GC-NICI-MS-SIM resulted in detection of adducts derived from anti-BPDE and r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydrochrysene. The results of this study demonstrate that GC-NICI-MS-SIM of tetramethyl ethers of tetraols formed by hydrolysis of PAH diol epoxide DNA and globin adducts is a promising approach for detection and quantitation of adducts derived from a broad range of PAH.

Full text of DOI:10.1021/tx950165z

508
Chem. Res. Toxicol. 1996, 9, 508-516
Detection of DNA a n d Globin Ad d u cts of P olyn u clea r
Ar om a tic Hyd r oca r bon Diol Ep oxid es by Ga s
Ch r om a togr a p h y-Ma ss Sp ectr om etr y a n d [3H]CH3I
P ostla belin g of Relea sed Tetr a ols
Assieh A. Melikian,* Peng Sun, Stuart Coleman, Shantu Amin, and
Stephen S. Hecht
American Health Foundation, Valhalla, New York 10595
Received September 25, 1995X
Gas chromatography-negative ion chemical ionization mass spectrometry-selected ion
monitoring (GC-NICI-MS-SIM) was employed to detect tetramethyl ether derivatives of tetraols
formed upon hydrolysis of DNA and globin adducts derived from diol epoxides of benzo[a]-
pyrene (BP) and other polynuclear aromatic hydrocarbons (PAH). The tetramethyl ether
derivatives could also be detected by [3H]CH3I postlabeling. The methodology involves the
following steps: (1) isolation of DNA or globin; (2) mild acid hydrolysis under vacuum; (3)
isolation of the resulting tetraols and derivatization to the corresponding tetramethyl ethers
3
using methyl sulfinyl carbanion and unlabeled or H-labeled CH3I; (4) analysis by GC-NICI-
MS-SIM or HPLC with radioflow detection. The optimum conditions for hydrolysis of adducts
and derivatization of the resulting tetraols as well as the feasibility of this approach for detecting
PAH adducts in mice and humans were explored. Using the set of four BP-tetraols that can
be formed upon hydrolysis of adducts formed from r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene (anti-BPDE) or r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo-
[a]pyrene (syn-BPDE) as models, the stability of the tetraols under the hydrolysis conditions
was investigated. Adducts derived from anti-BPDE yield predominantly the stable r-7,t-8,9-
c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BP-tetraol), while adducts
derived from syn-BPDE released cis-syn-BP-tetraol as a major hydrolysis product. Hydrolysis
under vacuum significantly increased the recovery of tetraols. Conditions for derivatization
of the BP-tetraols as well as tetraols derived from several other PAH anti-diol epoxides were
investigated. Tetramethyl ethers proved to be superior derivatives that were stable, easy to
prepare in high yields, and detectable with high sensitivity by GC-NICI-MS-SIM (1-50 fmol
per injection). Alternatively, these derivatives could be detected by HPLC with radioflow
detection if [3H]CH3I were employed for derivatization. The methodology was tested by
comparing levels of DNA and globin adducts in mice treated with either unlabeled or 3H-
labeled BP. Good agreement was obtained among the GC-NICI-MS-SIM, [3H]CH3I postlabeling,
and conventional radiometric methods. Moreover, analysis of human hemoglobin by GC-NICI-
MS-SIM resulted in detection of adducts derived from anti-BPDE and r-1,t-2-dihydroxy-t-3,4-
epoxy-1,2,3,4-tetrahydrochrysene. The results of this study demonstrate that GC-NICI-MS-
SIM of tetramethyl ethers of tetraols formed by hydrolysis of PAH diol epoxide DNA and globin
adducts is a promising approach for detection and quantitation of adducts derived from a broad
range of PAH.
over, quantitation of cellular macromolecular damage in
humans can assist in extrapolating results of animal
experiments to humans.
In tr od u ction
Polynuclear aromatic hydrocarbons (PAH)1 are ubiq-
uitous environmental carcinogens arising from incom-
plete combustion of organic materials and fossil fuels.
Therefore, human exposure to these compounds at some
levels is unavoidable. Many PAH are tumorigenic in
animal bioassays (1-4). Although there is no direct
evidence that an individual PAH causes cancer in hu-
mans, there is substantial evidence to suggest that
complex PAH mixtures such as those in tobacco smoke,
coal tar pitch, engine exhaust emissions, and shale oils
cause cancer in humans (1-4). Characterization and
measurement of PAH-macromolecular adducts in target
tissues or in readily available surrogate cells in humans
can provide qualitative and quantitative information
about biologically effective doses of PAH exposure. More-
PAH mediate their biological activities through their
metabolic activation to reactive diol epoxides that bind
covalently to cellular macromolecules (5-16). Methods
for detecting the resulting adducts in humans such as
32P-postlabeling (17), [35S]phosphorothioate labeling (18),
HPLC-linked fluorescence detection (19, 20), synchronous
fluorescence spectrophotometry (21), fluorescense line
narrowing (22), immunoassays (23-25), and GC-MS (26-
28) have been developed; however, few are quantitative
or specific for individual PAH. Each of these methods
has some limitation. Most of the techniques applied do
not provide full characterization of unknown PAH ad-
ducts or quantitative information on individual PAH.
Our long-term goal is to develop a mass spectrometric
method to characterize PAH diol epoxide-derived adducts
with hemoglobin and DNA in human tissues, to quantify
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0893-228x/96/2709-0508$12.00/0 © 1996 American Chemical Society
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Detection of PAH Adducts by GC-MS and [3H]CH3I
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 509
F igu r e 1. Structures of BP-tetraols and their corresponding tetramethyl ethers.
such adducts, and to incorporate them as biomarkers into
molecular epidemiology studies. In our initial studies,
described here, we have used mild acid hydrolysis of
globin or DNA under vacuum to release the PAH moiety
as a tetraol. This is followed by permethylation of the
PAH-tetraols via tritiated or unlabeled CH3I in the
presence of methyl sulfinyl carbanion (29, 30) and
analysis of the derivatized tetraols by HPLC or gas
chromatography-negative ion chemical ionization mass
spectrometry-selected ion monitoring (GC-NICI-MS-
SIM). The set of BP-tetraol isomers shown in Figure 1
has been used as a model system for investigation of the
methodology. Each BP diol epoxide forms adducts with
hemoglobin and DNA that can be hydrolyzed to two
isomeric tetraols (trans or cis). Methylation of each of
these tetraols gives the corresponding tetramethyl ethers.
Figure 1 shows the stereochemistry of the major tetraols
that can be formed upon acid hydrolysis of adducts from
r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo-
[a]pyrene (anti-BPDE) or r-7,t-8-dihydroxy-c-9,10-epoxy-
7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE). It also
shows the structures of the corresponding tetramethyl
ethers.
1
Abbreviations: PAH, polynuclear aromatic hydrocarbons; BP,
benzo[a]pyrene; anti-BPDE, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene; syn-BPDE, r-7,t-8-dihydroxy-c-9,10-epoxy-
7,8,9,10-tetrahydrobenzo[a]pyrene; anti-chrysene-DE, r-1,t-2-dihydroxy-
t-3,4-epoxy-1,2,3,4-tetrahydrochrysene; anti-5-MeCDE, r-1,t-2-dihydroxy-
t-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene; anti-6-MeCDE, r-1,t-
2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene; anti-
B[a]ADE, r-3,t-4-dihydroxy-t-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]an-
thracene; anti-B[c]PDE, r-3,t-4-dihydroxy-t-1,2-epoxy-1,2,3,4-tetrahy-
drobenzo[c]phenanthrene; trans-anti-BP-tetraol, r-7,t-8,9,c-10-tetrahy-
droxy-7,8,9,10-tetrahydrobenzo[a]pyrene; trans-syn-BP-tetraol, r-7,t-
8,10,c-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-anti-BP-
tetraol, r-7,t-8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene;
cis-syn-BP-tetraol, r-7,t-8,c-9,10-tetrahydroxy-7,8,9,10-tetrahydroben-
zo[a]pyrene; trans-anti-chrysene-tetraol, r-1,t-2,3,c-4-tetrahydroxy-
1,2,3,4-tetrahydrochrysene; trans-anti-5-MeC-tetraol, r-1,t-2,3,c-4-
tetrahydroxy-1,2,3,4-tetrahydro-5-methylchrysene; trans-anti-6-MeC-
tetraol, r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydro-6-methylchrysene;
trans-anti-B[a]A-tetraol, r-1,t-2,3-c-4-tetrahydroxy-1,2,3,4-tetrahy-
drobenz[a]anthracene; trans-anti-B[c]P-tetraol, r-1,t-2,3-c-4-tetrahy-
droxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; trans-anti-TME-BP-
tetraol, r-7,t-8,9,c-10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene;
trans-syn-TME-BP-tetraol, r-7,t-8,10,c-9-tetramethoxy-7,8,9,10-tet-
rahydrobenzo[a]pyrene; cis-anti-TME-BP-tetraol, r-7,t-8,9,10-tetra-
methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-syn-TME-BP-tetraol,
r-7,t-8,c-9,10-tetramethoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; TME-
chrysene-tetraol, r-1,t-2,3,c-4-tetramethoxy-1,2,3,4-tetrahydrochrysene;
GC-NICI-MS, gas chromatogaphy-negative ion chemical ionization
mass spectrometry; GC-NICI-MS-SIM, gas chromatogaphy-negative
ion chemical ionization mass spectrometry-selected ion monitoring.
This paper describes (a) the stability of BP-tetraols
under acid hydrolysis conditions; (b) optimal hydrolysis
conditions for release of BP-tetraols from DNA and
globin; (c) derivatization of individual BP-tetraols and
other PAH-tetraols to tetramethyl ethers; (d) GC-NICI-
MS analysis of the tetramethyl ethers; (e) application of
the assay for quantifying globin or DNA adducts from
mice treated with [3H]BP or unlabeled BP and compari-
son of the GC-NICI-MS-SIM results to those obtained
with conventional scintillation counting methodology or
by postlabeling via [3H]CH3I; and (f) detection of BP-
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510 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Melikian et al.
Sch em e 1. Exp er im en ta l Design of in Vivo Stu d ies
w ith DNA a n d Globin of Mice
tetraols and chrysene-tetraols in hydrolysates of human
globin.
Exp er im en ta l Section
Ca u tion . PAH and many of their metabolites are mutagens
and/ or carcinogens. They should be handled with extreme care,
using appropriate safetywear and ventilation at all times.
Ch em ica ls. r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahy-
drobenzo[a]pyrene (trans-anti-BP-tetraol), r-7,t-8,10,c-9,-tet-
rahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-syn-BP-
tetraol), r-7,t-8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]-
pyrene (cis-anti-BP tetraol), r-7,t-8,c-9,10-tetrahydroxy-7,8,9,-
10-tetrahydrobenzo[a]pyrene (cis-syn-BP-tetraol), and (()-anti-
BPDE (0.53 Ci/mmol) were supplied by the National Cancer
Institute’s Chemical Carcinogen Reference Standard Repository,
Midwest Research Institute (Kansas City, MO). r-1,t-2-Dihy-
droxy-t-3,4-epoxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-
DE); r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydro-5-methyl-
chrysene (anti-5-MeCDE); r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4-
tetrahydro-6-methylchrysene (anti-6-MeCDE); r-1,t-2-dihydroxy-
t-3,4-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene (anti-B[a]ADE);
and r-1,t-2-dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydrobenzo[c]-
phenanthrene (anti-B[c]PDE) were synthesized as described
previously (31, 32). [3H]BP, specific activity 25 Ci/mmol (diluted
with unlabeled BP to give a specific activity of 67 mCi/mmol),
and [3H]CH3I, 80 Ci/mmol (in toluene solution), were purchased
from Amersham Corp. (Arlington Heights, IL). CH3I and NaH
(60% dispersion in mineral oil) were obtained from Aldrich
Chemical Co. (Milwaukee, WI). HCl (6 N), DMSO, and vacuum
reaction tubes were bought from Pierce Chemical Co. (Rockford,
IL).
P r ep a r a tion of (()-a n ti-BP DE-DNA Ad d u cts. A solu-
tion of about 50 µg of (()-BPDE in 0.1 mL of acetone was added
to purified calf thymus DNA (1 mg/mL in 0.01 M Tris buffer,
pH 7.4) and incubated at 37 °C for 6 h. The unreacted BPDE
that hydrolyzed to BP-tetraols was extracted five times with 2
volumes of EtOAc. DNA was precipitated by adding EtOH.
Sta bility of BP -Tetr a ols u n d er Acid Hyd r olysis Con d i-
tion s. Five micrograms of each isomer of BP-tetraol (purified
by HPLC) in 500 µL of MeOH was transferred into vials. After
removal of the MeOH, 0.5 mL of 0.1 N HCl was added to each
vial and the mixtures were incubated at either 80 or 90 °C for
3 h. After cooling, each sample was adjusted to pH 5 and
extracted five times with 2 volumes of EtOAc. The solvent was
removed under N2, and the residue was dissolved in MeOH for
HPLC analysis (system 1).
P r ep a r a tion of P AH-Tetr a ols via Hyd r olysis of P AH-
Diol Ep oxid es. Each PAH-diol epoxide, anti-5-MeCDE, anti-
6-MeCDE, anti-B[a]ADE, and anti-B[c]PDE (200 µg), was
dissolved in a minimal volume of EtOH (about 1-2 mL), added
to 10 mL of 0.1 N HCl, and stirred at room temperature
overnight. As an exception, anti-chrysene-DE was dissolved in
200 µL of THF and hydrolyzed in 0.9% formic acid overnight at
room temperature. After the pH was adjusted to 5, the PAH-
tetraols were extracted five times with 2 volumes of EtOAc and
purified by HPLC (system 4). The concentration of each tetraol
was determined by UV. The major product in each case was
the trans-anti-tetraol resulting from trans-ring opening as
previously reported (11-13, 33).
An im a l Tr ea tm en ts. Seven- to eight-week-old female Crl:
CD-1(1CR)BR mice were obtained from Charles River Breeding
Laboratories (North Wilmington, MA). In experiment 1, [3H]-
BP (0.012 mmol, 0.8 mCi) in 0.2 mL of corn oil was administered
by gavage to each of 28 mice. In experiment 2, each of 10 mice
received the same dose of unlabeled BP in 0.2 mL of corn oil by
gavage. Animals were sacrificed 24 h after treatment. Blood
samples were collected in EDTA-containing vacutainers, and
the livers and lungs were isolated and frozen immediately. DNA
was isolated from tissues and globin from blood cells. They were
analyzed as outlined in Scheme 1.
between Dr. David Ciavarella of the Hudson Valley Blood Bank
and Dr. Sharon E. Murphy of the American Health Foundation.
All procedures were approved by the Institutional Review
Boards of both institutions. Other samples were obtained
courtesy of Dr. Nancy Haley, Metropolitan Life Insurance Co.
Isola tion of DNA fr om Tissu es. DNA was isolated as de-
scribed previously (34). In brief, the tissues were homogenized
in 1 mM EDTA containing 1% sodium dodecyl sulfate. The
homogenates were incubated with proteinase K (500 µg/mL) and
extracted with phenol that contained 0.1% 8-hydroxyquinoline
as an antioxidant and then with phenol/CHCl3/isoamyl alcohol
and CHCl3/isoamyl alcohol. DNA was precipitated by adding
EtOH. Residual unbound radioactive material was removed by
repeated washing of the DNA with acetone and then with ether.
Isola tion of Globin fr om Mou se Blood . Red blood cells
were isolated by centrifugation (2000 rpm, 4 °C, 10 min) and
washed three times with 1 volume of physiological saline. Cells
were lysed by adding 1 volume of H2O and 1 volume of 0.67 M
sodium phosphate buffer (pH 6.5). Cell debris was pelleted by
centrifugation (13 500 rpm, 25 min). Globin was precipitated
with acetone as follows: the supernatant was added dropwise
to 20 volumes of ice-cold acetone/15 mM HCl, mixed vigorously,
and allowed to stand at -20 °C for 30 min. The globin
precipitate was separated by centrifugation (2000 rpm, 10 min),
washed three times with acetone, and then redissolved in a
small volume of H2O. The precipitation was repeated twice to
remove metabolites of [3H]BP and heme (21). At each step the
total radioactivity released in acetone was measured. After
three consecutive precipitations of globin, radioactive material
was no longer detected in the acetone wash.
Isola tion of Globin fr om Hu m a n Blood . Red blood cells
were isolated and lysed as described above. After centrifugation
to remove cellular debris, the supernatant was dialyzed against
H2O at 4°C for 48 h. Globin was precipitated by dropwise
addition of the hemoglobin solution to 20 volumes of ice-cold
acetone/15 mM HCl, with vigorous stirring. The precipitate was
filtered and washed with ice-cold acetone. An aliquot of each
globin sample was subjected to acid hydrolysis.
Hyd r olysis of DNA Ad d u cts. Calf thymus DNA samples
modified with (()-anti-BPDE in vitro or DNA isolated from the
lungs and livers of mice treated with [3H]BP (0.66 mg/mL of
H2O) were hydrolyzed in 0.1 N HCl (pre-extracted with EtOAc)
for up to 4 h. Hydrolysis was carried out at 80 °C under vacuum
(using vacuum reaction tubes) or under N2 or air. Aliquots were
taken hourly and, after cooling, the released tetraols were
extracted five times with 2 volumes of EtOAc. The radioactivity
in the extracts was measured and, after removal of solvent
under N2, the residue was dissolved in MeOH and analyzed by
HPLC (system 2).
Hu m a n Blood Sa m p les. Some samples were obtained from
smokers and nonsmokers through a collaborative agreement
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Detection of PAH Adducts by GC-MS and [3H]CH3I
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 511
isocratic for 25 min, and, finally, a linear gradient to 100%
MeOH, at a flow rate of 1 mL/min.
Sch em e 2. P r ep a r a tion of TME-P AH-Tetr a ols via
CH3I
System 3: isocratic elution with 100% H2O for 10 min, a
linear gradient to 50:50 MeOH/H2O in 5 min, isocratic for 30
min, a linear gradient to 80:20 MeOH/H2O in 40 min, and finally
a linear gradient to 100% MeOH in 20 min, at a flow rate of 1
mL/min.
System 4: isocratic elution with 40:60 MeOH/H2O for 10 min
followed by a linear gradient to 100% MeOH in 40 min at a
flow rate of 1 mL/min.
GC-NICI-MS An a lysis Con d ition s. Permethylated PAH-
tetraol standards or permethylated tetraols released from DNA
or globin were analyzed by GC-NICI-MS or GC-NICI-MS-SIM
on a Hewlett-Packard Model 5988A system using on-column
injection with a 10-µL syringe equipped with a 15-cm fused silica
needle. An SE-54 (30 m × 0.25 mm i.d. × 0.25 µm film
thickness) capillary GC column (Alltech Associates, Deerfield,
IL) was interfaced directly with the MS source. The GC oven
temperature was held at 50 °C for 2 min, followed by program-
ming to 300 °C at 20 °C/min. The temperature was then kept
at 300 °C for 20 min. MS conditions were as follows: ion source,
150 °C; emission current, 250 µA; electron energy, 110 eV.
Ultrahigh-purity methane was the reagent gas. Solvent blanks
were injected between runs to ensure that there was no carry-
over from one sample to the next.
Hyd r olysis of Globin Ad d u cts. An aqueous solution of
globin from mice treated with [3H]BP (5 mg/mL) was transferred
into vacuum reaction tubes and hydrolyzed in 0.05-0.1 N HCl
for 0-3 h at 80 or 90 °C. [3H]BP-tetraols liberated from globin
were extracted and analyzed as described above.
On the basis of the results of this experiment, the optimum
conditions for hydrolysis of globin adducts were established as
0.1 N HCl for 3 h at 80 °C under vacuum. These conditions
were used for hydrolysis of adducts in human globin samples.
The pH was adjusted to 5, and the hydrolysate was extracted
five times with 2 volumes of EtOAc. With each set of experi-
ments a control sample that consisted of all reagents except
globin was run.
P er m eth yla tion of P AH-Tetr a ols. PAH-tetraol standard
samples or PAH-tetraols released from biological samples were
converted to tetramethyl ether derivatives according to Scheme
2 as previously described (28). Briefly, methyl sulfinyl carban-
ion was prepared by heating 100 mg of NaH (60% suspension
in mineral oil) in 1 mL of DMSO at 90 °C under N2 for 20-30
min until generation of H2 ceased. Freshly prepared methyl
sulfinyl carbanion solution (50 µL) was added to approximately
20 µg of the specified PAH-tetraol isomer dissolved in 50 µL of
DMSO. The mixture was stirred for 2 min at room temperature,
and CH3I (50 µL, 0.8 mmol) was added. After another 2 min, 1
mL of H2O was added and the tetramethyl ether derivatives of
the PAH-tetraols were extracted with benzene.
For labeling the BP-tetraols as [3H]tetramethyl ether deriva-
tives, [3H]CH3I solution (50 µL, 0.05 mCi, 0.006 mmol) was
added to the mixture of BP-tetraols (estimated to be less than
0.1 µg) in 50 µL of DMSO and 50 µL of the methyl sulfinyl
carbanion solution. The [3H]CH3I derivatization was carried
out in well-ventilated hoods; benzene was removed from the
extracts of the [3H]tetramethyl ethers by a stream of N2.
Precautions were taken to avoid release of radioactive materials
into the atmosphere by passing the N2 stream through two cold
traps above the freezing point of benzene and finally through
H2O before it was released.
Resu lts a n d Discu ssion
Sta bility of BP -Tetr a ol Isom er s u n d er Acid Hy-
d r olysis Con d ition s. Shugart et al. have demonstrated
that BPDE adducts to globin or DNA are hydrolyzed to
BP-tetraols under mild acid hydrolysis conditions (35,
36). Four BP-tetraol isomers can be liberated. We have
studied the stability of these tetraols under the hydrolysis
conditions. The results are summarized in Table 1.
trans-anti-BP-tetraol (Figure 1) was the most stable
tetraol under these hydrolysis conditions; about 80% of
the original material was recovered unchanged. cis-anti-
BP-tetraol was unstable; approximately 70% of it was
converted to trans-anti-BP-tetraol. Similarly, trans-syn-
BP-tetraol was unstable and epimerized to cis-syn-BP-
tetraol (59-67%) and only about 16% of the original
compound remained. In contrast, cis-syn-BP-tetraol did
not epimerize but decomposed to unknown products
which eluted from the HPLC column after cis-syn-BP-
tetraol (data not shown). The instability of cis-syn-BP-
tetraol was reported previously (37, 38). J ansen et al.
have shown a similar epimerization of BP-tetraols under
acid hydrolysis conditions (39). Epimerization of BP-
tetraols occurred immediately after 0.1 N HCl was added;
heating was not a major factor. However, when the
hydrolysis temperature was increased from 80 to 90 °C,
the decomposition of cis-syn-BP-tetraol into unknown
products increased from 23% to 38%.
cis-anti-BP-tetraol exists in a half-chair conformation
with C7-H and C8-H being pseudodiequatorial and C10-
OH being pseudodiaxial (40). This causes an unfavorable
steric interaction between C10-OH and C8-OH, which is
relieved upon epimerization to trans-anti-BP-tetraol.
Similar relief of 1-3 transannular interactions between
hydroxyl groups can account for the epimerization of
trans-syn-BP-tetraol.
The results of these experiments indicate that trans-
anti-BP-tetraol will be the major product detected upon
hydrolysis of adducts formed from anti-BPDE, indepen-
dent of their being trans or cis adducts. Similarly, syn-
BPDE adducts will give mainly cis-syn-BP-tetraol.
Der iva tiza tion of Ra cem ic BP -Tetr a ols to Tet-
r a m eth yl Eth er s. The application of GC-MS for detect-
An a lysis of TME-BP -Tetr a ols fr om Mice. Benzene ex-
tracts containing 3H-labeled TME-BP-tetraols from experiment
2 (Scheme 1) were concentrated to dryness, redissolved in
MeOH, and analyzed by HPLC (system 3). One-milliliter
fractions were collected, and radioactivity was measured by
scintillation counting.
The benzene extracts were also analyzed by GC-NICI-MS-
SIM as described below.
An a lysis of TME-P AH-Tetr a ols fr om Hu m a n Globin .
The extracts were concentrated to dryness, redissolved in
MeOH, and further purified by HPLC (system 4). The fraction
eluting from 25 to 45 min, which included the elution positions
of four and five ring tetraols, was collected and concentrated
under a stream of N2, and the residue was redissolved in
benzene and analyzed by GC-NICI-MS-SIM.
HP LC An a lysis Con d ition s. Four solvent systems were
employed for reversed-phase HPLC on a 5 µm Ultrasphere ODS
C18 column (25 cm × 4.6 mm, Beckman, Fullerton, CA).
System 1: isocratic elution with 55:45 MeOH/H2O for 30 min
followed by a linear gradient to 100% MeOH in 20 min, at a
flow rate of 1 mL/min.
System 2: isocratic elution with 15:85 MeOH/H2O for 30 min,
a linear gradient to 45:55 MeOH/H2O in 10 min, isocratic for
35 min, a linear gradient to 55:45 MeOH/H2O in 10 min,
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512 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Ta ble 1. Sta bility of BP -Tetr a ols u n d er Acid Hyd r olysis Con d ition sa ,b
% of each tetraol recovered after hydrolysis at 80 °C (90 °C)
tetraol subjected to hydrolysis trans-anti-BP-tetraol cis-anti-BP-tetraol trans-syn-BP-tetraol cis-syn-BP-tetraol unknown products
Melikian et al.
trans-anti-BP-tetraol
cis-anti-BP-tetraol
trans-syn-BP-tetraol
cis-syn-BP-tetraol
80(72)
68(78)
4
12(19)
25(13)
3
1
6(9)
7(9)
16(16)
14
2
67(59)
55(50)
10 (25)
23 (38)
7(11)
a
b
Hydrolysis was performed in 0.1 N HCl for 3 h at 80 or 90 °C in open air. BP-tetraols were assigned on the basis of HPLC retention
times only.
F igu r e 2. HPLC elution profiles (system 3) of tetramethyl
ethers of BP-tetraols prepared by derivatization with CH3I: (A)
trans-anti-TME-BP-tetraol; (B) cis-anti-TME-BP-tetraol; (C)
trans-syn-TME-BP-tetraol; and (D) cis-syn-TME-BP-tetraol.
F igu r e 3. GC-NICI-MS of trans-anti-TME-BP-tetraol.
Ta ble 2. HP LC a n d GC Reten tion Tim es of BP -Tetr a ols
a n d Th eir Cor r esp on d in g Tetr a m eth yl Eth er s
times of their corresponding tetramethyl ethers, are
shown in Table 2. Two of the tetramethyl ethers, trans-
anti-TME-BP-tetraol and cis-syn-TME-BP-tetraol, had
GC retention times of 22.54 min, yet they can be
separated by HPLC (85.7 and 91.2 min, respectively;
Table 2). Thus, applying HPLC before GC-NICI-MS can
give some information about the stereochemistry of the
liberated tetraols.
retention time (min)
compound
HPLCa
GCb
trans-anti-BP-tetraol
cis-anti-BP-tetraol
trans-syn-BP-tetraol
cis-syn-BP-tetraol
38.0
42.6
39.6
51.6
trans-anti-TME-BP-tetraol
cis-anti-TME-BP-tetraol
trans-syn-TME-BP-tetraol
cis-syn-TME-BP-tetraol
85.7
86.5
92.0
91.2
22.54
23.43
21.67
22.54
The NICI-MS of trans-anti-TME-BP-tetraol is illus-
trated in Figure 3. The characteristic MS shows a
molecular ion at m/ z 376 (M)- which loses one molecule
of CH3OH to produce m/ z 344 (M - 32)-. The ion at
m/ z 344 again eliminates CH3OH, resulting in m/ z 312
(M - 64)-, or loses two CH3O, producing m/ z 314 (M -
62)-. Subsequently, the ion at m/ z 312 loses one
molecule of HCHO to form m/ z 282 (M - 94)- or two
molecules of HCHO to form m/ z 252 (M - 124)-. Similar
fragmentation patterns were observed for the other TME-
BP-tetraols. These data are consistent with previously
reported MS data for TME-BP-tetraols (28).
a
b
Using HPLC elution system 3. The retention times change
as portions of the GC column are broken off to remove contamina-
tion.
ing and quantifying PAH-tetraols requires derivatization
to increase volatility and, potentially, to increase sensi-
tivity. In the course of developing the method several
perfluorinated derivatives of BP-tetraols were prepared
and analyzed. Under the derivatization conditions, the
tetraols tended to aromatize or oxidize, yielding more
than a single product. However, methylation via CH3I
(Scheme 2), described by Hakomori for glycolipids, is
applicable to the derivatization of PAH-tetraols and
yields only a single product (28, 29).
HPLC profiles of the tetramethyl ethers obtained via
derivatization of the four BP-tetraols are illustrated in
Figure 2. The HPLC analyses demonstrate that deriva-
tization of each tetraol was complete without formation
of appreciable amounts of side products. Each tetraol
was derivatized primarily to a single less polar product
(Figures 2 and Table 2), and each showed a pyrene
absorption in its UV spectrum, similar to the UV spectra
of the BP-tetraols (data not shown). HPLC elution times
of the BP-tetraols, as well as GC and HPLC retention
Each major tetraol product, formed by trans-ring
opening in the hydrolysis of several other PAH anti-diol
epoxides, was converted to its corresponding tetramethyl
ether derivative. Their GC retention times and frag-
mentation patterns are summarized in Table 3.
Tetramethyl ethers of PAH-tetraols were superior to
the other derivatives studied because (a) the availability
of [3H]CH3I at high specific activity (80 Ci/mmol) comple-
ments the GC-NICI-MS-SIM analysis; (b) they are stable
with shelf lives of greater than 2 months, whereas other
derivatives such as trimethylsilyl ethers are labile (27);
(c) their preparation is relatively simple and rapid and
proceeds in good yield (80-90% for conversion of 1 ng of
corresponding tetraol); and (d) they can be detected with
+
+

Detection of PAH Adducts by GC-MS and [3H]CH3I
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 513
Ta ble 3. GC Reten tion Tim es a n d NICI-MS Da ta for Tetr a m eth yl Eth er s of P AH-Tetr a ols
compound
GC retention time (min)
NICI-MS m/ z (rel intensity)
TME-chrysene-tetraola
TME-B[a]A-tetraol
TME-B[c]P-tetraol
TME-5-MeC-tetraol
TME-6-MeC-tetraol
18.6
18.3
18.8
19.4
19.5
353 (M- + 1, 28), 352 (M-, 10), 351 (M- - 1, 20), 336 (34), 298 (11), 287 (100), 257 (17)
352 (M-, 100)
352 (M-, 100), 320 (17)
367 (M- + 1, 33), 366 (M-, 23), 365 (M- - 1, 76), 350 (32), 301 (100), 271 (25), 229 (4)
367 (M- + 1, 60), 366 (M-, 26), 365 (M- - 1, 84), 350 (40), 301 (100), 304 (30), 271 (10),
257 (100), 229 (4)
a
EI-MS (70 eV), 352 (M+, 3), 254 (M+ - [CH3OCHOCH3], 30), 249 (M+ - [CH3OCHOCH3], 45); similar to that reported in ref 28.
Ta ble 4. Bin d in g of [3H]BP Meta bolites to th e DNA of Mou se Lu n g, Liver , a n d Globin a n d Tim e Cou r se of
EtOAc-Extr a cta ble Meta bolites Relea sed u p on Acid Hyd r olysisa ,b
EtOAc-extractable [3H]BP metabolites released from DNA
upon HCl hydrolysis (pmol/mg of DNA or globin) after hydrolysis time of
total binding of [3H]BP metabolites to
tissue
DNA or globin (pmol/mg of DNA or globin)
1 h
2 h
3 h
4 h
liver
lung
blood
113
14.6
3.9c
9.3
7.6
0.32
12.3
9.1
0.41
12.6
8.8
0.41
12.5
9.2
0.40
a
DNA was isolated from a group of mice 24 h after oral administration of 0.012 mmol of [3H]BP/mouse (experiment 1, Scheme 1);
hydrolysis was carried out with 0.1 N HCl at 80 °C under vacuum. Mean of two measurements. c Estimated on the basis of the weight
b
of globin.
high sensitivity by GC-NICI-MS-SIM. The GC-NICI-MS-
SIM detection limit for trans-anti-TME-BP-tetraol was
about 1 fmol per injection, and intensities of selected ions
(m/ z 376, 282, 252) increased linearly at concentrations
between 1 fmol and 1 pmol. The detection limit of BP-
tetraols by [3H]CH3I postlabeling was approximately 1
fmol (500 dpm). The GC-NICI-MS-SIM detection limits
for TME-chrysene-tetraol and TME-B[a]A-tetraol were
approximately 50 and 1 fmol per injection, respectively.
Ap p lica tion of Meth od ology to Mice Tr ea ted w ith
BP . As summarized in Scheme 1, two experiments were
carried out to test the proposed methodology for DNA
and globin analysis. In experiment 1, mice were treated
F igu r e 4. HPLC radiochromatogram of EtOAc-extractable
products released upon acid hydrolysis of DNA isolated from
with [3H]BP and the released [3H]BP-tetraols were either
analyzed by HPLC or derivatized and analyzed by GC-
NICI-MS-SIM. In experiment 2, mice were treated with
unlabeled BP and the unlabeled tetraols were derivatized
with [3H]CH3I and analyzed by HPLC.
lungs of mice treated with [3H]BP (experiment 1, Scheme 1).
a . Detection of BP -Tetr a ols by Ra d ioflow HP LC
or GC-MS. The total binding of [3H]BP metabolites to
DNA and globin (experiment 1, Scheme 1) and the levels
of released EtOAc-extractable metabolites as a function
of hydrolysis time are summarized in Table 4. Maximal
release of EtOAc-extractable radioactivity was achieved
after 2 h. Total binding of [3H]BP metabolites to liver
DNA exceeded that to lung DNA. However, only about
10% of the bound material was released as EtOAc-
extractable from liver DNA or globin, while hydrolysis
of lung DNA released about 60% of the total radioactivity.
These observations are in agreement with previous
studies which indicate that about 90% of the radioactivity
associated with liver DNA is from unknown products (41,
42).
F igu r e 5. GC-NICI-MS-SIM of tetramethyl ethers of (A) trans-
anti-BP-tetraol standards; (B) [3H]BP-tetraol released from the
lung DNA of mice treated with [3H]BP (experiment 1, Scheme
1); and (C) [3H]BP-tetraol released from mouse globin treated
with [3H]BP (experiment 1, Scheme 1).
The HPLC profile of EtOAc-extractable materials from
lung DNA is illustrated in Figure 4. [3H]BP-tetraols
contributed about half of the products in the extract and
the remaining were unknowns (peak X of Figure 4.).
These unidentified products were also present in liver
DNA and globin but to different extents (data not shown).
Recovery of [3H]BP-tetraols was enhanced by about 25%
when hydrolysis was performed under N2 or vacuum.
Lowering the hydrolysis temperature also improved the
yield of tetraols. For example, EtOAc-extractable ma-
terials released from globin incubated under vacuum at
80 or 90 °C were about the same, but the recovery of
tetraols at 80 °C was about 10% greater than at 90 °C.
The [3H]BP-tetraols released from globin and DNA
were derivatized to their corresponding tetramethyl
ethers and also were analyzed by GC-NICI-MS-SIM
(Figure 5B,C). The selected ions monitored were m/ z
376, 282, and 252. The results shown in Figure 5 confirm
the identity of the [3H]BP-tetraols released from mouse
DNA and globin. The concentrations of these analytes
were estimated from the intensities of total selected ions
and were comparable with levels determined by the
conventional radioactive tracer method (Table 5).
b. Detection of BP -Tetr a ols by [3H]CH3I P ostla -
b elin g. The HPLC radiogram of the [3H]tetramethyl
+
+

514 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Melikian et al.
Ta ble 5. Levels of BP -Tetr a ols Relea sed fr om Mou se
Globin a n d Lu n g a n d Liver DNA
pmol BP-tetraols/mg DNA or globin
by radioflow
HPLC of
by radioflow
HPLC of
by GC-NICI-MS-SIM
[3H]BP-tetraolsa of TME-BP-tetraolsa [3H]TME-BP-tetraolsb
lung
liver
globin
3.4 ( 0.1c
2.9d
NAf
0.3g
2.9e
4.1e
3.7e
0.2
a
b
Experiment 1, Scheme 1. Experiment 2, Scheme 1. c Mean
( SE, n ) 3. Estimated amount. e Mean of two analyses. f Not
analyzed. Using d12-BP-tetraol as internal standard.
d
g
F igu r e 7. GC-NICI-MS-SIM (selected ions for TME-BP-tetraol)
of HPLC-purified permethylated derivatives of globin hydroly-
sates from a nonsmoker. The inset shows the chromatogram of
the same sample for the major selected ion before HPLC
purification.
F igu r e 6. HPLC radiochromatograms of BP-tetraols after
derivatization with [3H]CH3I: (A) [3H]trans-anti-TME-BP-tet-
raol standard; (B) [3H]TME-BP-tetraols obtained upon hydroly-
sis of calf thymus DNA modified with (()-anti-BPDE; and (C)
[3H]TME-BP-tetraols obtained upon hydrolysis of lung DNA
after treatment of mice with BP (experiment 2, Scheme 1).
F igu r e 8. GC-NICI-MS-SIM (selected ions for TME-chrysene-
tetraol) of HPLC-purified permethylated derivatives of globin
hydrolysates from a nonsmoker. The inset shows the chromato-
gram of the same sample for the major selected ion before HPLC
purification.
ethers of BP-tetraols released from mouse lung DNA
(experiment 2, Scheme 1) is shown in Figure 6C. This
profile had the identical peaks 1 and 2 that were observed
upon HPLC analysis of the standard (Figure 6A) or
analysis of [3H]TME-tetraols released from calf thymus
DNA modified with anti-BPDE (Figure 6B). Peak 1 of
Figure 6C coeluted with trans-anti-TME-BP-tetraol stan-
dard (Figure 2A). This indicates that the major BP-
tetraol released from mouse lung DNA is derived from
anti-BPDE. Quantitative results, which are summarized
in Table 5, show good agreement between the conven-
tional radioactive tracer method (experiment 1), GC-
NICI-MS-SIM, and postlabeling via [3H]CH3I (experi-
ment 2). This demonstrates the feasibility of postlabeling
via [3H]CH3I for animal model studies. Levels of BP-
tetraols quantified from experiment 1 or 2 were similar
to the concentration of [3H]BPDE-DNA adducts reported
previously in a similar bioassay (41, 42).
which demonstrate the presence of TME-BP-tetraol and
TME-chrysene-tetraol in the hydrolysates. These ana-
lytes cochromatographed with standard samples (data
not shown). TME-BP-tetraol could be either trans-anti-
or cis-syn. The insets in Figures 7 and 8 show the results
of the GC-NICI-MS-SIM analysis without prepurification
by HPLC. BP-tetraol was detected in all 20 samples,
while chrysene-tetraol was detected in 7 of 10 smokers
and 2 of 5 nonsmokers. Control samples had no detect-
able amounts of BP-tetraols or chrysene-tetraols. Fur-
ther studies are in progress on quantitation of these
adducts in human hemoglobin. Some samples showed
evidence for the presence of B[a]A-tetraol, but this
requires confirmation.
These results are consistent with previous analyses of
human hemoglobin by GC-NICI-MS-SIM of trimethylsi-
lylated tetraols, in which tetraols derived from BP and
chrysene have been detected (27, 43, 44). The tetra-
Ap p lica tion of GC-NICI-MS-SIM Meth od ology to
Hu m a n Hem oglobin . Results of analysis of hemoglobin
from a nonsmoker are illustrated in Figures 7 and 8,
+
+

Detection of PAH Adducts by GC-MS and [3H]CH3I
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 515
(13) Peltonen, K., Cheng, S. C., Hilton, B. D., Lee, H., Cortez, C.,
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methyl ether derivatives employed in our work may have
advantages over trimethylsilylated tetraols because of
their greater stability.
Con clu sion . We have defined the optimum conditions
for hydrolysis of BP DNA and globin adducts to the
corresponding tetraols and have established the stabili-
ties of these tetraols under the hydrolysis conditions. We
have found that derivatization of the tetraols to their
tetramethyl ethers is a superior method with many
advantages. These stable derivatives can be detected
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GC-NICI-MS-SIM. The method also appears to be ap-
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postlabeling and GC-NICI-MS-SIM methods by compar-
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determination of BP DNA and globin adducts in mice.
Moreover, the GC-NICI-MS-SIM method is applicable to
hydrolysates of human hemoglobin, in which we have
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will ultimately be applicable to quantitation of a broad
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Grants CA-29580 and CA-44377 from the U.S. National
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