Cytochrome P450-mediated metabolism and DNA binding of 2-Amino-1,7- dimethylimidazo[4,5-g]quinoxaline and its carcinogenic isomer 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in mice

Article abstract of DOI:10.1021/tx2004536

2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA) that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), a rodent carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its metabolism and carcinogenic potential is lacking. Metabolism studies on MeIgQx and MeIQx were conducted with human and mouse liver microsomes, and recombinant human P450s. DNA binding studies were also investigated in mice to ascertain the genotoxic potential of MeIgQx in comparison to MeIQx. Both HAAs underwent comparable rates of N-oxidation to form genotoxic N-hydroxylated metabolites with mouse liver microsomes (0.2-0.3 nmol/min/mg protein). The rate of N-oxidation of MeIQx was 4-fold greater than the rate of N-oxidation of MeIgQx with human liver microsomes (1.7 vs 0.4 nmol/min/mg protein). The rate of N-oxidation, by recombinant human P450 1A2, was comparable for both substrates (6 pmol/min/pmol P450 1A2). MeIgQx also underwent N-oxidation by human P450s 1A1 and 1B1 at appreciable rates, whereas MeIQx was poorly metabolized by these P450s. The potential of MeIgQx and MeIQx to form DNA adducts was assessed in female C57BL/6 mice given [¹⁴C]-MeIgQx (10 μCi, 9.68 mg/kg body wt) or [¹⁴C]-MeIQx (10 μCi, 2.13 mg/kg body wt). DNA adduct formation in the liver, pancreas, and colorectum was measured by accelerator mass spectrometry at 4, 24, or 48 h post-treatment. Variable levels of adducts were detected in all organs. The adduct levels were similar for both HAAs, when adjusted for dose, and ranged from 1 to 600 adducts per 10⁷ nucleotides per mg/kg dose. Thus, MeIgQx undergoes metabolic activation and binds to DNA at levels that are comparable to MeIQx. Given the high amounts of MeIgQx formed in cooked meats, further investigations are warranted to assess the carcinogenic potential of this HAA.

Full text of DOI:10.1021/tx2004536

Article
pubs.acs.org/crt
Cytochrome P450-Mediated Metabolism and DNA Binding of
2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline and Its Carcinogenic
Isomer 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline in Mice
Robert J. Turesky,*,† Erin E. Bessette,† Deborah Dunbar,† Rosa G. Liberman,‡ and Paul L. Skipper‡
†Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health, Albany, New York 12201,
United States
‡Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: 2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA)
that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f ]quinoxaline (MeIQx), a rodent
carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its
metabolism and carcinogenic potential is lacking. Metabolism studies on MeIgQx and MeIQx were conducted with human and mouse
liver microsomes, and recombinant human P450s. DNA binding studies were also investigated in mice to ascertain the genotoxic potential
of MeIgQx in comparison to MeIQx. Both HAAs underwent comparable rates of N-oxidation to form genotoxic N-hydroxylated
metabolites with mouse liver microsomes (0.2−0.3 nmol/min/mg protein). The rate of N-oxidation of MeIQx was 4-fold greater than the
rate of N-oxidation of MeIgQx with human liver microsomes (1.7 vs 0.4 nmol/min/mg protein). The rate of N-oxidation, by recombinant
human P450 1A2, was comparable for both substrates (6 pmol/min/pmol P450 1A2). MeIgQx also underwent N-oxidation by human
P450s 1A1 and 1B1 at appreciable rates, whereas MeIQx was poorly metabolized by these P450s. The potential of MeIgQx and MeIQx to
form DNA adducts was assessed in female C57BL/6 mice given [14C]-MeIgQx (10 μCi, 9.68 mg/kg body wt) or [14C]-MeIQx (10 μCi,
2.13 mg/kg body wt). DNA adduct formation in the liver, pancreas, and colorectum was measured by accelerator mass spectrometry at 4,
24, or 48 h post-treatment. Variable levels of adducts were detected in all organs. The adduct levels were similar for both HAAs, when
adjusted for dose, and ranged from 1 to 600 adducts per 107 nucleotides per mg/kg dose. Thus, MeIgQx undergoes metabolic activation
and binds to DNA at levels that are comparable to MeIQx. Given the high amounts of MeIgQx formed in cooked meats, further
investigations are warranted to assess the carcinogenic potential of this HAA.
to be human carcinogens.7 Thus, there is much concern about the
health risk associated with the exposure to these chemicals.
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is
INTRODUCTION
■
More than 20 heterocyclic aromatic amines (HAAs) have been
identified in cooked beef, poultry, and fish.1,2 The formation of
one of the most abundant HAAs formed in cooked meats.
HAAs is dependent upon the type of meat and method of
MeIQx is a powerful bacterial mutagen and induces tumors
cooking: the concentrations of HAAs can range from less than 1
at multiple sites in rodents during long-term feeding studies.1 It
part-per-billion (ppb), up to 500 ppb in meat or poultry that is
cooked well done.2−4 The HAAs thus far assayed have been
is noteworthy that MeIQx and other known HAAs account for
shown to induce tumors at multiple sites in rodents.1 Several
less than 30% of the mutagenicity attributed to this class of
genotoxicants in well-done grilled meats and other uncharac-
prevalent HAAs are classified as probable or possible human
carcinogens (Group 2A and 2B), based on toxicity data reviewed
by the International Agency for Research on Cancer.5,6 The Report
on Carcinogens, 11th edition, of the National Toxicology Program,
also concluded that prevalent HAAs are “reasonably anticipated”
terized HAAs are undoubtedly present.8 We recently discovered
Received: October 18, 2011
Published: November 25, 2011
© 2011 American Chemical Society
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2-Hydroxyamino-3,8-dimethylimidazo[4,5-f ]quinoxaline (HONH-
MeIQx) was prepared by the reduction of 2-nitro-3,8-dimethylimi-
dazo[4,5-f ]quinoxaline with hydrazine and Pd/C serving as a
catalyst.12 2-Amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f ]-
quinoxaline (8-CH2OH-IQx) was prepared by the oxidation of MeIQx
with SeO2 as previously described.13 N-(Deoxyguanosin-8-yl)-MeIQx
(dG-C8-MeIQx), [13C10]-dG-C8-MeIQx, and 5-(deoxyguanosin-N2-yl)-
MeIQx (dG-N2-MeIQx) were synthesized by reaction of the N-acetoxy
derivative of MeIQx with either dG or [13C10]-dG as previously reported.14
Microsomal Metabolism Studies. The metabolism studies were
conducted with liver microsomes prepared from female C57BL/6J
mice,15 the mouse strain used for in vivo DNA binding studies, or with
human liver microsomes, or recombinant human P450s 1A1, 1A2,
and 1B1. Liver microsomal protein (1 mg/mL) or recombinant P450
(100 pmol/mL), in 100 mM potassium phosphate (pH 7.6)
containing 0.5 mM EDTA, 5 mM glucose 6-phosphate, glucose
6-phosphate dehydrogenase (1 unit/mL), 1 mM NADPH, and 1 mM
NAD+ were incubated with MeIgQx or MeIQx (200 μM); the
N-oxidation rates are at Vmax for MeIQx at this substrate concentra-
tion.15 The enzymes plus cofactors were incubated for 3 min prior to the
addition of HAA substrates. The metabolism was done for 5 or 10 min, and
metabolite formation was a linear function with time. Some metabolism
studies with human liver microsomes were conducted for time periods up
to 30 min, to produce large amounts of metabolites for characterization by
mass spectrometry. The enzyme reactions were terminated by the addition
of 2 vol CH3OH. The precipitated proteins were removed by centrifuga-
tion, and the supernatants were assayed by HPLC with an Agilent 1100
Chemstation equipped with an UV photodiode array detector. The
metabolites were resolved with an Aquasil C18 column (4.6 × 250 mm,
5 μm particle size, Thermo Scientific), employing a linear gradient
starting from 10% CH3CN in 50 mM NH4CH3CO2 (pH 6.8) and
reaching 100% CH3CN at 20 min at a flow rate of 1 mL/min.
The estimates of formation of 2-hydroxyamino-1,7-dimethyl-
imidazo[4,5-g]quinoxaline (HONH-MeIgQx) and 2-hydroxyamino-
3,8-dimethylimidazo[4,5-f ]quinoxaline (HONH-MeIQx), and other
oxidation products were determined by HPLC with UV detection. The
metabolites of MeIgQx were monitored at 370 or 390 nm, and
metabolites of MeIQx were monitored at 274 nm. We assumed that
the molar extinction coefficients of N-hydroxylated and ring-oxidized
metabolites were the same values as the molar extinction coefficients
of the parent compounds at their respective maximal absorbance
wavelengths. The UV responses at these wavelengths were used to
estimate rates of metabolite formation.
2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx), a
linear tricyclic ring isomer of MeIQx in the urine of meat-
eaters,9 and subsequently identified MeIgQx in cooked beef.9−11
MeIgQx is the most mass-abundant HAA formed in cooked
ground beef, steaks, and gravies;4 it occurs at 5-fold or greater
levels than the amounts of MeIQx or 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP). Both MeIQx and PhIP
are believed to contribute to diet related cancers.1 MeIgQx is a
less potent bacterial mutagen than MeIQx and PhIP; however,
studies have not been conducted on the metabolism of MeIgQx,
and the carcinogenic potential of MeIgQx is unknown.11
In this investigation, we have examined the capacities of
human and mouse liver microsomes, and recombinant human
cytochrome P450s to metabolize MeIgQx and MeIQx to
electrophilic metabolites that are capable of binding to DNA.
We also compared the potential of [14C]-MeIgQx and [14C]-
MeIQx to bind to DNA in the liver, pancreas and colorectum,
potential target organs of HAA carcinogenicity, in C57BL/6
mice. These data provide a preliminary estimate of the genotoxic
potential of MeIgQx in vivo.
MATERIALS AND METHODS
■
Chemicals. MeIQx and [2-14C]-MeIQx (50 mCi/mmol, >98%
radiochemical purity) were purchased from Toronto Research
Chemicals (Ontario, Canada). KCN and Br2 were was purchased
from Sigma (St. Louis, MO). K[14C]N (54 mCi/mmol) was
purchased from Moravek Chemicals (Brea, CA). Human liver samples
were from Tennessee Donor Services, Nashville, TN, and kindly
provided by Dr. F. P. Guengerich, Vanderbilt University. Recombinant
human P450s 1A1, 1A2, and 1B1 contained in supersomes from
baculovirus-infected insect cells and furafylline were purchased from Becton
Dickinson (San Jose, CA).
General Methods. Mass spectra of synthetic derivatives and
metabolites were acquired on a Finnigan TSQ Quantum Ultra triple
stage quadrupole mass spectrometer (Thermo Fisher, San Jose, CA).
The MS analyses were conducted in the positive ionization mode
and employed an Advance CaptiveSpray source from Michrom
Bioresource Inc. (Auburn, CA). Typical instrument tune parameters
used were as follows: capillary temperature, 200 °C; source spray
voltage, 1.4 kV; tube lens offset, 95 V; capillary offset, 35 V; and source
fragmentation, 5 V. Argon, set at 1.5 mTorr, was used as the collision
gas, and the collision energy was set between 15 to 30 eV; no sheath
gas, sweep gas, or auxiliary gas was employed.
Liquid Chromatography−Electrospray Ionization/Mass Spec-
trometry (LC-ESI/MS) Characterization of Metabolites. The
N-oxidized and ring-oxidized metabolites were collected from the HPLC
effluent and directly characterized by electrospray ionization tandem
mass spectrometry (ESI/MS2) with the triple stage quadrupole mass
spectrometer. The metabolites were also characterized by ESI/MS2
following the reaction with nitrosobenzene (10 μg, in 0.1% HCO2H in
CH3OH) at 50 °C for 1 h.
In Vivo DNA Binding Studies. DNA binding studies were con-
ducted with female C57BL/6Ntac mice (Taconic Farm, Albany, NY).
Animals were housed in polycarbonate cages and allowed to acclimate
for 1 week prior to the commencement of the study.
Synthesis. The synthesis of [2-14C]-MeIgQx was done as
previously described for the synthesis of the unlabeled compound
with minor modifications.11 K[14C]N (0.093 mmol, 54 mCi/mmol)
was diluted with KCN (0.4 mmol) in CH3OH (0.6 mL), sonicated
for 10 min, and then slowly added to Br2 (0.50 mmol) in CH3OH
(0.5 mL) on ice. The reaction was allowed to proceed for 20 min.
Thereafter, 4-fluoro-5-nitrobenzene-1,2-diamine (41 mg, 0.24 mmol)
in C2H5OH (0.5 mL) was added to the newly formed [14C]NBr, and
the reagents were mixed overnight at room temperature. The 2-[14C]-
5-fluoro-6-nitro-1H-benzimidazole-2-amine intermediate was reacted
with NH4OH to displace the fluorine moiety, followed by reduction
with NaBH4, ring-closure with methylglyoxal, and last by methyla-
tion with CH3I, as previously described.11 [2-14C]-MeIgQx and its
6-methylated homologue were resolved by semipreparative HPLC
employing a Waters XBridge phenyl column (10 × 250 mm, 5 μm
particle size). An isocratic solvent composed of 23% CH3OH in 5 mM
NH4CH3CO2 (pH 8.7) was used for chromatography. The flow rate
was 3 mL/min, and the run time was 70 min. The chemical and
radiochemical purities of MeIgQx exceeded 98.5% as judged by HPLC
with UV diode array detection and liquid scintillation counting. The
specific activity of MeIgQx was determined by ESI-MS, by measuring
the ratio of the 14C/12C isotopes of the protonated molecules [M+H]+ at
m/z 216.1 and 214.1: the specific activity was estimated at 11 mCi/mmol.
Animal Dosing. Mice (20 g, N = 5 animals per time point) were
dosed by gavage with 14C-MeIgQx (10 μCi, 9.68 mg/kg body wt) or
14C-MeIQx (10 μCi, 2.13 mg/kg body wt) in a volume of 0.25 mL
water. At 4, 24, or 48 h, animals were sacrificed by asphyxiation with
CO2 gas, followed by cervical dislocation; blood was retrieved via the
inferior vena cava. The liver and pancreas were harvested, rinsed with
chilled PBS, and snap-frozen in liquid nitrogen. The colorectal tissue
(ileo-cecal junction to the rectum) was retrieved, the fecal contents
were removed, and the tissue segment was incubated with cold,
freshly prepared EDTA buffer (phosphate-buffered saline containing
1.5 mM Na2-EDTA, 6000 U sodium heparin, DTT (8 mg), and
phenylmethanesufonyl fluoride (4 mg, 100 μL of 40 mg/mL DMSO,
per 100 mL of buffer). After 20 min, the mucosal layer containing the
colonocytes was retrieved by placing the intestine segment in a Petri
dish, and the loosened mucosal layer was displaced by gently scraping
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+
the outer intestinal segment with the edge of a glass slide to eject the
colonocytes. The colonocytes were centrifuged at 3,000g at 4 °C.
Pelleted colonocytes were resuspended in EDTA buffer (2 mL) and
centrifuged again. This washing procedure was repeated a third time,
and the colonocytes were quick-frozen in liquid nitrogen.
Radioactivity in Plasma. Radioactivity was measured with a
Beckman Coulter liquid scintillation counter (Fullerton, CA) with
external calibration.
The MS/MS scan mode produced the aglycone ion BH2 adducts
[M+H-116]+ or [M+H-121]+ from the protonated dG-C8-MeIQx
and dG-C8-[14C]-MeIQx adducts or [13C10]dG-C8-MeIQx adduct, respec-
tively. The ions monitored were as follows: dG-C8-MeIQx (m/z 479.1 >
363.1 >); dG-C8-[14C]-MeIQx (m/z 481.1 > 365.1 >); and [13C10]-dG-
C8-C8-MeIQx (m/z 489.1 > 368.1 >). The product ion spectra of the
aglycone adducts at the MS3 scan stage were acquired from m/z 125 to
400. The normalized collision energies were set at 24 and 34 eV, and the
isolation widths were set at an m/z = 4.0 and 1.0, respectively, for the MS2
and MS3 scan modes. The activation Q was set at 0.35, and the activation
time was 10 ms for both transitions. Helium was used as the collision
damping gas in the ion trap and was set at a pressure of 1 mTorr.
One μscan was used for data acquisition. The same mass spectral param-
eters were employed to screen for putative dG-C8 adducts of MeIgQx.
Isolation of DNA. The entire liver, pancreas, or colonocytes were
homogenized in 2 mL of TE buffer (50 mM Tris-HCl and 10 mM
EDTA, pH 8.0) and centrifuged for 10 min at 3,000g. DNA was
isolated from the nucelar pellets of the liver and kidney (equivalent of
200 mg of tissue) and from the entire nuclear pellet of the colorectum,
by solvent extraction as described by Gupta.16 The purified DNA under-
went a second solvent extraction procedure and ethanol precipitation
step, to further remove noncovalently bound radioactivity.
The DNA was dissolved in distilled−deionized H2O, and the con-
centration was determined by UV spectroscopy, assuming a con-
centration of DNA (50 μg/mL) is equal to the 1.0 absorbance unit at
260 nm. The A260:280 ratios were on average greater than 1.75.
Accelerator Mass Spectrometry. AMS analyses were conducted
at the MIT BEAMS Lab using procedures described elsewhere in
detail.17 Sample aliquots (1.50 μL) were applied directly to a CuO
matrix and introduced into a laser-induced combustion interface
for subsequent AMS analysis. Each run contained two quantitation
standards ([14C-methyl] bovine serum albumin, 0.003 dpm/μL),
followed by two blanks (1 μg/μL human serum albumin), 10 samples,
and another two standards. Quantitation was performed by integrating
peaks produced in the continuous trace of 14C detector count rate
versus time generated during operation of the combustion interface,
which produces and delivers the CO2 of combustion to the AMS ion
source, and taking the product of the sample/standard peak area ratio
multiplied by the standard concentration as the concentration of the
sample. Blank values, if measurable, were subtracted from the sample
peak areas prior to calculating sample concentration.
RESULTS
■
Metabolism of MeIQx and MeIgQx with Human and
Mouse Liver Microsomes and Recombinant Human
P450s. HAAs undergo bioactivation by cytochrome P450-
mediated N-oxidation of the exocyclic amine group, to form
electrophilic N-hydroxy-HAA metabolites that covalently bind
to DNA.20−22 The liver is the most active organ in the meta-
bolism of HAAs,20−22 and P450 1A2 is the major hepatic
P450 involved in the N-oxidation of most HAAs.15,23,24
We previously characterized the principal oxidation products
of MeIQx formed by rat and human liver microsomes, rat P450
1A2, and recombinant human P450 1A2.15 In this study, we
have examined the pathways of P450-mediated metabolism and
compared the rates of N-oxidation of MeIQx and MeIgQx,
by employing microsomes prepared from liver of female
C57BL/6 mice, the species used for in vivo DNA adduct
binding studies. The capacities of human liver microsomes,
recombinant human P450 1A2, P450 1A1, and P450 1B1 to
bioactivate both compounds were also investigated. Both P450
1A1 and P450 1B1 are prominently expressed in extrahepatic
tissues25,26 and catalyze the N-oxidation of several HAAs.27,28
The chemical structures of MeIQx and MeIgQx and their
proposed pathways of metabolism by human P450 1A2 (vide
infra) are depicted in Figure 1.
The HPLC-UV profiles of the metabolites of MeIQx and
MeIgQx formed by human liver microsomes are shown in
Figure 2, along with the UV spectra of the parent amines and
their principal N-oxidized metabolites. The major pathway of
metabolism of MeIQx occurs by N-oxidation of the exocyclic
amine group, to form HONH-MeIQx. The UV spectrum of
HONH-MeIQx is identical to the spectra of the metabolite and
synthetic derivative previously reported;29 the absorbance
maximum (272 nm) displays a slight hypsochromic shift relative
to the absorbance maximum of MeIQx (274 nm) (Figure 2).29
The dihydroxylated product, 2-(hydroxyamino)-8-hydroxymeth-
yl-3-methylimidazo[4,5-f ]quinoxaline (HONH-8-CH2OH-IQx)
is the second most abundant metabolite, followed by the
alcohol, 8-CH2OH-IQx, which is a minor metabolite.30 Human
liver microsomes also produced three oxidation products (M-1,
M-2, and M-3) of MeIgQx (Figure 2). Their UV and mass
spectral data are discussed below.
DNA Adduct Measurements by LC-ESI/MS3. DNA (100 μg) in
5 mM Bis-Tris-HCl buffer (pH 7.1, 50 μL) underwent enzymatic
digestion with DNase I for 1.5 h, followed by incubation with nuclease
P1 for 3 h, and then digested with alkaline phosphatase and phos-
phodiesterase for 18 h.18 These enzyme digestion conditions were
shown to be highly efficient in the recovery of the dG-C8-MeIQx from
calf thymus DNA modified with N-acetoxy-MeIQx.14,19 The digest was
added to 2 vol of chilled C2H5OH and placed on ice for 1 h, followed
by centrifugation at 15,000g to pellet salt and protein. The supernatants
were transferred to silylated capillary LC vials and concentrated to
dryness by vacuum centrifugation. The extracts were dissolved in 1:1
H2O/DMSO (20 μL).
The adducts were separated from nonmodified nucleosides with a
NanoAcquity UPLC system (Waters Corporation, Milford, MA)
interfaced with a linear quadrupole ion trap mass spectrometer (LTQ
MS, Thermo Fisher, San Jose, CA). A Waters Symmetry trap column
(180 μm × 20 mm, 5 μm particle size) was employed for online solid
phase enrichment of the DNA adducts. The analytical column was
a Michrom C18 AQ (0.3 × 150 mm, 3 μm particle size, Michrom
Bioresources Inc., Auburn, CA). The DNA digests were injected onto
the trap column and washed with 0.2% HCO2H in 5% CH3CN at a
flow rate of 12 μL/min for 5 min. Thereafter, the DNA adducts were
back-flushed onto the C18 AQ Michrom AQ column. A linear gradient
was employed to resolve the DNA adducts, starting at 0.01% HCO2H
containing 5% CH3CN and arriving at 0.01% HCO2H in 95% CH3CN
at 20 min. The flow rate was set at 5 μL/min.
Mass spectrometric measurements were performed at the MS3 scan
stage in the positive ionization mode. The ion source was an Advance
CaptiveSpray source from Michrom Bioresource Inc. (Auburn, CA).
Representative optimized instrument tuning parameters were as
follows: capillary temperature, 200 °C; source spray voltage, 2.0 kV;
no sheath gas, sweep gas or auxiliary gas was employed; capillary
voltage, 40 V; tube lens voltage, 110 V; and in-source fragmentation,
10 V. The automatic gain control settings were full MS target 30000
and MSn target 10000, and the maximum injection time was 10 ms.
The metabolites of MeIQx and MeIgQx were collected from
the HPLC effluent and directly infused into the triple stage
quadrupole mass spectrometer. The product ion spectra of
HONH-MeIQx and its azoxy derivative, obtained by reaction of
HONH-MeIQx with nitrosobenzene, are shown in Figure 3.
The product ion spectrum of HONH-MeIQx ([M+H]+ at m/z
230.1) showed prominent fragment ions at m/z 213.1 and
212.1, indicative of the loss of 17 and 18 Da, which are
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Figure 1. Chemical structures of MeIQx and MeIgQx and proposed pathways of P450-mediated metabolism. The asterisk depicts the site of
incorporation of the 14C isotope.
attributed to the loss of HO• and H2O. These fragment ions are
typically observed in product ion spectra of HONH-HAA
metabolites, but the product ion spectra of ring-oxidized HAA
metabolites often display a neutral loss of 18 Da (H2O) or
28 Da (CO).30,31 The product ion spectrum of the azoxy
derivative confirms the structure as HONH-MeIQx.29,32 The
product ion spectra of HONH-8-CH2OH-IQx and 8-CH2OH-
IQx (Figure 4) are also in excellent agreement to the spectra of
the reference compounds previously reported.30
A large bathochromic shift was observed in the UV spectrum
of HONH-MeIgQx compared to that in the spectrum of
MeIgQx. The maximum of absorbance of M-3 was at 385 nm,
whereas the maximum absorbance of MeIgQx occurred at 365 nm
(Figure 2). This large bathochromic shift is not observed in the
UV spectrum of HONH-MeIQx, nor is this bathochromic shift
observed in the UV spectra of the structurally related N-hydroxy-
HAA derivatives of 2-amino-3-methylimidazo[4,5-f ]quinoline or
PhIP. However, the UV spectrum of the proposed N3-glucuronide
conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]-
pyridine (HONH-PhIP), which exists as an oxime, also displays a
bathochromic shift of ∼20 nm in its maximum absorbance in
comparison to HOHN-PhIP.31,33 We surmise that HONH-
MeIgQx exists preferentially as the oxime and not as the
N-hydroxylamine tautomer. The carcinogen 4-hydroxyamino
quinoline 1-oxide was also reported to preferentially exist in its
4-hydroxyimino tautomeric form.34
The mass spectrum of the principal metabolite of MeIgQx
(M-3) displayed a protonated ion [M+H]+ at m/z 230.1, a
mass which is 16 Da greater than MeIgQx. The ESI product ion
spectra of M-3 and its adduction product with nitrosobenzene
were very similar to the product ion spectra of HONH-MeIQx
and its azoxy adduct (Figure 3). M-3 underwent oxidation
with time, as did HONH-MeIQx, to form the presumed nitroso
derivative: a prominent ion [M+H]+ was observed at m/z
228.1. The product ion spectra of these intermediates displayed
prominent fragment ions at m/z 213.1 and 198.1, which are
The second most abundant metabolite of MeIgQx (M-2)
displayed a UV spectrum similar to that of MeIgQx. The mass
spectrum of M-2 displayed a protonated ion at [M+H]+ at m/z
230.0, indicative of another mono-oxidation metabolite.
Prominent ions were observed in the product ion spectrum
at m/z 201.0, 200.0, and 186.1 (Figure 4). These fragment ions
•
ascribed, respectively, to the loss of CH3 and NO moieties
(data not shown). Thus, the structure of M-3 is assigned as
2-(hydroxyamino)-1,7-dimethylimidazo[4,5-f ]quinoxaline
(HONH-MeIgQx).
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Figure 2. HPLC UV profiles of MeIQx and MeIgQx metabolites formed with human liver microsomes, following a 30 min incubation. The UV
spectra of the parent compounds and major N-oxidized metabolites of each HAA are presented.
Figure 3. Product ion spectra of HONH-MeIQx and HONH-MeIgQx (M-3) (right panel) and product ion spectra of the azoxy conjugates formed
with nitrosobenzene (left panel).
are proposed to arise through losses of HCO•, H2CO, and
protonated ion [M+H]+ at m/z 246.1, indicating that the
metabolite is a dihydroxylated product of MeIgQx (Figure 4).
Prominent ions are observed in the product ion spectrum at
m/z 228.1 and 229.1, indicative of the loss of 17 and 18 Da and
suggestive of the presence of the N-hydroxylamine moiety
(Figure 4). The base peak, observed at m/z 200.1, is ascribed to
the loss H2O, followed by the loss of CO. Thus, the product
ion spectrum is consistent with the structure of a dihydroxy-
lated metabolite of MeIgQx that contains the N-hydroxylamine
moiety with the second site of hydroxylation possibly occurring
at the C-4 or C-9 position of the quinoxaline ring.
•
HCO• + CH3 . The product ion spectrum of M-2 is consistent
with a ring-hydroxylated metabolite of MeIgQx: the site of oxida-
tion may have occurred at the C-4 or C-9 position of the quinoxa-
line skeleton (Figure 1). If oxidation had occurred at the 7-CH3-
group, a prominent fragment ion would have been expected at
m/z 212.1 [M+H-18]+, due to the loss of H2O, as is observed
in the product ion spectrum of 8-CH2OH-IQx (Figure 4).
The third metabolite of MeIgQx (M-1) displayed a UV
spectrum that was very similar to the spectrum of HONH-
MeIgQx (M-3), with a maximum absorbance situated at
385 nm. The full scan mass spectrum of M-1 displayed a
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Figure 4. ESI/MS/MS product ion spectra of 8-CH2OH-IQx, 8-CH2OH-HONH-IQx, and the monohydroxylated (M-2) and dihydroxylated (M-1)
metabolites of MeIgQx.
Table 1. Estimated N-Oxidation and C-Oxidation Rates of MeIgQx and MeIQx with Liver Microsomes or Recombinant
a
Human P450s
nmol/min/mg microsomal protein or 100 pmol P450
enzyme
HONH-MeIgQx
HO-MeIgQx
HONH-(HO)-MeIgQx
HONH-MeIQx
HONH-8-CH2OH-IQx
8-CH2OH-IQx
mouse liver microsomes
0.3 0.1
0.4 0.02
14.1 4.9
5.4 2.1
3.9 0.6
0.5 0.2
0.1 0.01
6.9 2.4
ND
ND
0.2 0.01
1.7 0.2
0.3 0.1
6.1 0.9
ND
ND
ND
ND
ND
ND
ND
b
human liver microsomes
ND
0.09 0.01
ND
recombinant human P4501A1
recombinant human P4501A2
recombinant human P4501B1
ND
ND
ND
1.7 0.5
ND
2.5 0.3
a
[Substrate] = 200 μM; mean SD, N = 3 or 4 independent measurements. ND = not detected (<0.05 nmol/min/mg protein or <0.05 nmol/min/
b
100 pmol P450), only observed following 30 min incubation.
8-CH2OH-HONH-IQx and the dihydroxylated metabolite of
MeIgQx (M-1) were reacted with nitrosobenzene to form azoxy
adducts. Protonated molecules [M+H]+ were observed at m/z
335.2, a mass that is consistent with the molecular weight of the
proposed azoxy conjugates (Figure S-1, Supporting Informa-
tion). The product ion spectrum of the azoxy derivative of
8-CH2OH-HONH-IQx displays prominent ions at m/z 317.1
and 299.3, which are ascribed to the loss of one and two
molecules of H2O, whereas the product ion spectrum of the
azoxy derivative of M-1 displays prominent fragment ions at
m/z 317.2 and 289.2, which are consistent with the loss of H2O
and H2O+CO. These mass spectral data provide additional
support for the secondary site of oxidation proposed to occur at
the quinoxaline ring and not the 7-methyl group of MeIgQx.
Estimated Rates of P450-Mediated Oxidation of MeIQx
and MeIgQx. The estimated rates of N- and C-oxidation of
MeIQx and MeIgQx, by mouse and human liver microsomes
and recombinant human P450s, are summarized in Table 1.
The N-oxidation rates of MeIgQx and MeIQx with mouse liver
microsomes were comparable, whereas the rate of N-oxidation
of MeIQx was about 4-fold higher than that for MeIgQx with
human liver microsomes. MeIgQx underwent N-oxidation by
recombinant human P450 1A2 but also by extrahepatic P450s
1A1 and 1B1. Moreover, the rate of MeIgQx N-oxidation by
P450 1A1 was ∼3-fold greater than the rate of N-oxidation
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carried out by P450 1A2. Human P450 1A2 appears to be
involved only in N-oxidation (bioactivation), whereas P450
1A1 and P450 1B1 catalyze both N-oxidation and ring-
oxidation (detoxication) of MeIgQx. In contrast to MeIgQx, the
N-oxidation of MeIQx by P450s 1A1 and 1B1 was low to not
detectable.
In Vivo DNA Binding Studies in Mice. Plasma Clear-
ance. The study design was not intended to completely
describe the rate of absorption and clearance profile of these
chemicals. However, the data obtained from the 3 time points
show that the kinetics of absorption and elimination of both
14C-MeIgQx and 14C-MeIQx were rapid. The peak plasma levels
in radioactivity associated with both compounds occurred at 4 h,
and greater than 95% of the radioactivity was cleared from
plasma within 48 h (Figure 5).
Figure 5. Plasma clearance of [14C]-MeIQx and [14C]-MeIgQx. Data
represent the means and the standard deviations of 5 animals per time
point.
DNA Adducts. The apparent levels of [14C]-MeIgQx- and
[14C]-MeIQx-DNA adduct formation and removal in the liver,
pancreas, and colorectal DNA are presented in Figure 6. The
clearance of 14C-isotope associated with DNA was less rapid
than the clearance of 14C-radioactivity associated with either
carcinogen from plasma. The [14C]-MeIgQx isotope associated
with liver DNA at 24 and 48 h was 33% and 27% of the 14C-
isotope detected at 4 h, whereas the isotope level associated
with pancreas DNA was 44% and 17% of the level of 14C-isotope
bound to DNA at 4 h. The [14C]-MeIQx isotope associated with
liver and pancreas DNA also appeared to persist: up to 74% and
25% of the 14C-isotope observed in DNA of these organs at 4 h
remained, respectively, at 48 h. The [14C]-MeIgQx isotope in
colorectal DNA reached its maximum at 24 h, while the highest
level of the [14C]-MeIQx isotope in colorectal DNA was
observed at 4 h. The disposition and elimination of MeIgQx in
the mouse is not known. The fecal content may contain high
levels of 14C isotope, resulting in high levels of exposure to this
carcinogen, perhaps at greater levels than most other tissues.
LC-ESI/MS3 of DNA Adducts. The formation of DNA
adducts of [14C]-MeIQx and [14C]-MeIgQx was examined by
LC-ESI/MS3 with a linear quadrupole ion trap mass spectro-
meter. The ion trap MS permits multistage tandem scanning
for structural characterization and identification of the aglycone
adducts [BH2]+.18,35 Two isomeric dG adducts of MeIQx, dG-
C8-MeIQx, and dG-N2-MeIQx are formed in vivo in the liver of
rats19 and in human hepatocytes.36 In addition, a third adduct is
formed with dA; the structure has been tentatively assigned as
5-(deoxyadenosin-N6-yl)-MeIQx (dA-N6-MeIQx) (Figure 7).37
The stable isotope dilution method was used to measure the
levels of dG-C8-MeIQx formed in mouse liver at the 24 h time
Figure 6. Level of apparent [14C]-MeIQx and [14C]-MeIgQx DNA
adduct formation in the liver, pancreas, and colon as a function of
time. Data represent the means and the standard error of the means of
5 animals per time point. The values of adduct formation were
normalized to adducts per nucleotide/mg HAA/kg body weight .
point. Reconstructed ion chromatograms for dG-C8-MeIQx,
dG-C8-[14C]-MeIQx, [13C10]-dG-C8-MeIQx, and a putative
isomeric dG-C8-MeIgQx at the MS3 scan stage are presented in
Figure 8. Both the dG-C8-MeIQx and dG-C8-[14C]-MeIQx
adducts are observed. The product ion spectra of the unlabeled
and labeled adducts, at the MS3 scan stage, confirmed the
structure of the adducts.19,35
The estimate of total [14C]-MeIQx-DNA adduct formation
in the liver 24 h post-treatment, based upon AMS measure-
ments, was 11.4
7.6 adducts per 108 nucleotides versus
3.8 0.6 dG-C8-MeIQx adducts per 108 nucleotides, based on
quantitative LC-ESI/MS3 measurements. The contribution of
dG-C8-MeIQx to total 14C bound to DNA ranged from 13−
72% of the radioactivity. The relative response of the signals for
dG-N2-MeIQx and dA-N6-MeIQx adducts, by LC-ESI/MS3,
are ∼5% of the response of the signal observed for dG-C8-
MeIQx.37 The dG-N2-MeIQx and dA-N6-MeIQx adducts are
formed in rat liver at levels that are respectively ∼30−50% and
∼5% of the level of dG-C8-MeIQx, 24 h following oral dosing
with MeIQx (10 mg/kg).19,37 If the relative level of formation
of isomeric dG-MeIQx and dA-MeIQx adducts are similar in
rat and mouse liver, the weak response in signals of dG-N2-
MeIQx and dA-N6-MeIQx would preclude their detection, by
LC-ESI/MS3, in mouse liver DNA. Thus, the contribution of
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Figure 7. Chemical structures of dG-C8-MeIQx, dG-N2-MeIQx, and dA-N6-MeIQx.
Figure 8. Reconstructed ion chromatograms at the MS3 scan stage for (A) dG-C8-MeIQx, dG-C8-[14C]-MeIQx, and [13C10]-dG-C8-MeIQx, and
(B) putative dG-C8-MeIgQx and dG-C8-[14C]-MeIgQx with [13C10]-dG-C8-MeIQx employed as an internal standard. The product ion spectra at
the MS3 scan stage of dG-C8-MeIQx, dG-C8-[14C]-MeIQx, and [13C10]-dG-C8-MeIQx are depicted in panel C.
dG-N2-MeIQx and dA-N6-MeIQx adduct formation to the
amount of [14C]-MeIQx bound to DNA has not been taken
into account, and the 14C-isotope associated with the DNA as
covalently bound adducts likely exceeds the contribution made
by the dG-C8-MeIQx adduct. In a previous AMS study con-
ducted with male C57BL/6 mice treated with [14C]-MeIQx,
the formation of liver adducts was shown to be linearly depen-
dent on dose from an exposure of 5 mg down to 500 ng per kg
of body weight).38 The amount [14C]-MeIQx bound to liver
DNA was estimated at about 20 adducts per 108 nucleotides at
24 h post-treatment with [14C]-MeIQx (5 mg/kg). This estimate
in 14C-adduct formation is remarkably similar to the level of [14C]-
MeIQx bound to liver DNA, when adjusted per dose, in our study.
In contrast to MeIQx, a putative dG-C8-MeIgQx adduct was
not detected, when scanning at the MS2 or MS3 scan stages
(Figure 8B). The potential formation of a dA adduct of
MeIgQx ([M+H]+ at m/z 463.2) was also monitored, but no
peaks were detected above background level. The data-dependent
constant neutral loss-triple stage mass spectrometry scanning
mode (CNL/MS3) was employed to monitor for the loss of the
deoxyribose moiety, followed by MS3 of the putative aglycone
adducts [BH2]+.37 However, the CNL/MS3 mode also failed to
detect any adducts of MeIgQx.
Carcinogen Binding Index (CBI). The CBI is a measure
of the amount of adduct formed from a given dose; it is
calculated by dividing the amount of adduct per unit of DNA by
the dose per unit of body mass (μmol adduct bound per mole
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nucleotide)/(mmol chemical administered per kg animal).39
The CBI of MeIgQx and MeIQx in the liver, pancreas, and
colorectum are presented in Table 2. The CBI data are reported
value of MeIQx in this mouse strain is 24 mg/kg (http://
potency.berkeley.edu/chempages/MeIQx.html).
The CBI approach has been used in rodent models as a means
to assess the carcinogenic potential of genotoxic chemicals.43
A
a
CBI value of 1−10 is characteristic of weak carcinogens, while
moderate carcinogens display CBI values of about 100.39 The
CBI for both MeIQx and MeIgQx in the liver were comparable
at 4 and 24 h (CBI = 7−28). These findings suggest that
MeIgQx would be a weak to moderate liver carcinogen in this
animal model. The [14C]-MeIgQx isotope is also associated with
DNA in pancreas and colorectal tissue, suggesting that tumors
could form in these organs as well. However, the presence of
DNA adducts alone is insufficient to predict the development of
cancer as carcinogenesis involves numerous biological events.44
Moreover, dietary, environmental, and genetic factors can greatly
impact the biological potency of HAAs and other genotox-
icants.1,45,46
Table 2. Covalent Binding Index of MeIQx and MeIgQx
liver: (μmol/mol dNp)/(mmol carcinogen/kg bw)
time
MeIgQx
MeIQx
4
21.7 8.6
7.0 3.2
5.8 10.6
27.7 8.3
11.5 7.7
20.5 8.4
24
48
pancreas: (μmol/mol dNp)/(mmol carcinogen/kg bw)
time
MeIgQx
MeIQx
4
2.4 2.4
1.1 0.6
0.2 0.2
0.8 0.2
0.2 0.1
0.1 0.1
24
48
colon: (μmol/mol dNp)/(mmol carcinogen/kg bw)
Thus far, we have not successfully identified MeIgQx-DNA
adducts by LC-ESI/MSn. We surmise that the linear tricyclic
ring structure of MeIgQx inhibits the efficacy of enzymatic
digestion of the MeIgQx-adducted to DNA, and the MeIgQx
adducts may be present as incompletely digested oligomers,
which escaped detection by LC-ESI/MSn. Alternatively, the
instrument tuning parameters of the mass spectrometer may
not have been optimal for detection of MeIgQx-DNA adducts,
or the sensitivity in response of MeIgQx-DNA adducts is at least
10-fold weaker than the signal of response for dG-C8-MeIQx.
We have not been able to synthesize HONH-MeIgQx by
methods previously employed for the production of other
HONH-HAAs.12 Studies that examine the reactivity of synthetic
HONH-MeIgQx with deoxynucleosides and DNA are required
to identify and elucidate the structures of MeIgQx-DNA
adducts.
HAAs have been classified as probable human carcinogens by
IARC and by the National Toxicology Program, sponsored by
NIEHS.6,7 MeIgQx is the most mass abundant HAA formed in
cooked beef.4,11 MeIgQx is a bacterial mutagen; however, its
potency in the Ames reversion assay is about 4,000-fold lower
than the potency of MeIQx, in S. typhimurium tester strain TA98.
The genotoxic potency of MeIgQx is 16-fold greater in the
S. typhimurium strain YG1024 than in the S. typhimurium strain
TA98. The YG1024 strain is derived from strain TA98 and
contains high O-acetyltransferase activity that enhances the
mutagenicity of some HAAs, presumably through the formation
of reactive N-acetoxy intermediates, which covalently bind to
DNA.47 The higher potency of MeIgQx in the YG1024 bacterial
tester strain compared to TA98, indicates that the HONH-
MeIgQx undergoes O-acetylation to form the N-acetoxy
intermediate, as does MeIQx.11 MeIgQx displays about 10-fold
weaker mutagenic potency than the structurally related carcinogen
PhIP (3,015 rev/μg) in S. typhimurium strain YG1024. It is
noteworthy that the mutagenic potency of MeIgQx is
comparable to the potency of 4-aminobiphenyl, a known
human carcinogen, in TA98 and YG1024 (frameshift-specific)
and YG1029 (primarily point mutation-specific) tester
strains.48−50 The strong mutagenic potencies of MeIQx and
several other HAAs of angular tricyclic ring structure in the
S. typhimurium TA98 strain have been attributed to the ability
of these HAAs to frequently induce reversions about 9 base
pairs upstream of the original CG deletion in the hisD+ gene in
a run of GC repeats.51 This sequence context may not be a hot-
spot for MeIgQx and PhIP adduct formation, or the capacities
of MeIgQx and PhIP adducts to induce mutations are weaker
time
MeIgQx
MeIQx
4
12.0 16.5
138 114
28.7 18.5
113 119
10.4 11.9
2.1 0.9
24
48
a
The mean SD of 4 or 5 animals per time point.
at the 4, 24, and 48 h time points. The CBI values for liver DNA
adduct formation of MeIgQx and MeIQx were not significantly
different at 4 and 24 h (P > 0.4, Student’s unpaired t test);
however, the CBI values were significantly different at 48 h (P <
0.005). These data are suggestive of a faster rate of apparent
MeIgQx-DNA adduct removal than for MeIQx-DNA adducts
in the liver; additional time points are required to accurately
determine the rate of adduct elimination. For extrahepatic tissues,
there was a significant difference (P < 0.01) between MeIgQx
and MeIQx CBI values of the pancreas at 24 h and colorectal
tissue at 48 h.
DISCUSSION
■
This study was designed to examine the capacity of MeIgQx to
undergo metabolic activation to form DNA adducts in
comparison with its isomer, MeIQx, a multisite carcinogen in
rodents.1 The metabolism and DNA binding data provide a
preliminary assessment of the genotoxic potential of MeIgQx
relative to MeIQx, which also induces lacI mutations in the liver
and colon40 and aberrant crypt foci in the colon of C57BL/6N
mice,41 the species employed for our DNA binding studies. The
estimated rates of P450-mediated N-oxidation of MeIgQx with
mouse liver microsomes and the in vivo 14C-binding data of
MeIgQx, when adjusted for dose, are comparable to those data
obtained for MeIQx. We employed the highly sensitive AMS
method for adduct detection,17,38 which enabled us to estimate
DNA adduct formation based upon 14C isotope bound to
DNA, since the structures of the MeIgQx adducts are unknown.
MeIQx and other structurally related HAAs studied induce
tumors at multiple sites in rodents during long-term feeding
studies. The target organs include the oral cavity, liver, stomach,
colon, pancreas, and the prostate gland in males and the
mammary gland in females.1,42 The total dose required to
induce tumor formation (TD50) varies for each HAA and is host
species-dependent. The TD50 values of the individual HAAs
have been reported to range from 0.1 to 64.6 mg/kg/day in
rodents.1 MeIQx is a liver carcinogen in CDF1 mice:1 the TD50
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Chemical Research in Toxicology
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than the potencies of adducts formed by MeIQx and other
angular tricyclic HAAs. The mutagenicity of MeIgQx has not
been assessed in mammalian cell systems. Such studies would
provide additional data about the biological effects of MeIgQx
in comparison to carcinogenic HAAs, such as MeIQx and PhIP,
and aid in assessing the health risk of this newly discovered
HAA.
HONH-8-CH2OH-IQx, 2-(hydroxyamino)-8-hydroxymethyl-3-
methylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine; dG-C8-MeIQx, N-(deoxyguano-
sin-8-yl)-MeIQx; dG-N2-MeIQx, 5-(deoxyguanosin-N2-yl)-
MeIQx; dA-N6-MeIQx, 5-(deoxyadenosin-N6-yl)-MeIQx; ppb,
part per billion
Both MeIgQx and MeIQx undergo N-oxidation by mouse
liver microsomes and by recombinant human P450 1A2 at
similar rates. Moreover, recombinant human P450s 1A1 and
1B1, which are expressed in extrahepatic tissues,25,26 are far
more efficient at catalyzing the N-oxidation of MeIgQx than the
N-oxidation of MeIQx (Table 1). The differences in chemical
structures of MeIgQx and MeIQx appear to affect the
regioselectivities of P450-mediated N-oxidation of the exocyclic
amine groups, as well as the quinoxaline ring structures and
methyl groups of both HAAs. The higher CBI values of putative
MeIgQx adducts formed in the pancreas in comparison to
MeIQx may be attributed to more efficient bioactivation of
MeIgQx by extrahepatic P450s.
Historically, short-term bacterial mutagenesis assays have
been an effective screening tool for the identification of some
mutagenic HAAs in complex food matrices, but they do not
reliably predict carcinogenic potency in mammals.52 MeIgQx
forms apparent DNA adducts at levels that are comparable to
the levels of adducts formed by its carcinogenic isomer, MeIQx,
when adjusted for dose. Given the elevated concentrations
of MeIgQx formed in cooked meats, further studies on the
genetic toxicology and health risk of this novel HAA are clearly
warranted.
REFERENCES
■
(1) Sugimura, T., Wakabayashi, K., Nakagama, H., and Nagao, M.
(2004) Heterocyclic amines: Mutagens/carcinogens produced during
cooking of meat and fish. Cancer Sci. 95, 290−299.
(2) Felton, J. S., Jagerstad, M., Knize, M. G., Skog, K., and
Wakabayashi, K. (2000) Contents in Foods, Beverages and
Tobacco, in Food Borne Carcinogens Heterocyclic Amines (Nagao, M.,
and Sugimura, T., Eds.) pp 31−71, John Wiley & Sons Ltd.,
Chichester, England.
(3) Sinha, R., Rothman, N., Salmon, C. P., Knize, M. G., Brown,
E. D., Swanson, C. A., Rhodes, D., Rossi, S., Felton, J. S., and Levander,
O. A. (1998) Heterocyclic amine content in beef cooked by different
methods to varying degrees of doneness and gravy made from meat
drippings. Food Chem. Toxicol. 36, 279−287.
(4) Ni, W., McNaughton, L., LeMaster, D. M., Sinha, R., and
Turesky, R. J. (2008) Quantitation of 13 heterocyclic aromatic amines
in cooked beef, pork, and chicken by liquid chromatography-electrospray
ionization/tandem mass spectrometry. J. Agric. Food Chem. 56,
68−78.
(5) International Agency for Research on Cancer (1986) IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans: Tobacco
Smoking, International Agency for Research on Cancer, Lyon, France.
(6) International Agency for Research on Cancer (1993) IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans: Some
Naturally Occurring Substances: Food Items and Constituents, Heterocyclic
Aromatic Amines and Mycotoxins, International Agency for Research on
Cancer, Lyon, France.
(7) National Toxicology Program (2005) Report on Carcinogenesis,
11th ed., U.S. Department of Health and Human Services, Public
Health Service, Research Triangle Park, NC.
(8) Knize, M. G., Dolbeare, F. A., Carroll, K. L., Moore, D. H., and
Felton, J. S. (1994) Effect of cooking time and temperature on the
heterocyclic amine content of fried beef patties. Food Chem. Toxicol.
32, 595−603.
ASSOCIATED CONTENT
* Supporting Information
■
S
ESI/MS/MS product ion spectra of human liver microsomal
metabolites 8-CH2OH-HONH-MeIQx and HO-HONH-MeIgQx
derivatized with nitrosobenzene to form the respective azoxy
conjugates. This material is available free of charge via the Internet
at http://pubs.acs.org.
(9) Holland, R. D., Taylor, J., Schoenbachler, L., Jones, R. C.,
Freeman, J. P., Miller, D. W., Lake, B. G., Gooderham, N. J., and
Turesky, R. J. (2004) Rapid biomonitoring of heterocyclic aromatic
amines in human urine by tandem solvent solid phase extraction liquid
chromatography electrospray ionization mass spectrometry. Chem. Res.
Toxicol. 17, 1121−1136.
(10) Turesky, R. J., Taylor, J., Schnackenberg, L., Freeman, J. P., and
Holland, R. D. (2005) Quantitation of carcinogenic heterocyclic
aromatic amines and detection of novel heterocyclic aromatic amines
in cooked meats and grill scrapings by HPLC/ESI-MS. J. Agric. Food
Chem. 53, 3248−3258.
(11) Turesky, R. J., Goodenough, A. K., Ni, W., McNaughton, L.,
LeMaster, D. M., Holland, R. D., Wu, R. W., and Felton, J. S. (2007)
Identification of 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline: an
abundant mutagenic heterocyclic aromatic amine formed in cooked
beef. Chem. Res. Toxicol. 20, 520−530.
AUTHOR INFORMATION
Corresponding Author
*Phone: 518-474-4151. Fax: 518-473-2095. E-mail: Rturesky@
wadsworth.org.
■
Funding
This research was supported by grant R03ES016613 (to E.E.B.
and R.J.T.) from the National Institute of Environmental
Health Sciences, by grant number 05B025 from the American
Institute for Cancer Research (to R.J.T.), and by 2-PO1-
ES006052 (to R.L. and P.L.S.) from the National Institute of
Environmental Health Sciences.
ABBREVIATIONS
■
AMS, accelerator mass spectrometry; CBI, carcinogen binding
index; CNL/MS3, constant neutral loss-triple stage mass spect-
rometry scanning mode; HAA, heterocyclic aromatic amine;
LC-ESI/MS, Liquid chromatography-electrospray ionization/
mass spectrometry; MeIgQx, 2-amino-1,7-dimethylimidazo-
[4,5-g]quinoxaline; HONH-MeIgQx, 2-hydroxyamino-1,7-
dimethylimidazo[4,5-g]quinoxaline; MeIQx, 2-amino-3,
8-dimethylimidazo[4,5-f]quinoxaline; HONH-MeIQx, 2-hydrox-
yamino-3,8-dimethylimidazo[4,5-f]quinoxaline; 8-CH2OH-IQx,
2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline;
(12) Turesky, R. J., Lang, N. P., Butler, M. A., Teitel, C. H., and
Kadlubar, F. F. (1991) Metabolic activation of carcinogenic hetero-
cyclic aromatic amines by human liver and colon. Carcinogenesis 12,
1839−1845.
(13) Langouet, S., Welti, D. H., Kerriguy, N., Fay, L. B., Huynh-Ba,
̈
T., Markovic, J., Guengerich, F. P., Guillouzo, A., and Turesky, R. J.
(2001) Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
in human hepatocytes: 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-
carboxylic acid is a major detoxification pathway catalyzed by cytochrome
P450 1A2. Chem. Res. Toxicol. 14, 211−221.
419
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Chemical Research in Toxicology
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(14) Turesky, R. J., Rossi, S. C., Welti, D. H., Lay, J. O. Jr., and
Kadlubar, F. F. (1992) Characterization of DNA adducts formed in
vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]-
quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]-
quinoxaline at the C-8 and N2 atoms of guanine. Chem. Res. Toxicol.
5, 479−490.
metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo-
[4,5-f]quinoxaline in rat hepatocytes. Chem. Res. Toxicol. 3, 524−535.
(30) Turesky, R. J., Parisod, V., Huynh-Ba, T., Langouet, S., and
̈
Guengerich, F. P. (2001) Regioselective differences in C(8)- and
N-oxidation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by human
and rat liver microsomes and cytochromes P450 1A2. Chem. Res.
Toxicol. 14, 901−911.
(15) Turesky, R. J., Constable, A., Richoz, J., Varga, N., Markovic, J.,
Martin, M. V., and Guengerich, F. P. (1998) Activation of heterocyclic
aromatic amines by rat and human liver microsomes and by purified
rat and human cytochrome P450 1A2. Chem. Res. Toxicol. 11, 925−
936.
(31) Langouet, S., Paehler, A., Welti, D. H., Kerriguy, N.,
̈
Guillouzo, A., and Turesky, R. J. (2002) Differential metabolism of
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rat and human
hepatocytes. Carcinogenesis 23, 115−122.
(16) Gupta, R. C. (1993) 32P-postlabelling analysis of bulky aromatic
adducts. IARC Sci. Publ. 124, 11−23.
(32) Yamazoe, Y., Abu-Zeid, M., Manabe, S., Toyama, S., and Kato,
R. (1988) Metabolic activation of a protein pyrolysate promutagen
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat liver microsomes
and purified cytochrome P-450. Carcinogenesis 9, 105−109.
(33) Kadlubar, F. F., Kaderlik, K. R., Mulder, G. J., Lin, D.-X., Butler,
M. A., Teitel, C. H., Minchin, R. F., Ilett, K. F., Friesen, M. D., Bartsch,
H., Nagao, M., Esumi, E., Sugimura, T., and Lang, N. P. (1995)
Metabolic Activation and DNA Adduct Detection of PhIP in Dogs,
Rats, And Humans in Relation to Urinary Bladder and Colon
Carcinogenesis, in Heterocyclic Amines in Cooked Foods: Possible Human
Carcinogens (Adamson, R. H., Gustafsson, J.-A., Ito, N., Nagao, M.,
Sugimura, T., Wakabayashi, K, and Yamazoe, Y., Eds.) 23rd
Proceedings of the Princess Takamatusu Cancer Society, pp 207−
213, Princeton Scientific Publishing Co., Inc., Princeton, NJ.
(34) Kawazoe, Y., Ogawa, O., and Huang, G.-F. (1980) Studies
on chemical carcinogens XVII: Structure of carcinogenic 4-
hydroxaminoquinoline N-1 oxide derivatives. Tetrahedron 36, 2933−
2938.
(35) Bessette, E. E., Spivack, S. D., Goodenough, A. K., Wang, T.,
Pinto, S., Kadlubar, F. F., and Turesky, R. J. (2010) Identification of
carcinogen DNA adducts in human saliva by linear quadrupole ion
trap/multistage tandem mass spectrometry. Chem. Res. Toxicol. 23,
1234−1244.
(36) Nauwelaers, G., Bessette, E. E., Gu, D., Tang, Y., Rageul, J.,
Fessard, V., Yuan, J. M., Yu, M. C., Langouet, S., and Turesky, R. J.
(2011) DNA adduct formation of 4-aminobiphenyl and heterocyclic
aromatic amines in human hepatocytes. Chem. Res. Toxicol. 24, 913−
925.
(37) Bessette, E. E., Goodenough, A. K., Langouet, S., Yasa, I.,
Kozekov, I. D., Spivack, S. D., and Turesky, R. J. (2009) Screening for
DNA adducts by data-dependent constant neutral loss-triple stage
mass spectrometry with a linear quadrupole ion trap mass
spectrometer. Anal. Chem. 81, 809−819.
(38) Turteltaub, K. W., Felton, J. S., Gledhill, B. L., Vogel, J. S.,
Southon, J. R., Caffee, M. W., Finkel, R. C., Nelson, D. E., Proctor,
I. D., and Davis, J. C. (1990) Accelerator mass spectrometry in
biomedical dosimetry: relationship between low-level exposure and
covalent binding of heterocyclic amine carcinogens to DNA. Proc.
Natl. Acad. Sci. U.S.A. 87, 5288−5292.
(39) Lutz, W. K. (1979) In vivo covalent binding of organic
chemicals to DNA as a quantitative indicator in the process of
chemical carcinogenesis. Mutat. Res. 65, 289−356.
(17) Liberman, R. G., Tannenbaum, S. R., Hughey, B. J., Shefer, R. E.,
Klinkowstein, R. E., Prakash, C., Harriman, S. P., and Skipper, P. L.
(2004) An interface for direct analysis of (14)C in nonvolatile samples
by accelerator mass spectrometry. Anal. Chem. 76, 328−334.
(18) Goodenough, A. K., Schut, H. A., and Turesky, R. J. (2007)
Novel LC-ESI/MS/MSn method for the characterization and
quantification of 2′-deoxyguanosine adducts of the dietary
carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by 2-D
linear quadrupole ion trap mass spectrometry. Chem. Res. Toxicol. 20,
263−276.
(19) Paehler, A., Richoz, J., Soglia, J., Vouros, P., and Turesky, R. J.
(2002) Analysis and quantification of DNA adducts of 2-amino-3,8-
dimethylimidazo[4,5-f]quinoxaline in liver of rats by liquid
chromatography/electrospray tandem mass spectrometry. Chem. Res.
Toxicol. 15, 551−561.
(20) Kato, R., and Yamazoe, Y. (1987) Metabolic activation and
covalent binding to nucleic acids of carcinogenic heterocyclic amines
from cooked foods and amino acid pyrolysates. Jpn. J. Cancer Res. 78,
297−311.
(21) Kato, R. (1986) Metabolic activation of mutagenic heterocyclic
aromatic amines from protein pyrolysates. CRC Crit. Rev. Toxicol. 16,
307−348.
(22) Turesky, R. J. (2005) Interspecies metabolism of heterocyclic
aromatic amines and the uncertainties in extrapolation of animal
toxicity data for human risk assessment. Mol. Nutr. Food Res. 49, 101−
117.
(23) Butler, M. A., Iwasaki, M., Guengerich, F. P., and Kadlubar, F. F.
(1989) Human cytochrome P-450PA (P450IA2), the phenacetin
O-deethylase, is primarily responsible for the hepatic 3-demethylation
of caffeine and N-oxidation of carcinogenic arylamines. Proc. Natl.
Acad. Sci. U.S.A. 86, 7696−7700.
(24) Guengerich, F. P., and Turvy, C. G. (1991) Comparison of
levels of several human microsomal cytochrome P-450 enzymes and
epoxide hydrolase in normal and disease states using immunochemical
analysis of surgical liver samples. J. Pharmacol. Exp. Ther. 256, 1189−
1194.
(25) Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y.-Y. P., Jabs, E. W.,
Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) Complete
cDNA sequence of a human dioxin-inducible mRNA identifies a new
gene subfamily of cytochrome P450 that maps to chromosome 2*.
J. Biol. Chem. 269, 13092−13099.
(26) Ding, X., and Kaminsky, L. S. (2003) Human extrahepatic
cytochromes P450: function in xenobiotic metabolism and tissue-
selective chemical toxicity in the respiratory and gastrointestinal tracts.
Annu. Rev. Pharmacol. Toxicol. 43, 149−173.
(40) Itoh1, T, Suzuki, T., Nishikawa, A., Furukawa, F., Takahashi, M.,
Xue, W., Sofuni, T., and Hayashi, M. (2000) In vivo genotoxicity of
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in lacI transgenic (Big
Blue) mice. Mutat. Res. 468, 19−25.
(41) Okonogi, H., Ushijima, T., Shimizu, H., Sugimura, T., and
Nagao, M. (1997) Induction of aberrant crypt foci in C57BL/6N mice
by 2-amino-9H-pyrido[2,3-b]indole (AalphaC) and 2-amino-3,8-
dimethylimidazo[4,5-f]quinoxaline (MeIQx). Cancer Lett. 111, 105−
109.
(42) Shirai, T., Sano, M., Tamano, S., Takahashi, S., Hirose, M.,
Futakuchi, M., Hasegawa, R., Imaida, K., Matsumoto, K., Wakabayashi,
K., Sugimura, T., and Ito, N. (1997) The prostate: A target for
carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) derived from cooked foods. Cancer Res. 57, 195−198.
(27) Hammons, G. J., Milton, D., Stepps, K., Guengerich, F. P., and
Kadlubar, F. F. (1997) Metabolism of carcinogenic heterocyclic and
aromatic amines by recombinant human cytochrome P450 enzymes.
Carcinogenesis 18, 851−854.
(28) Crofts, F. G., Sutter, T. R., and Strickland, P. T. (1998)
Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by
human cytochrome P4501A1, P4501A2 and P4501B1. Carcinogenesis
19, 1969−1973.
(29) Turesky, R. J., Bracco-Hammer, I., Markovic, J., Richli, U., Kappeler,
A.-M., and Welti, D. H. (1990) The contribution of N-oxidation to the
420
dx.doi.org/10.1021/tx2004536 | Chem. Res. Toxicol. 2012, 25, 410−421

Chemical Research in Toxicology
Article
(43) Otteneder, M., and Lutz, W. K. (1999) Correlation of DNA
adduct levels with tumor incidence: carcinogenic potency of DNA
adducts. Mutat. Res. 424, 237−247.
(44) Sugimura, T. (1992) Multistep carcinogenesis: A 1992
perspective. Science 258, 603−607.
(45) Ghoshal, A., Preisegger, K. H., Takayama, S., Thorgeirsson, S. S.,
and Snyderwine, E. G. (1994) Induction of mammary tumors in
female Sprague-Dawley rats by the food-derived carcinogen 2-amino-
1-methyl-6-phenylimidazo[4,5-b]pyridine and effect of dietary fat.
Carcinogenesis 15, 2429−2433.
(46) Ubagai, T., Ochiai, M., Kawamori, T., Imai, H., Sugimura, T.,
Nagao, M., and Nakagama, H. (2002) Efficient induction of rat large
intestinal tumors with a new spectrum of mutations by intermittent
administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
in combination with a high fat diet. Carcinogenesis 23, 197−200.
(47) Schut, H. A., and Snyderwine, E. G. (1999) DNA adducts of
heterocyclic amine food mutagens: implications for mutagenesis and
carcinogenesis. Carcinogenesis 20, 353−368.
(48) Scribner, J. D., Fisk, S. R., and Scribner, N. K. (1979) Mechanisms
of action of carcinogenic aromatic amines: an investigation using muta-
genesis in bacteria. Chem.- Biol. Interact. 26, 11−25.
(49) Thompson, P. A., DeMarini, D. M., Kadlubar, F. F., McClure, G.
Y., Brooks, L. R., Green, B. L., Fares, M. Y., Stone, A., Josephy, P. D.,
and Ambrosone, C. B. (2002) Evidence for the presence of mutagenic
arylamines in human breast milk and DNA adducts in exfoliated breast
ductal epithelial cells. Environ. Mol. Mutagen. 39, 134−142.
(50) Radomski, J. L., Hearn, W. L., Radomski, T., Moreno, H., and
Scott, W. E. (1977) Isolation of the glucuronic acid conjugate of
N-hydroxy-4-aminobiphenyl from dog urine and its mutagenic activity.
Cancer Res. 37, 1757−1762.
(51) Fuscoe, J. C., Wu, R., Shen, N. H., Healy, S. K., and Felton, J. S.
(1988) Base-change analysis of revertants of the hisD3052 allele in
Salmonella typhimurium. Mutat. Res. 201, 241−251.
(52) Sugimura, T. (1988) Successful use of short-term tests for
academic purposes: Their use in identification of new environmental
carcinogens with possible risk for humans. Mutat. Res. 205, 33−39.
421
dx.doi.org/10.1021/tx2004536 | Chem. Res. Toxicol. 2012, 25, 410−421