Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline in humans

Article abstract of DOI:10.1021/tx9701891

The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in five human volunteers given a dietary equivalent of ¹⁴C-labeled MeIQx. The amount of the dose excreted in urine ranged from 20.2% to 58.6%, with unmetabolized MeIQx accounting for 0.7-2.8% of the dose. Five principal metabolites were detected in urine, and four of the derivatives were characterized by on-line UV spectroscopy and by HPLC-MS following immunoaffinity chromatography. Two metabolites were identified as the phase II conjugates N²,(3,8-dimethylimidazo[4,5-f]quinoxalin-2- yl)sulfamic acid (MeIQx-N²-SO₃^-) and N²-(β-1-glucosiduronyl)-2-amino- 3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx-N²-Gl). Two other metabolites were the cytochrome P450-mediated (P450) oxidation products 2-amino-8- (hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH₂OH-MeIQx), and N²- (β-1-glucosi duronyl)-N-hydroxy-2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (NOH-MeIQx-N²-Gl). The latter product is a conjugate of the genotoxic metabolite 2-(hydroxyamino)-3,8-dimethylimidazo- [4,5- f]quinoxaline (NHOH-MeIQx). A large interindividual variation was observed in the metabolism and disposition of MeIQx; these four metabolites and unchanged MeIQx combined accounted for 6.3-26.7% of the total dose. The remaining principal metabolite found in all subjects accounted for 7.6-28% of the dose. It has not been previously identified in rodents or nonhuman primates, and its structure remains unknown. P450-mediated ring oxidation of MeIQx at the C-5 position, a major pathway of detoxication in rodents, was not detected in humans. Both 8-CH₂OH-MeIQx formation and NHOH-MeIQx formation are catalyzed by P450 1A2 and may be useful biomarkers of P450 1A2 activity in humans. The levels of NHOH- MeIQx-N₂-Gl found in human urine ranged from 1.4% to 10.0% of the dose, which is significantly higher than that formed in rodents and nonhuman primates undergoing cancer bioassays. Thus, bioactivation of MeIQx by P450-mediated N-oxidation is extensive in humans.

Full text of DOI:10.1021/tx9701891

Chem. Res. Toxicol. 1998, 11, 217-225
217
Meta bolism of th e F ood -Bor n e Mu ta gen
2-Am in o-3,8-d im eth ylim id a zo[4,5-f]qu in oxa lin e in
Hu m a n s
Robert J . Turesky,*,† R. Colin Garner,‡ Dieter H. Welti,† J anique Richoz,†
Steve H. Leveson,§ Karen H. Dingley,‡,| Kenneth W. Turteltaub, and
Laurent B. Fay†
Nestle´ Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland,
J ack Birch Unit for Environmental Carcinogenesis, Department of Biology, University of York,
Heslington, York Y01 5DD, U.K., York District Hospital, Wiggington Road, York Y03 7HE, U.K.,
and Molecular and Structural Biology Division and Center for Accelerator Mass Spectrometry,
Lawrence Livermore National Laboratory, Livermore, California 94550
Received October 23, 1997
The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated
in five human volunteers given a dietary equivalent of 14C-labeled MeIQx. The amount of the
dose excreted in urine ranged from 20.2% to 58.6%, with unmetabolized MeIQx accounting for
0.7-2.8% of the dose. Five principal metabolites were detected in urine, and four of the
derivatives were characterized by on-line UV spectroscopy and by HPLC-MS following
immunoaffinity chromatography. Two metabolites were identified as the phase II conjugates
N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid (MeIQx-N2-SO3-) and N2-(â-1-
glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx-N2-Gl). Two other
metabolites were the cytochrome P450-mediated (P450) oxidation products 2-amino-8-
(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-MeIQx), and N2-(â-1-glucosi-
duronyl)-N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (NOH-MeIQx-N2-Gl). The
latter product is a conjugate of the genotoxic metabolite 2-(hydroxyamino)-3,8-dimethylimidazo-
[4,5-f]quinoxaline (NHOH-MeIQx). A large interindividual variation was observed in the
metabolism and disposition of MeIQx; these four metabolites and unchanged MeIQx combined
accounted for 6.3-26.7% of the total dose. The remaining principal metabolite found in all
subjects accounted for 7.6-28% of the dose. It has not been previously identified in rodents
or nonhuman primates, and its structure remains unknown. P450-mediated ring oxidation of
MeIQx at the C-5 position, a major pathway of detoxication in rodents, was not detected in
humans. Both 8-CH2OH-MeIQx formation and NHOH-MeIQx formation are catalyzed by P450
1A2 and may be useful biomarkers of P450 1A2 activity in humans. The levels of NHOH-
MeIQx-N2-Gl found in human urine ranged from 1.4% to 10.0% of the dose, which is significantly
higher than that formed in rodents and nonhuman primates undergoing cancer bioassays.
Thus, bioactivation of MeIQx by P450-mediated N-oxidation is extensive in humans.
gravies containing HAAs is associated with an increased
risk in certain types of cancers, including colorectal
cancer (8-12). Thus, there is concern that HAAs may
be human carcinogens.
The principal pathways of MeIQx metabolism and
detoxication have been elucidated in rodents (13-17) and
nonhuman primates (18) and include cytochrome P450
In tr od u ction
The cooking of meat, fish, and poultry results in the
formation of heterocyclic aromatic amines (HAAs)1 (1-
3). Nineteen HAAs have been identified in these cooked
foods, and thus far all HAAs examined induce tumors in
rodent carcinogen bioassays (3, 4). MeIQx is one of the
most abundant HAAs found in cooked foods, and it is
structurally representative of this class of chemicals (2,
5, 6). The diet is believed to be an important factor in
the etiology of cancer (7), and several studies have shown
that the frequent consumption of grilled meats and
1
Abbreviations: HAAs, heterocyclic aromatic amines; MeIQx,
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NHOH-MeIQx, 2-(hy-
droxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline; NO2-MeIQx, 2-ni-
tro-3,8-dimethylimidazo[4,5-f]quinoxaline; MeIQx-N2-SO3
,
N2-(3,8-
-
dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid; N2-acetyl-MeIQx,
2-(acetylamino)-3,8-dimethylimidazo[4,5-f]quinoxaline; MeIQx-N2-Gl,
N2-(â-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxa-
line; 8-CH2OH-MeIQx, 2-amino-8-(hydroxymethyl)-3-methylimidazo-
[4,5-f]quinoxaline; 5-HO-MeIQx, 2-amino-5-hydroxy-3,8-dimethylimidazo-
[4,5-f]quinoxaline; NHOH-8-(CH2OH)-MeIQx, 2-(hydroxyamino)-8-
(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline; NOH-MeIQx-N2-
Gl, N2-(â-1-glucosiduronyl)-N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-
f]quinoxaline; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; PCB,
polychlorinated biphenyls; UDPGA, uridine 5′-diphosphoglucuronic
acid; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; ANF, R-naph-
thoflavone; CID, collision-induced dissociation.
* To whom correspondence should be addressed: phone,
+41-21-785-8833;
fax,
+41-21-785-8553;
e-mail,
Turesky@
CHLSNR.NESTRD.CH.
† Nestec Ltd.
‡ University of York.
§ York District Hospital.
Lawrence Livermore National Laboratory.
| Current address: Molecular and Structural Biology Division and
Center for Accelerator Mass Spectrometry, Lawrence Livermore
National Laboratory, Livermore, CA.
S0893-228x(97)00189-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/20/1998

218 Chem. Res. Toxicol., Vol. 11, No. 3, 1998
Turesky et al.
human B lymphoblastoid cells were purchased from Gentest
Corp. (Woburn, MA). Human liver samples designated as
HL102, HL103, HL112, and HL127 were provided by Dr. F. P.
Guengerich, Vanderbilt University School of Medicine, Nash-
ville, TN, and human liver sample designated as HL-G was
kindly provided by Dr. F. F. Kadlubar, National Center for
Toxicological Research, J efferson, AR.
(P450)-mediated ring oxidation at the C-5 position,
followed by conjugation to sulfate or glucuronide. Direct
conjugation to the exocyclic amino group also occurs, and
N2-sulfamate and N2-glucuronide conjugates have been
detected in these species. In addition, glucuronidation
of MeIQx at the N1-imidazole moiety is a major pathway
of detoxication in nonhuman primates undergoing car-
cinogen bioassay (18). Metabolic activation of MeIQx
occurs by P450-mediated N-oxidation to form NHOH-
MeIQx, a direct-acting mutagen that readily binds to
DNA (19-21). NHOH-MeIQx is chemically unstable and
has not been detected in vivo; however, the N2-glu-
curonide conjugate of NHOH-MeIQx is stable and has
been detected in hepatocytes, bile, and urine of rodents
(16, 17).
Hu m a n Stu d y. Five human subjects who were undergoing
colorectal cancer surgery volunteered for this study. A written
protocol was provided to the subjects and followed-up by further
discussion on the nature of the study. All subjects participated
in the study after giving informed consent. The protocol of this
study was independently reviewed by and approved by the
Human Research Ethics Committee at York District Hospital,
the Department of Health Committee on the Administration of
Radioactive Substances to Persons-the Medicines (administra-
tion of radioactive substances) Regulation of 1978, United
Kingdom, and the Institutional Review Board at the Lawrence
Livermore National Laboratory. Each subject received [2-14C]-
MeIQx (4.3 µCi, 21 µg) which was placed in a gelatin capsule.
This dose is equivalent to the amount of MeIQx consumed in
several meals of well-done grilled beef and is comparable to the
level of total HAAs consumed in the daily diet (6, 27, 32).
Subjects took the capsule approximately 4-6 h prior to anes-
thesia and surgery (anesthetics varied with each patient and
were a combination of the following: thiopentone, propofol, or
etomidate; atracurium or naucuron was used as muscle relax-
ants). Dose adminstration was under the auspices of Dr. S. H.
Leveson, York District Hospital. Urine was collected up to 26
h posttreatment and stored frozen on dry ice.
Several pathways of MeIQx metabolism have been
characterized in vitro with human tissues. Human liver
microsomes N-hydroxylate MeIQx and other HAAs at
levels comparable to animal species which develop tu-
mors in long-term carcinogen bioassays (19, 20, 22-24).
Human liver and colon may further activate the N-
hydroxy-HAAs by N,O-acetyltransferase and sulfotrans-
ferase to form highly reactive esters that bind to DNA
(23, 25, 26). However, there is limited information on
the metabolism and disposition of MeIQx in humans.
Analysis of MeIQx in urine of humans following con-
sumption of fried meats has shown that the absorption
and metabolism of MeIQx are extensive with only 1-5%
of the oral dose excreted in urine as unchanged MeIQx
within 24 h (27-29). Furthermore, the amount of MeIQx
recovered in the urine of subjects who were treated with
the P450 1A2 inhibitor furafylline increased by as much
as 14-fold (30), indicating that P450 1A2 is an important
enzyme in metabolism of MeIQx in vivo. Another study
revealed that humans transform MeIQx by phase II
metabolism, and there was evidence of acid-labile MeIQx-
En zym e Assa ys. Liver microsomes were prepared by dif-
ferential centrifugation as previously described (17). Rates of
hepatic microsomal N-oxidation were determined using mi-
crosomal protein (1 mg/mL) and [2-14C]MeIQx (200 µM, 10 mCi/
mmol) in 100 mM potassium phosphate buffer (pH 7.6), con-
taining 0.5 mM EDTA, 5 mM glucose 6-phosphate, 1 unit/mL
glucose 6-phosphate dehydrogenase, 0.4 mM NADPH, and 0.3
mM NAD+. Incubation was conducted at 37 °C for 20 min.
Inhibition studies were conducted with R-naphthoflavone (ANF)
(10 µM) or furafylline (50 µM) where the inhibitor was pre-
incubated with microsomes for 3 or 8 min, respectively, prior
to the addition of MeIQx. The reaction was terminated by
addition of 3 volumes of chilled acetonitrile followed by removal
of the protein by centrifugation. The supernatant was concen-
-
N2-Gl and MeIQx-N2-SO3 conjugates present in urine
(29). The MeIQx content increased by 3-21-fold follow-
ing acid treatment of urine which quantitatively hydro-
lyzed these phase II conjugates to MeIQx, and these
products combined with MeIQx accounted for as much
as 14% of the ingested dose (29, 31).
trated under
a stream of nitrogen, and metabolites were
analyzed by HPLC with a Varian Vista 5000 system (Basel,
Switzerland) equipped with a Supelco C18 dB column (4 µm, 4.6
× 250 mm) (Buchs, Switzerland) connected to a Berthold LB
506 C-1 radioactivity monitor (Regensdorf, Switzerland). Me-
tabolites were eluted at a flow rate of 1 mL/min using a solvent
of 50 mM NH4CH3CO2 buffer (pH 6.8) and 20% (v/v) CH3OH
for 15 min, followed by a linear gradient to 100% CH3OH at 25
min, and held at 100% CH3OH for 5 min.
The direct spectroscopic characterization of MeIQx
metabolites in humans has not been reported, and the
major pathways of MeIQx metabolism in vivo remain to
be elucidated. In this study, we have investigated the
biotransformation of MeIQx in aged subjects who under-
went colorectal cancer surgery. Our objective was to
identify pathways of MeIQx metabolism in human urine
by UV and mass spectroscopic techniques following
immunoaffinity purification. The results show that
several urinary metabolites of MeIQx are derived from
P450 1A2-mediated oxidation and can be used as bio-
markers to aid in the human risk assessment of this
dietary mutagen.
Ur in a r y Meta bolite P u r ifica tion a n d An a lysis. Aliquots
of urine (10 mL) were added to 5 volumes of methanol-acetone
(1:1, v/v) on ice for 1 h. Protein and salt were removed by
centrifugation at 10000g for 20 min at 4 °C. The supernatant
was rotary evaporated to dryness and resuspended in 50 mM
NH4CH3CO2 buffer (pH 6.8) This extract was further purified
by solid-phase extraction with a C18 cartridge (29). The recovery
of radioactivity was 84 ( 14% (mean ( SD, n ) 10). After
evaporation to dryness, the extract was analyzed by HPLC with
a Varian Vista 5000 system equipped with a Supelco C18 dB
column (4 µm, 4.6 × 250 mm). Metabolites were eluted at a
flow rate of 1 mL/min using a solvent of 50 mM NH4CH3CO2
buffer (pH 5.0) and 11% (v/v) CH3OH for 10 min. This was
followed by a linear gradient which reached 20% (v/v) CH3OH
at 40 min, followed by an increase to 100% CH3OH at 51 min,
and held at 100% CH3OH for an additional 5 min. Fractions
were collected (30 s) and measured by liquid scintillation
counting.
Ma ter ia ls a n d Meth od s
Ca u tion : MeIQx and several of its derivatives are carcino-
genic to rodents and should be handled carefully.
Ch em ica ls a n d Rea gen ts. MeIQx and [2-14C]MeIQx (10
and 47 mCi/mmol) were purchased from Toronto Research
Chemicals, Inc. (Downsview, Ontario, Canada). Furafylline was
obtained from Ultrafine Chemicals (Manchester, U.K.). C18 EC
cartridges (1 g) were obtained from Machery Nagel (Du¨ren,
Germany). Human recombinant P450 1A2 microsomes (162
pmol of P450 1A2/mg of microsomal protein) derived from

MeIQx Metabolism in Humans
Chem. Res. Toxicol., Vol. 11, No. 3, 1998 219
Ta ble 1. P a tien t Diet, Dr u g, a n d Sm ok in g Histor ies
subject
1
age (years)
63
sex
M
drugs
smoking history
diet
no medication
exsmoker, does not come into
contact with other smokers
never smoked, does not come into
contact with other smokers
smoker, does not come into contact
with other smokers
exsmoker, regularly comes into
contact with other smokers
exsmoker, regularly comes into
contact with other smokers
medium-cooked meat, either fried
or roasted, >5 times/week
medium-done roasted meat in
casseroles, 3-5 times/week
well-done roasted meat in
casseroles, 3-5 times/week
well-done meat either grilled or
fried, every day
2
3
4
5
84
70
80
58
M
F
ferrous sulfate,
200 mg, 1/day
no medication
M
M
becotide v, intal,
salbutamol
aspirin, 1/day
well-done grilled meat,
3-5 times/week
Ta ble 2. P er cen t Dose Recover ed in Ur in e a s a F u n ction
of Tim ea
Urinary metabolites partially purified by solid-phase extrac-
tion were further purified by immunoaffinity chromatography
as previously reported (33). The immunopurified metabolites
were analyzed by HPLC as described above except that a
Hewlett-Packard 1090M HPLC (Geneva, Switzerland) equipped
with a diode array UV/vis detection system was employed for
on-line UV spectra acquistion. Metabolites were further char-
acterized by treatment with â-glucuronidase or arylsulfatase or
by acid hydrolysis in 1 or 6 N HCl as previously described (14,
16).
%
%
time 1
(h)
%
dose
time 2 cumulative time 3 cumulative
subject
(h)
dose
(h)
dose
1
2
3
4
5
6
4
4
5.5
3.5
12.6
12.6
36.4
47.6
26.0
12
6
12
12
12
19.6
36.8
37.6
58.6
35.6
24
26
25
24
25
20.2
46.0
41.4
58.6
38.6
a
Sp ectr oscop ic An a lyses. HPLC-MS analyses were carried
out on Finnigan MAT TSQ-700 and TSQ-7000 mass spectrom-
eters (Bremen, Germany) connected via a Finnigan electrospray
interface to a Waters 600-MS pump (Rupperswil, Switzerland)
or a Hewlett-Packard 1100 pump, respectively. Separation of
microsomal metabolites was done with the same HPLC condi-
tions as described above in Enzyme Assays. The solvent was
split 1:10 with a LC Packings Acurate microflow processor
(Omnilab, Commugny, Switzerland) before the mass spectrom-
eter and 100 µL of solvent entered the source. The electrospray
interface was operated with a high voltage of 4.5 kV and a
capillary temperature of 200 °C. Nitrogen was used as sheath
gas at a pressure of 80 psi. The mass spectrometer operated in
positive detection mode, using full scan mode from 50 to 400
Da. Analysis of human urinary metabolites following immuno-
affinity purification was performed as described above for
radiolabeled analyses except that the concentration of NH4-
CH3CO2 buffer was decreased to 10 mM. The effluent was also
split 1:10 before entering the MS, and a solution of acetic acid
(10%, v/v) was added via a two-way mixing tee post HPLC
column at a flow rate of 2 µL/min. MS/MS analyses of the
metabolites were carried out on the Finnigan TSQ-7000 mass
spectrometer after collision-induced dissociation (CID) of the
protonated molecular ions of each of the metabolites. A collision
energy of 20 eV was used for metabolites and 30 eV for MeIQx.
Argon was used as the collision gas at a pressure of 2.5 mTorr.
The capillary temperature was set at 220 °C. The capillary
voltage and nitrogen sheath gas were operated under the
conditions described above.
Time after the administration of MeIQx. The first time point
was obtained prior to surgery.
with 30 mg of liver S-9 protein from rats pretreated with PCB
in 4 mL of 100 mM KH2PO4 buffer (pH 7.6) containing 5 mM
glucose 6-phosphate, 1 mM NADPH, 1 mM NAD+, and 10 U of
glucose 6-phosphate dehydrogenase for 3 h at 37 °C. The 5-O-
glucuronide and 5-sulfate conjugates of 5-HO-MeIQx were
prepared by incubating MeIQx with rat liver S-9 protein as
above except that 10 mM PAPS or 20 mM UDPGA was added
as cofactors. The metabolites were purified and characterized
under conditions previously described (16). Comparable experi-
ments were conducted with MeIQx and human liver S-9 protein
which showed that the 5-O-glucuronide and 5-sulfate conjugates
of 5-HO-MeIQx were not formed.
1H NMR was conducted on approximately 40 µg of 8-CH2OH-
MeIQx obtained from the rat S-9 incubation. The 1H NMR
spectrum supported this proposed structure; the C-8 methyl
group of MeIQx at 2.74 ppm was absent, and a new signal which
integrated to two protons was found at 4.80 ppm. It occurred
as a singlet due to fast hydroxyl hydrogen exchange in solution.
All other protons of the MeIQx moiety (16) were detected [3.66
ppm, s, 3H, NCH3; 6.56 ppm, s, ca. 2H, D2O exchangeable, NH2;
7.59 ppm, d, 1H, J ) 8.6 Hz, H-5 (or H-4); 7.77 ppm, d, 1H, J )
8.7 Hz, H-4 (or H-5); 8.85 ppm, s, 1H, H-7]. The small amount
of sample did not permit NOE experiments to make the
assignments of the H-5 and H-4 protons unequivocal. The
electron impact mass spectrum also supported the structure
with a molecular ion detected at m/z 229.
Electron impact mass spectra were recorded from 20 to 500
Da at 70 eV on a Finnigan MAT 8430 double focusing mass
spectrometer working with an EI source heated at 180 °C.
Resu lts
1H NMR characterization of 8-CH2OH-MeIQx was performed
at 360 MHz with a Bruker AM-360 spectrometer (Spectrospin
AG, Fa¨llanden, Switzerland) equipped with a 5-mm 1H probe-
head under conditions previously described (16). The NMR
sample was prepared in DMSO-d6 with TMS as an internal
standard.
The diet, drug, and smoking histories of the subjects
are summarized in Table 1. All of the subjects consumed
cooked meat at least 3-5 times per week. Several
subjects were on medication, and four of the five volun-
teers have a smoking record. The absorption of [14C]-
MeIQx was rapid in all five subjects, and between 20.2%
and 58.6% of the administered dose was eliminated in
urine within 24 h, with 90% of the recovered dose
excreted within 12 h of treatment (Table 2). The rapid
absorption and elimination of MeIQx are in agreement
with previous reports (27-29).
Refer en ce Sta n d a r d Meta bolites. NHOH-MeIQx was
prepared by reduction of NO2-MeIQx with ascorbic acid and
-
purified as previously described (23). MeIQx-N2-SO3 was
prepared by treatment of MeIQx with chlorosulfonic acid (14).
N2-Acetyl-MeIQx was prepared by reaction of MeIQx with acetic
anhydride as previously described (23). NOH-MeIQx-N2-Gl and
MeIQx-N2-Gl were prepared by incubating 2 mg of NHOH-
MeIQx with 30 mg of liver S-9 protein from rats pretreated with
PCB (17) in 5 mL of 100 mM KH2PO4, 0.5 mM EDTA (pH 7.6)
containing 5 mM MgCl2 and 20 mM UDPGA for 3 h at 37 °C.
8-CH2OH-MeIQx was prepared by incubating 1 mg of MeIQx
Five principal metabolites were found in urine of all
subjects (Figure 1). Four of these metabolites were
previously identified in rodents and nonhuman primates
(13-18), and their structures are shown in Chart 1. The

220 Chem. Res. Toxicol., Vol. 11, No. 3, 1998
Turesky et al.
MeIQx and its metabolites were characterized by UV
and mass spectroscopy following immunoaffinity purifi-
cation in order to unambiguously confirm the proposed
structures. MeIQx and four of the metabolites were
recognized by the monoclonal antibodies, and purified by
immunoaffinity chromatography. Metabolite 1 was not
recognized by the monoclonal antibodies, and it was
resistant to enzymatic hydrolysis with â-glucuronidase
and arylsulfatase (14, 16, 17). Acid treatment (1 or 6 N
HCl at 100 °C for 18 h) did not modify the retention time
of this derivative by HPLC, whereas MeIQx-N2-Gl and
-
MeIQx-N2-SO3 were hydrolyzed under the same condi-
tions with quantitative regeneration of MeIQx.
The identities of the immunopurified metabolites were
substantiated by HPLC comigration with standards
prepared chemically or biosynthetically from rodent
tissues. The on-line UV spectra of the human metabo-
lites are superimposed with their rodent counterparts
and are in excellent agreement (Figure 2). The spectra
F igu r e 1. HPLC radiochromatogram of metabolites of MeIQx
in urine of a subject collected over 26 h. Under these chromato-
graphic conditions, the retention times of the 5-O-glucuronide
and 5-sulfate conjugates of 5-HO-MeIQx and N2-acetyl-MeIQx
were, respectively, 14, 23, and 47 min.
-
of MeIQx-N2-SO3 and 8-CH2OH-MeIQx are readily
Ch a r t 1. Ch em ica l Str u ctu r es of MeIQx a n d
Kn ow n Meta bolites F ou n d in Hu m a n Ur in e
distinguished from each other and from the glucuronide
metabolites, with respect to their maxima and minima.
However, the UV spectra of MeIQx-N2-Gl and NOH-
MeIQx-N2-Gl are nearly identical except that the ab-
sorbance maximum of NOH-MeIQx-N2-Gl has been shifted
by 2 nm from 274 to 272 nm.
The metabolites’ identities were further corroborated
by HPLC-MS/MS using selective reaction monitoring.
Table 4 summarizes the characteristic ions in the MS/
MS spectra of the reference standard MeIQx metabolites
obtained after CID of their protonated molecular ions.
The MS/MS spectrum of 8-CH2OH-MeIQx is character-
ized by an intense fragment at m/z 212 corresponding to
the loss of water from the protonated molecular ion. In
contrast, the CID of NOH-MeIQx-N2-Gl leads to the loss
of OH from the protonated molecular ion at m/z 406 as
well as loss of the glucuronide ([M + H - 176]+) to
produce protonated NHOH-MeIQx at m/z 230 as the base
peak. For MeIQx-N2-Gl and MeIQx-N2-SO3-, the CID
process generated the protonated parent MeIQx molecule
at m/z 214 as the base peak of both MS/MS spectra.
These specific fragmentations were used to scan the
metabolites purified from human urine, taking into
account the 2 amu mass increase due to the 14C-label.
Figure 3 shows the electrospray HPLC-MS/MS detection
of the MeIQx metabolites in human urine following
immunoaffinity purification.
Metabolism studies were undertaken in vitro using
human liver microsomes and microsomes of recombinant
human P450 1A2 (lymphoblastoid cells) to determine
whether the unknown urinary metabolite 1 is derived
from P450-mediated metabolism and to determine if
P450 1A2, the principal P450 involved in N-oxidation of
MeIQx and other HAAs (22-24, 34), is also involved in
the hydroxylation of the 8-CH3 position of MeIQx. Three
metabolites were formed by human liver microsomes and
microsomes containing recombinant human P450 1A2.
The HPLC profile and full scan mass spectra of the
metabolites derived from human liver microsomes are
shown in Figure 4. NHOH-MeIQx was the major me-
tabolite formed in both microsomal preparations which
is in agreement with previous reports (23, 24). The minor
metabolite was identified as 8-CH2OH-MeIQx, while the
structure of the remaining metabolite was tentatively
assigned as NHOH-8-(CH2OH)-MeIQx. The chemical
assignments of NHOH-MeIQx and 8-CH2OH-MeIQx were
major metabolite (metabolite 1) found in urine of all
subjects has not been detected in other species. The 5-O-
glucuronide and 5-sulfate conjugates of 5-HO-MeIQx,
which are prominent metabolites in urine and bile of rats
and nonhuman primates (15, 17, 18), were not detected
in human urine. In addition, N2-acetyl-MeIQx, which has
been detected as a minor metabolite in urine of rats (13),
was not excreted in urine of these human subjects.
There was a large interindividual variation in the
quantities of urinary excretion of MeIQx and its metabo-
lites (Table 3). Unchanged MeIQx accounted for 0.7-
2.8% of the dose. Metabolite 1, the prominent metabolite
in all subjects, accounted for 7.6-28.0% of the dose. The
-
ranges of MeIQx-N2-Gl and MeIQx-N2-SO3 excreted in
urine were 1.6-6.3% and 0.6-3.1% of the dose, respec-
tively. The amount of the NOH-MeIQx-N2-Gl excreted
in urine accounted for 1.4-10.0% of the dose. The levels
of this metabolite were highest in subjects 2, 4, and 5
who also had the highest amounts of 8-CH2OH-MeIQx
(1.0-4.4% of the dose).

MeIQx Metabolism in Humans
Chem. Res. Toxicol., Vol. 11, No. 3, 1998 221
Ta ble 3. Distr ibu tion of MeIQx Meta bolites in Hu m a n Ur in e: P er cen t of Dose Excr eted in Ur in e over 26 h a
% dose
in urine
-
subject
unknown 1
MeIQx-N2-Gl
NOH-MeIQx-N2-Gl
MeIQx-N2-SO3
8-CH2OH-MeIQx
MeIQx
1
2
3
4
5
7.6
23.5
28.0
18.6
17.6
1.6
5.8
3.7
6.3
4.5
1.9
3.5
1.4
10.0
4.1
0.6
1.9
0.8
3.1
2.2
1.0
2.2
1.3
4.4
2.5
1.2
1.2
0.7
2.8
1.7
20.2
46.0
41.4
58.6
38.6
a
The recovery of radioactivity from urine of five subjects (five urine samples in duplicate) determined following protein precipitation
and solid-phase extraction was 84 ( 14% (mean ( SD, n ) 10). The metabolite estimates were the average from two independent
measurements per subject and are normalized to 100% recovery. The duplicate values were within 10%. Spiking experiments of radiolabeled
metabolites at comparable levels in urine revealed that all metabolites were recovered at levels g85%.
F igu r e 2. On-line HPLC UV spectra of four known human urinary metabolites (dashed lines) superimposed with rodent metabolites
(solid lines). The HPLC buffer contained 50 mM NH4CO2 (pH 5.0) and CH3OH.
Ta ble 4. MS/MS Sp ectr a of th e Refer en ce Sta n d a r d Meta bolites
other
daughter ions
metabolitea,b
[M + H]+
[M + H - H2O]+
[M + H - OH]+
[M + H - Gl]+
[M + H - SO3]+
MeIQx-N2-Gl
390 (61)
406 (40)
230 (20)
294 (63)
372 (5)
214 (100)
230 (100)
286 (5), 256 (18)
242 (70), 213 (30)
197 (9)
NOH-MeIQx-N2-Gl
8-CH2OH-MeIQx
MeIQx-N2-SO3H
389 (20)
212 (100)
214 (100)
a
b
Spectra were obtained after CID at 20 eV of the protonated molecular ions. Values as m/z (relative intensity). The spectrum of
MeIQx was obtained at 30 eV with [M + H]+ at 45% relative intensity and [M + H - CH3]+ m/z ) 199 detected as the base peak.
based upon comigration and by comparison of the mass
spectra and UV spectra with synthetic standards (Figure
4). The assignment of NHOH-8-(CH2OH)-MeIQx was
based upon the mass spectrum which displayed a pro-
tonated molecular ion at m/z 246 associated with adduct
ions at m/z 268 ([M + Na]+) and 284 ([M + K]+). This
indicates incorporation of two oxygen atoms in the MeIQx
molecule. The mass spectrum showed a loss of oxygen
([M + H - OH]+) at m/z 229 which is characteristic of
FAB and electrospray mass spectra of NHOH-MeIQx and
not characteristic of the spectra of metabolites hy-
droxylated directly to the heterocyclic ring of MeIQx (16,
17, 35). The base peak was observed at m/z 214 ([M +
H - 32]+) which is suggestive of a loss of CH3OH;
however, this loss of M - 32 was not seen in the spectrum
of 8-CH2OH-MeIQx under these solvent conditions [50
mM NH4CH3CO2 buffer (pH 6.8), 20% CH3OH]. Further
support for this structure was based upon the UV spectra.
Hydroxylation of MeIQx at the 8-methyl position shifted
the absorbance maximum of MeIQx by 2 nm from 276 to
278 nm. The absorbance maximum of the proposed
NHOH-8-(CH2OH)-MeIQx also increased by 2 nm rela-
tive to NHOH-MeIQx from 274 to 276 nm. The low-
intensity maximum of this new metabolite at 345 nm and
the spectral changes observed at the minimum centered
at 300-310 nm are characteristic of NHOH-MeIQx. In
contrast, UV spectra of metabolites hydroxylated directly
to the heterocyclic ring system of MeIQx result in other
spectral perturbations (14, 16). The proposed NHOH-8-
(CH2OH)-MeIQx metabolite was present at higher levels
than 8-CH2OH-MeIQx in the microsomal incubations,
irrespective of the incubation time (5-180 min) or
substrate concentration (10-200 µM), suggesting that it
is derived from NHOH-MeIQx. Metabolite 1 in human

222 Chem. Res. Toxicol., Vol. 11, No. 3, 1998
Turesky et al.
F igu r e 3. Selected reaction monitoring electrospray HPLC-
MS/MS profile of MeIQx metabolites immuopurified from hu-
man urine. The daughter ions of MeIQ-N2-Gl and NOH-MeIQx-
N2-Gl were [M + H - Gl]+, MeIQx-N2-SO3H [M + H - SO3]+,
8-CH2OH-MeIQx [M + H - H2O]+, and MeIQx [M + H - CH3]+.
The [M + H]+ and daughter ions were selected at the m/z for
the corresponding 14C-labeled isotope metabolites, or 2 Da
higher than the mass of the unlabeled metabolites (see Table
3).
urine elutes at tR 3.5-4 min under the chromatographic
conditions employed for the in vitro microsomal metabo-
lite analysis. Therefore, the proposed NHOH-8-(CH2OH)-
MeIQx metabolite cannot be metabolite 1. Furthermore,
NHOH-(8-CH2OH)-MeIQx and NHOH-MeIQx are chemi-
cally unstable and oxidize overtime, while urinary me-
tabolite 1 is chemically stable.
F igu r e 4. HPLC-UV profile of MeIQx metabolites generated
in vitro from human liver microsomes after a 30-min incubation
at 37 °C and electrospray full scan mass spectra of microsomal
metabolites NHOH-MeIQx, 8-CH2OH-MeIQx, and NHOH-8-
(CH2OH)-MeIQx* whose proposed structure is based upon mass
and UV spectral data.
The rates of N-oxidation and 8-hydroxylation of MeIQx
by five different human liver microsome preparations
varied by approximately 4-fold (Table 5). The presence
of ANF (10 µM) or furafylline (50 µM), reported selective
inhibitors of P450 1A2 (30, 36), decreased the formation
of NHOH-MeIQx, 8-CH2OH-MeIQx, and NHOH-8-
(CH2OH)-MeIQx by greater than 95% in human liver
microsomes and in recombinant human P450 1A2 prepa-
rations. Thus, P450 1A2 is involved in the hydroxylation
of the 8-methyl group and N-oxidation of the exocyclic
amino group of MeIQx.
Ta ble 5. Micr osom a l Meta bolism of MeIQx w ith Hu m a n
Liver Micr osom es a n d Micr osom es of Hu m a n B
Lym p h obla stoid Cells Con ta in in g Recom bin a n t P 450 1A2
nmol of metabolite formed/min/mg
of microsomal protein
NHOH-(8-CH2OH)- 8-CH2OH-
NHOH-
MeIQx
sample code
MeIQx
MeIQx
HL102
HL103
HL112
HL127
0.15 ( 0.04
0.11 ( 0.07
0.16 ( 0.01
0.15 ( 0.01
0.37 ( 0.04
0.12 ( 0.03
0.04 ( 0.01 0.76 ( 0.08
0.02 ( 0.01 0.65 ( 0.05
0.05 ( 0.01 1.34 ( 0.10
0.04 ( 0.01 0.95 ( 0.03
0.07 ( 0.01 2.23 ( 0.12
0.03 ( 0.01 0.98 ( 0.11
HLG
recombinanta
P450 1A2
Discu ssion
Humans are exposed to HAAs daily through the diet
(1-4), and epidemiology studies indicate that frequent
consumption of grilled meats and gravies containing
HAAs increases the risk in certain types of cancer (8-
12). It is difficult to assess the contribution of HAAs to
the etiology of human cancers, and biomarkers are
required to assess metabolism, the biologically effective
dose, and the impact of polymorphisms on HAA geno-
toxicity in vivo. Our data provide the first direct spec-
a
Recombinant P450 1A2 microsomes contained 162 pmol of
P450 1A2/mg of microsomal protein.
troscopic characterization of MeIQx metabolites in hu-
man urine which can be used as biomarkers for
interspecies comparisons and risk assessment.
The extrapolation of the results from this study to data
obtained from rodents, nonhuman primates, and healthy

MeIQx Metabolism in Humans
Chem. Res. Toxicol., Vol. 11, No. 3, 1998 223
human subjects must be made with caution. In this
study, the volunteers underwent colorectal cancer sur-
gery and several of the subjects were elderly. MeIQx was
given in capsule form and not as part of the diet. In
addition, some of the subjects were under medication
prior to surgery. All of these factors, combined with the
anesthetics and surgery 4-6 h following treatment with
MeIQx, could have influenced the metabolism and dis-
position of MeIQx. Despite these caveats, several fea-
tures on the biodisposition of MeIQx in these subjects
are similar to those reported on healthy human subjects
(27-29) and other species (15, 17, 18). In this study, the
amount of unchanged MeIQx recovered in urine 24 h
following an oral dose of MeIQx ranged from 0.7% to
2.8%, which is comparable to the percent of unmetabo-
lized MeIQx excreted in urine of subjects who consumed
this HAA as part of a meat or fish diet (27-29). The
urinary MeIQx-N2-Gl and MeIQx-N2-SO3- accounted for,
respectively, 4.4 ( 1.9% and 1.7 ( 1.0% (mean ( SD, n
) 5 subjects) of the dose which is comparable to the levels
found in urine of healthy subjects measured indirectly
as acid-labile conjugates of MeIQx following consumption
of grilled meats (29, 31). The amounts of unchanged
purify this metabolite from larger volumes of urine to
fully characterize it by H NMR and mass spectrosopic
methods.
1
Another important metabolic feature which shows
strong interspecies differences is P450-mediated ring
oxidation of MeIQx at the C-5 position. This pathway of
MeIQx detoxication occurs in rodents and nonhuman
primates (15-18), but it does not appear to occur in
humans. In rats, the 5-sulfate and 5-O-glucuronide
conjugates of 5-hydroxy-MeIQx combined accounted for
approximately 50% of the MeIQx dose excreted in bile
and urine (17). We observed that both conjugates were
readily formed in vitro by incubation of MeIQx with rat
liver S-9 preparations fortified with PAPS and UDPGA.
In contrast, 5-HO-MeIQx was not formed by oxidation
of MeIQx with human liver microsomes (23, 24) or
recombinant human P450 1A2 (Table 5), and neither the
5-O-glucuronide nor 5-sulfate conjugates of 5-HO-MeIQx
was detected following incubation of MeIQx with human
liver S-9 preparations fortified with PAPS and UDPGA.
In agreement with these data, neither conjugate was
detected in the five human urine samples examined
(Figure 1, Table 2). The absence of this oxidation
pathway in human liver suggests that P450 1A1, which
is active in aromatic ring hydroxylation (37) but not
expressed in human liver (36-38), may be involved.
Alternatively, the regioselectivity of human and rat P450
1A2 may be different, and rat P450 1A2 may catalyze
both N-oxidation and 5-hydroxylation of MeIQx. This
question is currently under investigation.
N-Oxidation of MeIQx and other HAAs by P450 1A2
is the metabolic pathway responsible for DNA adduct
formation and mutagenesis (19-21, 34). The C-8 deoxy-
guanosine DNA adduct of MeIQx has been detected in
animals (39) and recently identified in human kidney and
colon biopsy samples by 32P-postlabeling (40). This
adduct is currently the only biomarker which can directly
measure the genetic damage of MeIQx. However, this
procedure requires a biopsy sample and cannot be
performed routinely on a large number of healthy sub-
jects. Therefore, the measurement of NOH-MeIQx-N2-
Gl which can be obtained noninvasively and can assess
the capacity of individuals to metabolically activate this
procarcinogen is of critical importance.
Glucuronidation of N-hydroxyarylamines is a means
of transport of the carcinogenic N-hydroxy metabolites
to tissues such as the colon and urinary bladder where
hydrolysis may occur, and the liberated N-hydroxy
derivatives can then react with DNA (41). In contrast
to acid-abile N2-glucuronides of N-hydroxyarylamines,
NOH-MeIQx-N2-Gl is stable and not hydrolyzed under
the mildly acidic pH of urine (16). The range of NOH-
MeIQx-N2-Gl recovered in urine of humans was 1.4-
10.0% of the total dose (Table 2), which is 5-10-fold
greater than the amount excreted in urine of rodents (17)
and nonhuman primates undergoing carcinogen bioassay
with MeIQx (18) or the structurally related analogue IQ
(42). Thus, P450 1A2-mediated N-oxidation of MeIQx in
humans appears to be considerably higher than in other
species; however, interspecies differences in glucuronida-
tion rates of NHOH-MeIQx (43) or in the proportion of
NOH-MeIQx-N2-Gl excreted in urine must also be con-
sidered.
MeIQx, MeIQx-N2-Gl, and MeIQx-N2-SO3 excreted in
-
urine of male rats given an oral dose of MeIQx (10 µg/
kg) (17) were also similar. Thus, the relative contribution
of these phase II pathways in detoxication of MeIQx is
similar in rats and humans.
There are several important differences in the metabo-
lism of MeIQx by humans, rodents, and nonhuman
primates. The prominent metabolite of MeIQx found in
the urine of human subjects, which accounts for as much
as 28% of the dose, has not been detected in other species.
Pharmacokinetic studies with the selective P450 1A2
inhibitor furafylline revealed that P450 1A2-catalyzed
metabolism of MeIQx in humans accounts for as much
as 91% of the elimination of ingested MeIQx (30),
suggesting that this novel metabolite 1 may be a P450
1A2-derived species. However, this product is not readily
formed in vitro by oxidation of MeIQx with human
microsomes (Figure 4, Table 5) or human liver homogen-
ates fortified with various cofactors. Mercapturic acid
conjugates derived from reaction with the carcinogenic
metabolites NHOH-MeIQx or N-acetoxy-MeIQx were
prepared; however, the reaction products with N-acetyl-
cysteine (or cysteine) do not have a similar HPLC
retention time as metabolite 1 (R. Turesky, unpublished
data).
Metabolite 1 was not recognized by the monoclonal
antibodies, and it could not be purified from urine by
immunoaffinity chromatography. We isolated metabolite
1 from 1 L of human urine using five different HPLC
purification steps and attempted characterization by
electron impact and FAB/MS, techniques which have
been successfully used for the characterization of other
MeIQx metabolites (35). However, meaningful spectra
were not obtained. Using LC/MS, a full scan spectrum
was acquired in positive ionization mode (HPLC solvent
A, 2% CH3CN in 0.1% CF3COOH; solvent B, 0.1%
CF3COOH in 95% CH3CN; gradient from 10% to 90% B
in 20 min), which displayed a protonated molecular ion
at m/z 244 and an associated adduct at m/z 285 ([M +
CH3CN + H]+). In addition, 14C-isotopic peaks were
detected, respectively, at m/z 246 and 287. These data
are suggestive of a dioxygenated metabolite, and different
structures could be postulated. It will be necessary to
Metabolic polymorphisms are believed to be an impor-
tant determinant of carcinogen susceptibility (44-47).
There is considerable human variability in the expression

224 Chem. Res. Toxicol., Vol. 11, No. 3, 1998
Turesky et al.
capillary column gas chromatography electron capture negative
ion chemical ionisation mass spectrometry. Carcinogenesis 9,
321-325.
and enzyme activity of P450 1A2 which is known to
activate a variety of potential human carcinogens includ-
ing HAAs (34, 38, 45). Interindividual differences in
human hepatic microsomal P450 1A2-mediated N-oxida-
tion rates of the aromatic amines 4-aminobiphenyl and
2-naphthylamine have been reported where rates varied
by 44-fold (48). Similar results were reported on the
metabolic activation of several HAAs by human liver (22).
A large variability of P450 1A2 activity also has been
demonstrated in vivo where P450 1A2-mediated caffeine
3-demethylation and phenacetin O-deethylation varied
by 50-70-fold in different subjects (49-51). In this pilot
study with five human subjects, the urinary NOH-
MeIQx-N2-Gl levels differed by 7-fold (Table 3). This is
similar to the extent of variation of MeIQx N-oxidation
rates observed in vitro with five different human hepatic
microsome preparations (Table 5), which may be reflec-
tive of variable P450 1A2 levels.
(6) Sinha, R., Rothman, N., Brown, E. D., Mark, S. D., Hoover, R.
N., Caporaso, N. E., Levander, O. A., Knize, M. G., Lang, N. P.,
and Kadlubar, F. F. (1994) Pan-fried meat containing high levels
of heterocyclic aromatic amines but low levels of polycyclic
aromatic hydrocarbons induces cytochrome P4501A2 activity in
humans. Cancer Res. 54, 6154-6159.
(7) Doll, R., and Peto, R. (1981) The causes of cancer: Quantitative
estimates of avoidable risks of cancer in the United States today.
J . Natl. Cancer Inst. 66, 1191-1308.
(8) Schiffman, M. H., and Felton, J . S. (1990) Letter to the editor:
Re: “Fried foods and the risk of colon cancer” Am. J . Epidemiol.
131, 376-378.
(9) Willet, W. C., Stampger, M. J ., Graham, A., Colditz, G. A., Rosner,
B. A., and Speizer, F. E. (1990) Relation of meat, fat, and fiber
intake to the risk of colon cancer in a prospective study among
women. N. Engl. J . Med. 323, 1664-672.
(10) Gerhardsson de Verdier, M., Hagman, U., Peters, R. K., Steineck,
G., and O¨ vervik, E. (1991) Meat, cooking methods and colorectal
cancer: A case-referent study in Stockholm. Int. J . Cancer 49,
520-525.
Because the bioactivation of MeIQx appears higher in
these human subjects than in animal species which
succumb to tumorigenesis in long-term feeding studies,
the human health risk of MeIQx and other HAAs may
be underestimated (52, 53). Metabolism and bio-
monitoring studies with healthy individuals who con-
sume MeIQx and other HAAs as part of the daily diet
are warranted as part of the risk assessment process. The
urinary NHOH-MeIQx-N2-Gl metabolite provides a meas-
ure of the individual’s capacity to metabolically activate
this HAA and thus a measure of P450 1A2 activity. The
measurement of urinary NOH-MeIQx-N2-Gl and DNA
adduct formation as indices of P450 1A2 N-oxidation and
genetic damage, combined with phenotypic and genotypic
markers of enzyme polymorphisms, may aid in further
assessing the human health risk of these dietary mu-
tagens.
(11) Muscat, J . E., and Wynder, E. L. (1994) The consumption of well-
done red meat and the risk of colorectal cancer. Am. J . Public
Health 84, 856-858.
(12) Giovannucci, E., Rimm, E. B., Stampfer, M. J ., Colditz, G. A.,
Ascherio, A., and Willett, W. C. (1994) Intake of fat, meat, and
fiber in relation to risk of colon cancer in men. Cancer Res. 54,
2390-2397.
(13) Hayatsu, H., Kasai, H., Yokoyama, S., Miyazawa, T., Yamaizumi,
Z., Sato, S., Nishimura, S., Arimoto, S., Hayatsu, T., and Ohara,
Y. (1987) Mutagenic metabolites in urine and feces of rats fed
with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a carcino-
genic mutagen present in cooked meat. Cancer Res. 47, 791-794.
(14) Turesky, R. J ., Aeschbacher, H. U., Wu¨rzner, H. P., Skipper, P.
L., and Tannenbaum, S. R. (1988) Major routes of metabolism of
the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]-
quinoxaline in the rat. Carcinogenesis 9, 1043-1048.
(15) Sjo¨din, P., Wallin, H., Alexander, J . A., and J a¨gerstad, M. (1989)
Disposition and metabolism of the food mutagen 2-amino-3,8-
dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rats. Carcino-
genesis 10, 1269-1275.
(16) 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 metabolism of the food-borne carcinogen
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in rat hepatocytes.
Chem. Res. Toxicol. 3, 524-535.
(17) Turesky, R. J ., Markovic, J ., Bracco-Hammer, I., and Fay, L. B.
(1991) The effect of dose and cytochrome P450 induction on the
metabolism and disposition of the food-borne carcinogen 2-amino-
3,8-dimethylimidazo[4, 5-f]quinoxaline. Carcinogenesis 12, 1847-
1855.
(18) Snyderwine, E. G., Welti, D. H., Davis, C. D., Fay, L. B., and
Turesky, R. J . (1995) Metabolism of the food-derived carcinogen
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in non-
human primates. Carcinogenesis 16, 1377-1384.
(19) Kato, R. (1986) Metabolic activation of mutagenic heterocyclic
aromatic amines from protein pyrolysates. CRC Crit. Rev. Toxicol.
16, 307-348.
Ack n ow led gm en t. The excellent technical work of
Mme. J . Markovic, Nestec Ltd. Nestle´ Research Center,
is greatly appreciated. We thank H. Link and Y. Saudan,
Nestec Ltd., for providing some of the monoclonal anti-
bodies. The authors wish to thank Dr. R. Mauthe for
preparing and characterizing the MeIQx encapsulation
and also Dr. J . Felton (Lawrence Livermore University),
Drs. S. Tannenbaum, P. Skipper, W. G. Stillwell, and J .
Wishnok (MIT), and Dr. F. Kadlubar (NCTR) for their
valuable comments on this manuscript. R.C.G., S.H.L.,
and K.H.D. were partially funded by the U.K. Ministry
of Agriculture, Fisheries and Food (Contract IC008/
CSA3424) and York Against Cancer. K.W.D. and K.H.D.
were funded by NIH CY55861 and USAMRDC
MM4559FLB.
(20) 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 mi-
crosomes and purified cytochrome P-450. Carcinogenesis 9, 105-
109.
Refer en ces
(21) Turesky, R. J ., Rossi, S. C., Welti, D. H., Lay, O., J r., 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.
(1) Felton, J . S., and Knize, M. G. (1990) Heterocyclic amine
mutagens/carcinogens in foods. In Handbook of Experimental
Pharmacology (Cooper, C. S., and Grover, P. L., Eds.) pp 471-
502, Springer-Verlag, Berlin, Heidelberg.
(2) Gross, G. A., Turesky, R. J ., Fay, L. B., Stillwell, W. G., Skipper,
P. L., and Tannenbaum, S. R. (1993) Heterocyclic aromatic amine
formation in grilled bacon, beef and fish and in grill scrapings.
Carcinogenesis 14, 2313-2318.
(3) Wakabayashi, K., Nagao, M., Esumi, H., and Sugimura, T. (1992)
Food-derived mutagens and carcinogens. Cancer Res. 52 (Suppl.),
2092s-2098s.
(4) Sugimura, T., and Nagao, M. (1993) Carcinogenic factors in food
with relevance to colon cancer development. Mutat. Res. 290, 43-
51.
(5) Murray, S., Gooderham, N. J ., Boobis, A. R., and Davies, D. S.
(1988) Measurement of MeIQx and DiMeIQx in fried beef by
(22) Yamazoe, Y., Abu-Zeid, M., Yamauchi, K., and Kato, R. (1988)
Metabolic activation of pyrolysate arylamines by human liver
microsomes; possible involvement of a P-448-H type cytochrome
P-450. J pn. J . Cancer Res. (Gann) 79, 1159-1167.
(23) Turesky, R. J ., Lang, N. P., Butler, M. A., Teitel, C. H., and
Kadlubar, F. F. (1991) Metabolic activation of carcinogenic
heterocyclic aromatic amines by human liver and colon. Carcino-
genesis 12, 1839-1845.
(24) Rich, K. J ., Murray, B. P., Lewis, I., Rendell, N. B., Davies, D. S.,
Gooderham, N. J ., and Boobis, A. R. (1992) N-Hydroxy-MeIQx is
the major microsomal oxidation product of the dietary carcinogen
MeIQx with human liver. Carcinogenesis 13, 2221-2226.

MeIQx Metabolism in Humans
Chem. Res. Toxicol., Vol. 11, No. 3, 1998 225
(25) Chou, H.-C., Lang, N. P., and Kadlubar, F. F. (1995) Metabolic
activation of N-hydroxy arylamines and N-hydroxy heterocyclic
amines by human sulfotransferase(s). Cancer Res. 55, 525-529.
(26) Minchin, R. F., Reeves, P. T., Teitel, C. H., McManus, M. E.,
Mojarrabi, B., Ilett, K. F., and Kadlubar, F. F. (1992) N- and
O-acetylation of aromatic and heterocyclic amine carcinogens by
human monomorphic and polymorphic acetyltransferases ex-
pressed in COS-1 cells. Biochem. Biophys. Res. Commun. 185,
839-844.
(27) Ushiyama, H., Wakabayashi, K., Hirose, M., Itoh, H., Sugimura,
T., and Nagao, M. (1991) Presence of carcinogenic heterocyclic
amines in urine of healthy volunteers eating normal diet, but not
of inpatients receiving parenteral alimentation. Carcinogenesis
12, 1417-1422.
(28) Lynch, A. M., Knize, M. G., Boobis, A. R., Gooderham, N., Davies,
D. S., and Murray, S. (1992) Intra- and interindividual variability
in systemic exposure in humans to 2-amino-3,8-dimethylimidazo-
[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]-
pyridine, carcinogens present in food. Cancer Res. 52, 6216-6223.
(29) Stillwell, W. G., Turesky, R. J ., Gross, G. A., Skipper, P. L., and
Tannenbaum, S. R. (1994) Human urinary excretion of sulfamate
and glucuronide conjugates of 2-amino-3,8-dimethylimdazo[4,5-
f]quinoxaline (MeIQx). Cancer Epidemiol. Biomarkers Prevention
3, 399-405.
(39) Ochiai, M., Nagaoka, H., Wakabayashi, K., Tanaka, Y., Kim, S.-
B., Tada, A., Nukaya, H., Sugimura, T., and Nagao, N. (1993)
Identification of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethyl-
imidazo[4,5-f]quinoxaline 3′,5′-diphosphate, a major DNA adduct,
detected by nuclease P1 modification of the 32P-postlabeling
method, in the liver of rats fed MeIQx. Carcinogenesis 24, 2165-
2170.
(40) Totsuka, Y., Fukutome, K., Takahashi, M., Takashi, S., Tada, A.,
Sugimura, T., and Wakabayashi, K. (1996) Presence of N2-
(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxa-
line (dG.-C8-MeIQx) in human tissues. Carcinogenesis 17, 1029-
1034.
(41) Kadlubar, F. F., Butler, M. A., Kaderlik, K. R., Chou, H.-C., and
Lang, N. P. (1992) Polymorphisms for aromatic amine metabolism
in humans: Relevance for human carcinogenesis. Environ. Health
Perspect. 98, 69-74.
(42) Snyderwine, E. G., Welti, D. H., Fay, L. B., Wu¨rzner, H. P., and
Turesky, R. J . (1992) Metabolism of the food-mutagen 2-amino-
3-methylimidazo[4,5-f]quinoline in nonhuman primates undergo-
ing carcinogen bioassay. Chem. Res. Toxicol. 5, 843-851.
(43) Kaderlik, K. R., Mulder, G. J ., Turesky, R. J ., Lang, N. P., Teitel,
C. H., Chiarelli, M. P., and Kadlubar, F. F. (1994) Glucuronidation
of N-hydroxy heterocyclic amines by human and rat liver mi-
crosomes. Carcinogenesis 15, 1695-1701.
(30) Boobis, A. R., Lynch, A. M., Murray, S., de la Torre, R., Solans,
A., Farre´, M., Segura, J ., Gooderham, N. J ., and Davies, D. S.
(1994) CYP1A2-catalyzed conversion of dietary heterocyclic amines
to their proximate carcinogens is their major route of metabolism
in humans. Cancer Res. 54, 89-94.
(31) Stillwell, W. G., Kidd, L. C., Wishnok, J . S., Tannenbaum, S. R.,
and Sinha, R. (1997) Urinary excretion of unmetabolized and
phase II conjugates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-
pyridine and 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline in
humans: Relationship to cytochrome P450 1A2 and N-acetyl-
transferase activity. Cancer Res. 57, 3457-3464.
(44) Ilett, K. F., David, B. M., Detchon, P., Castleden, W. M., and Kwa,
R. (1987) Acetylation phenotype in colorectal carcinoma. Cancer
Res. 47, 1466-1469.
(45) Eaton, D. L., Gallagher, E. P., Bammler, T. K., and Kunze, K. L.
(1995) Role of cytochrome P4501A2 in chemical carcinogenesis:
implications for human variability in expression and enzyme
activity. Pharmacogenetics 5, 259-274.
(46) Robert-Thomson, I. C., Ryan, P., Khoo, K. K., Hart, W. J .,
McMichael, A. J ., and Butler, R. N. (1996) Diet, acetylator
phenotypes, and risk of colorectal neoplasia. Lancet 347, 1372-
1374.
(32) Sinha, R., Rothman, N., Brown, E. D., Salmon, C. P., Knize, M.
G., Swanson, C. S., Rossi, S. C., Mark, S. D., Levander, O. A.,
and Felton, J . S. (1995) High concentrations of the carcinogen
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) occur in
chicken but are dependent on the cooking method. Cancer Res.
55, 4516-4519.
(47) Lang, N. P., Butler, M. A., Massengill, J ., Lawson, M., Stotts, R.
C., Hauer-J ensen, M., and Kadlubar, F. F. (1994) Rapid metabolic
phenotypes for acetyltransferase and cytochrome P4501A2 and
putative expsoure to food-borne heterocyclic mines increase the
risk for colorectal cancer or polyps. Cancer Epidemiol. Biomarkers
Prevention 3, 675-682.
(33) Turesky, R. J ., Forster, C. M., Aeschbacher, H. U., Wu¨rzner, H.
P., Skipper, P. L., Trudel, L. J ., and Tannenbaum, S. R. (1989)
Purification of the food-borne carcinogens 2-amino-3-methyl-
imidazo[4,5-f]quinoline and 2-amino-3,8-dimethlyimidazo[4,5-f]-
quinoxaline in heated meat products by immunoaffinity chroma-
tography. Carcinogenesis 10, 151-56.
(34) Shimada, T., Iwasaki, M., Martin, M. V., and Guengerich, F. P.
(1989) Human liver microsomal cytochrome P-450 enzymes
involved in the bioactivation of procarcinogens detected by umu
gene response in Salmonella typhimurium TA 1535/pSK1002.
Cancer Res. 49, 3218-3228.
(35) Fay, L. B., and Turesky, R. J . (1992) Electron impact and fast
atom bombardment mass spectrometric analysis of the food-borne
carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-
3,8-dimethlyimidazo[4,5-f]quinoxaline and their metabolites. Biol.
Mass Spectrom. 21, 463-469.
(36) Guengerich, F. P., and Shimada, T. (1991) Oxidation of toxic and
carcinogenic chemicals by human cytochrome P-450 enzymes.
Chem. Res. Toxicol. 4, 391-407.
(48) Butler, M. A., Guengerich, F. P., and Kadlubar, F. F. (1989)
Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4′-
methylenebis(2-chloroaniline) by human hepatic microsomes and
by purified rat hepatic cytochrome P-450 monooxygenases. Cancer
Res. 49, 25-31.
(49) Butler, M. A., Lang, N. P., Young, J . F., Caporaso, N. E., Vineis,
P., Hayes, R. B., Teitel, C. H., Massengill, J . P., Lawsen, M. F.,
and Kadlubar, F. F. (1992) Determination of CYP1A2 and NAT2
phenotypes in human populations by analysis of caffeine urinary
metabolites. Pharmacogenetics 2, 116-127.
(50) Vistisen, K., Poulsen, H. E., and Loft, S. (1992) Foreign compund
metabolism capacity in man measured from metabolites of dietary
caffeine. Carcinogenesis 13, 1561-1568.
(51) Alvares, A. P., Kappas, A., Eiseman, J . L., Anderson, K. E.,
Pantuck, C. B., Pantuck, E. J ., Hsiao, K. C., Garland, W. A., and
Conney, A. H. (1979) Interindividual variation in drug metabo-
lism. Clin. Pharmacol. Ther. 26, 407-419.
(52) Layton, D. W., Bogen, K. T., Knize, M. G., Hatch, F. T., J ohnson,
V. M., and Felton, J . S. (1995) Cancer risk of heterocyclic amines
in cooked foods: An analysis and implications for research.
Carcinogenesis 16, 39-52.
(53) Bogen, K. T. (1994) Cancer potencies of heterocyclic amines found
in cooked foods. Food Chem. Toxicol. 32, 505-515.
(37) Gonzalez, F. J ., and Gelboin, H. V. (1994) Role of human
cytochrome P450 in the metabolic activation of chemical carcino-
gens and toxins. Drug Metab. Disp. 26, 165-183.
(38) Schweikl, H., Taylor, J . A., Kitareewan, S., Linko, P., Nagorney,
D., and Goldstein, J . A. (1993) Expression of CYP1A1 and
CYP1A2 genes in human liver. Pharmacogenetics 3, 239-249.
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